Quality control complete notes
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About This Presentation
Quality control complete notes Pharm d 4th professional
Slide Content
QUALITY CONTROL
4
th
PROFESSIONAL
GHULAM MURTAZA HAMAD
4
TH
PROFF. EVENING
PUNJAB UNIVERSITY COLLEGE OF PHARMACY, LAHORE
4
th
PROFESSIONAL
GHULAM MURTAZA HAMAD
4
TH
PROFF. EVENING
PUNJAB UNIVERSITY COLLEGE OF PHARMACY, LAHORE
GM Hamad
TABLE OF CONTENTS
Contents
1. Introduction
2. Quality Control of Solid Dosage Form
3. Quality Control of Syrups, Elixirs and Disperse System
4. Quality Control of Suppositories
5. Quality Control of Sterile Products
6. Biological Assays
7. Alcohol Determinations
8. Alkaloidal Drug Assay
9. Quality Assurance of Vaccines
10. Miscellaneous Determinations and Tests
11. Standardization of Pharmaceuticals
12. Statistical Quality Control Charts
TABLE OF CONTENTS
Contents
1. Introduction
2. Quality Control of Solid Dosage Form
3. Quality Control of Syrups, Elixirs and Disperse System
4. Quality Control of Suppositories
5. Quality Control of Sterile Products
6. Biological Assays
7. Alcohol Determinations
8. Alkaloidal Drug Assay
9. Quality Assurance of Vaccines
10. Miscellaneous Determinations and Tests
11. Standardization of Pharmaceuticals
12. Statistical Quality Control Charts
GM Hamad
INTRODUCTION
QUALITY MANAGEMENT SYSTEM (QMS)
• Quality Management System a set of interacting elements based on
procedures, policies, resources, and objectives that are established
collectively to guide an organization.
• Quality Management System takes into account all applicable guidelines
and regulations that are designed to maintain its robustness.
QUALITY
• Quality is the name of attributes. They are highly specific characters and
vary from dosage form to dosage form. They are pre-defined before the
preparation of dosage form. In-short we set standards (specifications).
These specifications are mentioned in official books like; BP, USP and
NF.
QA, GMP & QC INTER-RELATIONSHIP
QUALITY ASSURANCE (QA)
• It is the sum total of the organized arrangements with the
objective of ensuring that products will be of the quality
required for their intended use.
GOOD MANUFACTURING PRACTICE (GMP)
• Part of QA system aimed at ensuring that products are consistently
manufactured to a quality appropriate to their intended use.
QUALITY CONTROL
• QC Is that part of GMP concerned with sampling, specification and
testing, documentation and release procedures which ensure that the
necessary and relevant tests are performed and the product is released
for use only after ascertaining its quality.
• Quality is always a comparative study with the standard. Standards are
of high purity. The standards are mentioned in official books,
Pharmacopoeia.
Difference between Quality Control (QC) and Quality Assurance (QA)
QC QA
QA
GMP
QC
1
INTRODUCTION
QUALITY MANAGEMENT SYSTEM (QMS)
• Quality Management System a set of interacting elements based on
procedures, policies, resources, and objectives that are established
collectively to guide an organization.
• Quality Management System takes into account all applicable guidelines
and regulations that are designed to maintain its robustness.
QUALITY
• Quality is the name of attributes. They are highly specific characters and
vary from dosage form to dosage form. They are pre-defined before the
preparation of dosage form. In-short we set standards (specifications).
These specifications are mentioned in official books like; BP, USP and
NF.
QA, GMP & QC INTER-RELATIONSHIP
QUALITY ASSURANCE (QA)
• It is the sum total of the organized arrangements with the
objective of ensuring that products will be of the quality
required for their intended use.
GOOD MANUFACTURING PRACTICE (GMP)
• Part of QA system aimed at ensuring that products are consistently
manufactured to a quality appropriate to their intended use.
QUALITY CONTROL
• QC Is that part of GMP concerned with sampling, specification and
testing, documentation and release procedures which ensure that the
necessary and relevant tests are performed and the product is released
for use only after ascertaining its quality.
• Quality is always a comparative study with the standard. Standards are
of high purity. The standards are mentioned in official books,
Pharmacopoeia.
Difference between Quality Control (QC) and Quality Assurance (QA)
QC QA
QA
GMP
QC
1
GM Hamad
QC is that part of GMP which is
concerned with Sampling,
Specifications testing and within the
organization, documentation and
release procedures which ensure that
the necessary and relevant tests are
carried out.
QA is the sum total of organized
arrangements made with the object
of ensuring that product will be of
the quality required for their
intended use.
Operational laboratory techniques
and activities used to fulfill the
requirement of quality.
All those planned or systematic
actions necessary to provide
adequate confidence that a product
will satisfy the requirements for
quality.
QC is lab based. QA is company based.
CURRENT GOOD MANUFACTURING PRACTICES (cGMP)
• cGMP refers to the Current Good Manufacturing Practice regulations
enforced by the US Food and Drug Administration (FDA).
• cGMP provide for systems that assure proper design, monitoring and
control of manufacturing processes and facilities.
GOOD LABORATORY PRACTICES (GLP)
• GLP embodies a set of principles that provides a frame-work within
which laboratory studies are planned, performed, monitored and
archived and reported.
• GLP is an FDA regulation.
PURPOSE OF GLP
• GLP is to certify that every step of the analysis is valid or not.
• Assure the quality and integrity of data submitted to FDA in support of
the safety of regulated products.
• GLPs have heavy emphasis on data recording, record and specimen
retention.
GOOD LABORATORY PRACTICES PRINCIPLES
1. Test facility management.
2. Quality assurance program (QAP).
3. Facilities.
4. Apparatus, material and reagents.
5. Test systems.
6. Test and reference substances.
2
QC is that part of GMP which is
concerned with Sampling,
Specifications testing and within the
organization, documentation and
release procedures which ensure that
the necessary and relevant tests are
carried out.
QA is the sum total of organized
arrangements made with the object
of ensuring that product will be of
the quality required for their
intended use.
Operational laboratory techniques
and activities used to fulfill the
requirement of quality.
All those planned or systematic
actions necessary to provide
adequate confidence that a product
will satisfy the requirements for
quality.
QC is lab based. QA is company based.
CURRENT GOOD MANUFACTURING PRACTICES (cGMP)
• cGMP refers to the Current Good Manufacturing Practice regulations
enforced by the US Food and Drug Administration (FDA).
• cGMP provide for systems that assure proper design, monitoring and
control of manufacturing processes and facilities.
GOOD LABORATORY PRACTICES (GLP)
• GLP embodies a set of principles that provides a frame-work within
which laboratory studies are planned, performed, monitored and
archived and reported.
• GLP is an FDA regulation.
PURPOSE OF GLP
• GLP is to certify that every step of the analysis is valid or not.
• Assure the quality and integrity of data submitted to FDA in support of
the safety of regulated products.
• GLPs have heavy emphasis on data recording, record and specimen
retention.
GOOD LABORATORY PRACTICES PRINCIPLES
1. Test facility management.
2. Quality assurance program (QAP).
3. Facilities.
4. Apparatus, material and reagents.
5. Test systems.
6. Test and reference substances.
2
GM Hamad
7. Standard operating procedures (SOP).
8. Performance of the study.
9. Reporting of study results.
10. Storage and retention of records and materials.
VALIDATION
• Validation is the assessment of a process or instrument to assure that
the process and instrument is suitable for its intended use (FDA, 1987).
• Validation enables an efficient and productive use of the process and
instrumental variables.
• A new assay method, change in operator, laboratory and equipment
than the one in previous method requires validation.
STEPS IN VALIDATION
• Specificity
• Linearity
• Range
• Accuracy
• Precision
• Sensitivity
3
7. Standard operating procedures (SOP).
8. Performance of the study.
9. Reporting of study results.
10. Storage and retention of records and materials.
VALIDATION
• Validation is the assessment of a process or instrument to assure that
the process and instrument is suitable for its intended use (FDA, 1987).
• Validation enables an efficient and productive use of the process and
instrumental variables.
• A new assay method, change in operator, laboratory and equipment
than the one in previous method requires validation.
STEPS IN VALIDATION
• Specificity
• Linearity
• Range
• Accuracy
• Precision
• Sensitivity
3
GM Hamad
QUALITY CONTROL OF SOLID DOSAGE
FORM
• QC is strictly observed in order to ensure that the product is not only
meeting the requisite specifications but also a reproducible in term of
quality, hence therapeutically effective.
• Solid dosage forms are:
Tablets
Capsules
Powders
Granules
QUALITY CONTROL OF TABLETS
INTRODUCTION
• Tablets are solid preparation intended for oral administration containing
unit dose of one or more medicaments.
• Tablets are prepared by compressing uniform volume of particles
through:
Direct Compression
Wet Granulation
Dry Granulation. Also known as:
▪ Slugging and double compression.
• They are swallowed whole or dissolved/dispersed in water before
administration.
QUALITY CONTROL TESTS FOR TABLETS
Quality control tests for tablets includes:
PHYSICAL TESTS
• Tablet Hardness
• Thickness and diameter
• Friability
• Disintegration test
• Weight variation
4
QUALITY CONTROL OF SOLID DOSAGE
FORM
• QC is strictly observed in order to ensure that the product is not only
meeting the requisite specifications but also a reproducible in term of
quality, hence therapeutically effective.
• Solid dosage forms are:
Tablets
Capsules
Powders
Granules
QUALITY CONTROL OF TABLETS
INTRODUCTION
• Tablets are solid preparation intended for oral administration containing
unit dose of one or more medicaments.
• Tablets are prepared by compressing uniform volume of particles
through:
Direct Compression
Wet Granulation
Dry Granulation. Also known as:
▪ Slugging and double compression.
• They are swallowed whole or dissolved/dispersed in water before
administration.
QUALITY CONTROL TESTS FOR TABLETS
Quality control tests for tablets includes:
PHYSICAL TESTS
• Tablet Hardness
• Thickness and diameter
• Friability
• Disintegration test
• Weight variation
4
GM Hamad
CHEMICAL TESTS
• Content uniformity
• Assay of active ingredients
• Dissolution test
UNOFFICIAL TESTS
1. HARDNESS
• Hardness is related to solubility, proper hardness for tablets ensures that
tablet withstand the shock of handling, packing and shipping.
• Common hardness tester:
Strong-cobb
Monsanto tester
Eureka
Pfizer units
MECHANICAL TESTER
• Hardness is normally tested by mechanical tester now a days with
automatic operation. Mechanical tester measures resistance to
crushing of tablets.
• Force is applied by a beam. One end of beam is attached to pivot
controlled mechanically by a motor. The other end rests on
tablets. Motor moves the beam which applies force on tablets.
When the tablet breaks a micro switch stops the motor.
Mechanical strength is shown in the digital indicator.
HARDNESS SPECIFICATIONS
• Acceptable hardness range is 5 – 10 Kg/cm
-2
. Sometimes the scale is in
Newton (1 Newton = 9.8 kg)
2. THICKNESS AND DIAMETER
• Checking of thickness and diameter is usually an in-process quality
control check during production. Dimensional specifications of tablets
are very important because of many reasons:
Packaging requirements
Patient compliance
• Thickness is often related to tablet’s hardness.
• Thickness is set according to tablet weight.
APPARATUS
• The apparatus used for this purpose are:
5
CHEMICAL TESTS
• Content uniformity
• Assay of active ingredients
• Dissolution test
UNOFFICIAL TESTS
1. HARDNESS
• Hardness is related to solubility, proper hardness for tablets ensures that
tablet withstand the shock of handling, packing and shipping.
• Common hardness tester:
Strong-cobb
Monsanto tester
Eureka
Pfizer units
MECHANICAL TESTER
• Hardness is normally tested by mechanical tester now a days with
automatic operation. Mechanical tester measures resistance to
crushing of tablets.
• Force is applied by a beam. One end of beam is attached to pivot
controlled mechanically by a motor. The other end rests on
tablets. Motor moves the beam which applies force on tablets.
When the tablet breaks a micro switch stops the motor.
Mechanical strength is shown in the digital indicator.
HARDNESS SPECIFICATIONS
• Acceptable hardness range is 5 – 10 Kg/cm
-2
. Sometimes the scale is in
Newton (1 Newton = 9.8 kg)
2. THICKNESS AND DIAMETER
• Checking of thickness and diameter is usually an in-process quality
control check during production. Dimensional specifications of tablets
are very important because of many reasons:
Packaging requirements
Patient compliance
• Thickness is often related to tablet’s hardness.
• Thickness is set according to tablet weight.
APPARATUS
• The apparatus used for this purpose are:
5
GM Hamad
Micrometer screw gauge
Vernier caliper
• Now a days digital micrometers are available.
THICKNESS SPECIFICATION
• Thickness of tablet varies from 2 – 4 mm depending upon diameter of
tablet.
• A deviation of ± 5% from stated diameter is allowed except that for
exceeding 12.5 mm.
• For 12.5 mm or above deviation is ± 3%
3. FRIABILITY
• Friction and shock during tableting can cause tablet to chip, cap and
break. Loss of weight due to abrasion of friction is the measure of
tablet’s friability.
• The apparatus used for the purpose is:
Roche Friabilator
ROCHE FRIABILATOR
• It consist of hard plastic cylinder of 6-inch radius. Motor which
rotates cylinder at constant speed.
• A drum of transparent synthetic polymer with polished internal
surfaces. One side of the drum is removable. The tablets are
tumbled at each turn of the drum by a curved projection that
extends from the middle of the drum to the outer wall.
• The drum is attached to the horizontal axis of a device that rotates at 25
± 1 r/min. Thus, at each turn the tablets roll or slide and fall onto the
drum wall or onto each other.
• If tablet size or shape causes irregular tumbling, adjust the drum base so
that the base forms an angle of about 10° with the horizontal and the
tablets no longer bind together when lying next to each other, which
prevents them from falling freely.
• Effervescent tablets and chewable tablets may have different
specifications as far as friability is concerned. In the case of hygroscopic
tablets, a humidity-controlled environment is required for testing.
• For tablets with a unit mass equal to or less than 650 mg, take a sample
of whole tablets corresponding as near as possible to 6.5 g. For tablets
with a unit mass of more than 650 mg, take a sample of 10 whole
6
Micrometer screw gauge
Vernier caliper
• Now a days digital micrometers are available.
THICKNESS SPECIFICATION
• Thickness of tablet varies from 2 – 4 mm depending upon diameter of
tablet.
• A deviation of ± 5% from stated diameter is allowed except that for
exceeding 12.5 mm.
• For 12.5 mm or above deviation is ± 3%
3. FRIABILITY
• Friction and shock during tableting can cause tablet to chip, cap and
break. Loss of weight due to abrasion of friction is the measure of
tablet’s friability.
• The apparatus used for the purpose is:
Roche Friabilator
ROCHE FRIABILATOR
• It consist of hard plastic cylinder of 6-inch radius. Motor which
rotates cylinder at constant speed.
• A drum of transparent synthetic polymer with polished internal
surfaces. One side of the drum is removable. The tablets are
tumbled at each turn of the drum by a curved projection that
extends from the middle of the drum to the outer wall.
• The drum is attached to the horizontal axis of a device that rotates at 25
± 1 r/min. Thus, at each turn the tablets roll or slide and fall onto the
drum wall or onto each other.
• If tablet size or shape causes irregular tumbling, adjust the drum base so
that the base forms an angle of about 10° with the horizontal and the
tablets no longer bind together when lying next to each other, which
prevents them from falling freely.
• Effervescent tablets and chewable tablets may have different
specifications as far as friability is concerned. In the case of hygroscopic
tablets, a humidity-controlled environment is required for testing.
• For tablets with a unit mass equal to or less than 650 mg, take a sample
of whole tablets corresponding as near as possible to 6.5 g. For tablets
with a unit mass of more than 650 mg, take a sample of 10 whole
6
GM Hamad
tablets. The tablets are carefully dedusted prior to testing. Accurately
weigh the tablet sample and place the tablets in the drum. Rotate the
drum 100 times and remove the tablets. Remove any loose dust from
the tablets as before, and accurately weigh.
• Generally, the test is run once. If obviously cracked, cleaved, or broken
tablets are present in the tablet sample after tumbling, the sample fails
the test. If the results are difficult to interpret or if the weight loss is
greater than the targeted value, the test is repeated twice and the mean
of the 3 tests determined. A maximum loss of mass (obtained from a
single test or from the mean of 3 tests) not greater than 1.0 per cent is
considered acceptable for most products.
SPECIFICATIONS OF FRIABILITY
• The USP states that the friability should be 0.8 – 1%.
4. WEIGHT VARIATION TEST
• Compression weight, actual weight of tablet is determined by the
diameter of the die and weight adjustment cam on tablet compression
machine.
• Weight control on tablet is continuously checked and adjusted during
compression of whole batch. It is normally done on uncoated tablets.
APPARATUS
• Digital weighing balances are used for the purpose.
PROCEDURE FOR WEIGHT VARIATION
• Take 20 tablets at random from the given batch.
• Weigh the 20 tablets individually.
• Determine average weight of the tablets.
• Note down the deviation in weight for each tablet (may be + or -).
• Determine %age deviation for the individual tablet by using the formula:
%Deviation=
Deviation in weight
Average weight
×100
• Compare the percentage deviation with the specification given in official
table (B.P. or U.S.P.)
WEIGHT VARIATION SPECIFICATIONS
7
tablets. The tablets are carefully dedusted prior to testing. Accurately
weigh the tablet sample and place the tablets in the drum. Rotate the
drum 100 times and remove the tablets. Remove any loose dust from
the tablets as before, and accurately weigh.
• Generally, the test is run once. If obviously cracked, cleaved, or broken
tablets are present in the tablet sample after tumbling, the sample fails
the test. If the results are difficult to interpret or if the weight loss is
greater than the targeted value, the test is repeated twice and the mean
of the 3 tests determined. A maximum loss of mass (obtained from a
single test or from the mean of 3 tests) not greater than 1.0 per cent is
considered acceptable for most products.
SPECIFICATIONS OF FRIABILITY
• The USP states that the friability should be 0.8 – 1%.
4. WEIGHT VARIATION TEST
• Compression weight, actual weight of tablet is determined by the
diameter of the die and weight adjustment cam on tablet compression
machine.
• Weight control on tablet is continuously checked and adjusted during
compression of whole batch. It is normally done on uncoated tablets.
APPARATUS
• Digital weighing balances are used for the purpose.
PROCEDURE FOR WEIGHT VARIATION
• Take 20 tablets at random from the given batch.
• Weigh the 20 tablets individually.
• Determine average weight of the tablets.
• Note down the deviation in weight for each tablet (may be + or -).
• Determine %age deviation for the individual tablet by using the formula:
%Deviation=
Deviation in weight
Average weight
×100
• Compare the percentage deviation with the specification given in official
table (B.P. or U.S.P.)
WEIGHT VARIATION SPECIFICATIONS
7
GM Hamad
• Not more than two of individual weights should deviate by more than
given percentage.
• And none should deviate by more than twice that percentage.
OFFICIAL TABLE
Avg. weight of
tablet (USP)
%age Deviation
Avg. weight of
tablet (BP)
%age Deviation
130 mg or less ± 10.0 % 80 mg or less ± 10.0 %
For 130 mg to
324 mg
± 7.5 %
> 80 mg to < 250
mg
± 7.5 %
More than 324
mg
± 5.0 % 250 mg or more ± 5.0 %
OFFICIAL TESTS
1. DISINTEGRATION TEST
• It is the time required for the tablet to break into particles, the
disintegration test is a measure only to the time required under a given
set of conditions for a group of tablets to disintegrate into particles.
• Complete disintegration is defined as that state in which any residue of
the tablet, except fragments of insoluble coating remaining on the
screen of the test apparatus. Soft mess having no firm core remains.
• It is done because of following reasons:
To ensure product uniformity
Attempts are made to simulate in-vivo conditions. Actually, test
does not correlate with physiological conditions.
It is done as a process control.
DISINTEGRATION APPARATUS
• Basket rack assembly
• Discs
• Thermostat
• Suitable vessel of immersion fluid
• Immersion fluid
• A motoring device for raising and lowering the basket assembly in fluid
BASKET RACK ASSEMBLY
• It consist of six open ended glass tubes each 7.5 ± 0.25 cm long and
inside diameter approx. 21.5 mm and wall thickness is approx. 2mm.
8
• Not more than two of individual weights should deviate by more than
given percentage.
• And none should deviate by more than twice that percentage.
OFFICIAL TABLE
Avg. weight of
tablet (USP)
%age Deviation
Avg. weight of
tablet (BP)
%age Deviation
130 mg or less ± 10.0 % 80 mg or less ± 10.0 %
For 130 mg to
324 mg
± 7.5 %
> 80 mg to < 250
mg
± 7.5 %
More than 324
mg
± 5.0 % 250 mg or more ± 5.0 %
OFFICIAL TESTS
1. DISINTEGRATION TEST
• It is the time required for the tablet to break into particles, the
disintegration test is a measure only to the time required under a given
set of conditions for a group of tablets to disintegrate into particles.
• Complete disintegration is defined as that state in which any residue of
the tablet, except fragments of insoluble coating remaining on the
screen of the test apparatus. Soft mess having no firm core remains.
• It is done because of following reasons:
To ensure product uniformity
Attempts are made to simulate in-vivo conditions. Actually, test
does not correlate with physiological conditions.
It is done as a process control.
DISINTEGRATION APPARATUS
• Basket rack assembly
• Discs
• Thermostat
• Suitable vessel of immersion fluid
• Immersion fluid
• A motoring device for raising and lowering the basket assembly in fluid
BASKET RACK ASSEMBLY
• It consist of six open ended glass tubes each 7.5 ± 0.25 cm long and
inside diameter approx. 21.5 mm and wall thickness is approx. 2mm.
8
GM Hamad
• Tubes are held vertically with the help of two plastic plates each about 9
cm in diameter and 6 mm in thickness. Plastic plates consist of 6 holes
each about 24 mm diameter, equidistant from the center of plate and
equidistant from each other.
• 10 mesh (sieve opening 2 mm) and gauge woven stainless steel wire
cloth is attached with screws to the under surface of lower plate. Glass
tubes and upper plastic plates are screwed in position by means of
stainless-steel plate of diameter 9 cm and 1 mm thick. Central shaft 8 cm
in length upper end of which terminates in an eye through which a string
or wire may be inserted. Plates are assembled rigidly with bolts through
two plastic plates.
• Design of plastic assembly may vary between manufacturers but must
comply with specifications.
DISCS
• The decision to include plastic discs is based on the specific gravity of the
tablets to take care of floating tablets. Slotted and perforated discs of
9.5 ± 0.15 min thickness and 20.7 ± 0.15 mm in diameter.
• They are made up of transparent material, specific gravity between 1.18
and 1.20. Equally distant 4 V shaped notches in parallel to cylindrical
axis. All surfaces of the discs of smooth. Five 2 mm holes are drilled
perpendicular to the cylindrical axis. One-hole pass through cylindrical
axis other 4 are parallel to it with distance 2 mm apart.
THERMOSTAT
• For heating the fluid between 35℃ to 39℃.
DEVICE FOR LOWERING AND RAISING BASKET RACK ASSEMBLY
• Up and down cycles perforated at rate between 28 and 32 cycles per
minute through a distance of not less than 5 cm and not more than 6
cm.
• Volume of fluid in vessel in adjusted as such that the highest point of
upward stoke the wire mesh remains 2.5 cm from the bottom of vessel.
One of downward stoke do not descend to not less than cm from the
bottom of vessel.
• Time required for upward stoke should be equal to time require for
downward stoke. The change in stoke direction should be smooth.
9
• Tubes are held vertically with the help of two plastic plates each about 9
cm in diameter and 6 mm in thickness. Plastic plates consist of 6 holes
each about 24 mm diameter, equidistant from the center of plate and
equidistant from each other.
• 10 mesh (sieve opening 2 mm) and gauge woven stainless steel wire
cloth is attached with screws to the under surface of lower plate. Glass
tubes and upper plastic plates are screwed in position by means of
stainless-steel plate of diameter 9 cm and 1 mm thick. Central shaft 8 cm
in length upper end of which terminates in an eye through which a string
or wire may be inserted. Plates are assembled rigidly with bolts through
two plastic plates.
• Design of plastic assembly may vary between manufacturers but must
comply with specifications.
DISCS
• The decision to include plastic discs is based on the specific gravity of the
tablets to take care of floating tablets. Slotted and perforated discs of
9.5 ± 0.15 min thickness and 20.7 ± 0.15 mm in diameter.
• They are made up of transparent material, specific gravity between 1.18
and 1.20. Equally distant 4 V shaped notches in parallel to cylindrical
axis. All surfaces of the discs of smooth. Five 2 mm holes are drilled
perpendicular to the cylindrical axis. One-hole pass through cylindrical
axis other 4 are parallel to it with distance 2 mm apart.
THERMOSTAT
• For heating the fluid between 35℃ to 39℃.
DEVICE FOR LOWERING AND RAISING BASKET RACK ASSEMBLY
• Up and down cycles perforated at rate between 28 and 32 cycles per
minute through a distance of not less than 5 cm and not more than 6
cm.
• Volume of fluid in vessel in adjusted as such that the highest point of
upward stoke the wire mesh remains 2.5 cm from the bottom of vessel.
One of downward stoke do not descend to not less than cm from the
bottom of vessel.
• Time required for upward stoke should be equal to time require for
downward stoke. The change in stoke direction should be smooth.
9
GM Hamad
IMMERSION FLUID
• Water: use distilled water
• Hydrochloric acid: use ACS reagent code
• Sodium chloride: use ACS reagent code
• Pepsin
• Potassium phosphate, monobasic: use ACS reagent code
• Pancreatin: a USP grade
• Hydrochloride solution (0.1M): dilute 8.5 ml of HCl to 1000 ml with
water or dilute a commercial volumetric solution with water to obtain a
final concentration of 0.1M.
• Sodium hydroxide (0.2M): use ACS reagent grade. Dissolve 8g of sodium
hydroxide in and dilute to 1000 ml with carbon dioxide free water or
dilute a commercial volumetric solution with carbon dioxide free water
to give a final concentration of 0.2M.
• Simulated gastric fluid: dissolve 2.0g of sodium chloride and 3.2 g of
pepsin in 500 ml of water and 7.0 ml of HCl and dilute to 1000 ml with
water. The pH is about 1.2
• Simulated intestinal fluid: dissolve 68g of potassium phosphate
monobasic in 250 ml in water. Add 10.0g of pancreatin mix and adjust
the pH of the resulting solution to 7.5 ± 0.1 with NaOH (0.2M) dilute
with water to 1000 ml.
UNCOATED AND PLAIN COATED TABLETS
BP METHOD FOR UNCOATED AND PLAIN COATED TABLETS
• Assemble the apparatus when the devise for arising a lowering the
basket rack assembly in at rest and its cylinder in the extreme down
position.
• With 2.5 L or appropriate amount of water in the cylindrical jar, adjust
the apparatus until the level of fluid in the jar coincides approximately
with the mid-line of the upper plastic plate.
• Maintain the temperature of the fluid at 37 ± 2℃ by suitable means.
• Remove the basket rack assembly form the water and disassemble.
• Select at random six tablets from the sample and place one in each of
the tubes of the basket rack assembly.
• Place a plastic disk on each tablet according to the specific gravity of
tablet.
• Reinsert the assembly in the water and set the machine in motion.
10
IMMERSION FLUID
• Water: use distilled water
• Hydrochloric acid: use ACS reagent code
• Sodium chloride: use ACS reagent code
• Pepsin
• Potassium phosphate, monobasic: use ACS reagent code
• Pancreatin: a USP grade
• Hydrochloride solution (0.1M): dilute 8.5 ml of HCl to 1000 ml with
water or dilute a commercial volumetric solution with water to obtain a
final concentration of 0.1M.
• Sodium hydroxide (0.2M): use ACS reagent grade. Dissolve 8g of sodium
hydroxide in and dilute to 1000 ml with carbon dioxide free water or
dilute a commercial volumetric solution with carbon dioxide free water
to give a final concentration of 0.2M.
• Simulated gastric fluid: dissolve 2.0g of sodium chloride and 3.2 g of
pepsin in 500 ml of water and 7.0 ml of HCl and dilute to 1000 ml with
water. The pH is about 1.2
• Simulated intestinal fluid: dissolve 68g of potassium phosphate
monobasic in 250 ml in water. Add 10.0g of pancreatin mix and adjust
the pH of the resulting solution to 7.5 ± 0.1 with NaOH (0.2M) dilute
with water to 1000 ml.
UNCOATED AND PLAIN COATED TABLETS
BP METHOD FOR UNCOATED AND PLAIN COATED TABLETS
• Assemble the apparatus when the devise for arising a lowering the
basket rack assembly in at rest and its cylinder in the extreme down
position.
• With 2.5 L or appropriate amount of water in the cylindrical jar, adjust
the apparatus until the level of fluid in the jar coincides approximately
with the mid-line of the upper plastic plate.
• Maintain the temperature of the fluid at 37 ± 2℃ by suitable means.
• Remove the basket rack assembly form the water and disassemble.
• Select at random six tablets from the sample and place one in each of
the tubes of the basket rack assembly.
• Place a plastic disk on each tablet according to the specific gravity of
tablet.
• Reinsert the assembly in the water and set the machine in motion.
10
GM Hamad
• The plastic discs should travel and up and down freely exerting a gentle
rubbing action on each tablet. After 15 minutes remove the basket rack
assembly from the water.
• Uncoated tablets pass the test if each of the six uncoated tablets
disintegrates in not more than 15 minutes.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• Plain coated tablets pass the test if each of the six plain coated tablets
disintegrate is not more than 60 minutes. If any of the tablets has not
disintegrated at the end of 60 minutes, repeat the test of further six
plain coated tablets replacing the water in the cylindrical jar with HCl
(0.1M). The tablets pass the test if each of the six tablets disintegrates
within 60 minutes in the acid medium.
USP METHOD FOR UNCOATED TABLETS
• Start the disintegration test on 6 tablets.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
USP METHOD FOR COATED TABLETS
• To remove or dissolve the coat immerse the tablet in distilled water for 5
minutes. Put the tablet in the apparatus is water or HCl for 30 minutes at
37℃. If not disintegrated put in intestinal fluid.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
ENTERIC COATED TABLETS
BP METHOD FOR ENTERIC COATED TABLETS
11
• The plastic discs should travel and up and down freely exerting a gentle
rubbing action on each tablet. After 15 minutes remove the basket rack
assembly from the water.
• Uncoated tablets pass the test if each of the six uncoated tablets
disintegrates in not more than 15 minutes.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• Plain coated tablets pass the test if each of the six plain coated tablets
disintegrate is not more than 60 minutes. If any of the tablets has not
disintegrated at the end of 60 minutes, repeat the test of further six
plain coated tablets replacing the water in the cylindrical jar with HCl
(0.1M). The tablets pass the test if each of the six tablets disintegrates
within 60 minutes in the acid medium.
USP METHOD FOR UNCOATED TABLETS
• Start the disintegration test on 6 tablets.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
USP METHOD FOR COATED TABLETS
• To remove or dissolve the coat immerse the tablet in distilled water for 5
minutes. Put the tablet in the apparatus is water or HCl for 30 minutes at
37℃. If not disintegrated put in intestinal fluid.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
ENTERIC COATED TABLETS
BP METHOD FOR ENTERIC COATED TABLETS
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GM Hamad
• Assemble the apparatus as described using 2.5 L of simulated gastric
fluid in place of water. Remove the basket rack assembly from the
simulated fluid and disassemble.
• Select at random six tablets from the sample and place one in each of
the tubes of the basket rack assembly. Place a plunger in each tube as
specified (omitting the plastic disc). Insert the assembly in the simulated
gastric fluid and set the machine in motion.
• At the end of 30 minutes of operation, remove the basket rack assembly
from the fluid and gently rinse with water. Enteric coated tablets fail the
test if any of tablet show distant evidence of disintegration.
• Replace the simulated gastric fluid in the jar with 2.5 L of simulated
intestinal fluid. Remove the plungers, place a plastic disc on each tablet,
and re-insert the plunger. Continue the test by setting the machine in
motion.
• After 30 minutes remove the basket rack assembly from the fluid.
Enteric coated tablets pass the test each of the six tablets disintegrates
in not more than 30 minutes in the simulated intestinal fluid.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
USP METHOD FOR ENTERIC COATED TABLETS
• Put in distilled water for 5 minutes to dissolve the coat. Then put it in
simulated gastric fluid (0.1M HCl) for 1 hour. Then put it in simulated
intestinal fluid for 2 hours.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
BUCCAL TABLETS
• Apply the test for uncoated tablets, but omit the use of disc. After 4
hours, lift the basket from fluid and observe the tablets all of the tablets
should be disintegrated.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
12
• Assemble the apparatus as described using 2.5 L of simulated gastric
fluid in place of water. Remove the basket rack assembly from the
simulated fluid and disassemble.
• Select at random six tablets from the sample and place one in each of
the tubes of the basket rack assembly. Place a plunger in each tube as
specified (omitting the plastic disc). Insert the assembly in the simulated
gastric fluid and set the machine in motion.
• At the end of 30 minutes of operation, remove the basket rack assembly
from the fluid and gently rinse with water. Enteric coated tablets fail the
test if any of tablet show distant evidence of disintegration.
• Replace the simulated gastric fluid in the jar with 2.5 L of simulated
intestinal fluid. Remove the plungers, place a plastic disc on each tablet,
and re-insert the plunger. Continue the test by setting the machine in
motion.
• After 30 minutes remove the basket rack assembly from the fluid.
Enteric coated tablets pass the test each of the six tablets disintegrates
in not more than 30 minutes in the simulated intestinal fluid.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
USP METHOD FOR ENTERIC COATED TABLETS
• Put in distilled water for 5 minutes to dissolve the coat. Then put it in
simulated gastric fluid (0.1M HCl) for 1 hour. Then put it in simulated
intestinal fluid for 2 hours.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
• If more than 2 tablets (from 18) fail to disintegrate the batch must be
failed.
BUCCAL TABLETS
• Apply the test for uncoated tablets, but omit the use of disc. After 4
hours, lift the basket from fluid and observe the tablets all of the tablets
should be disintegrated.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
12
GM Hamad
SUBLINGUAL TABLETS
• Apply the test for uncoated tablets but omit the use of disc. Observe the
tablets within the time limit specified in individual monograph; all the
tablets have disintegrated.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
2. DISSOLUTION TEST
• It is a process by which solid enters into solution. It is one of the most
important QC test. Dissolution test represents in-vivo drug dissolution
however far from being understood properly. Therapeutic deficiency
cannot rely on dissolution test alone. In considering drug absorption one
must consider:
Total dose required
Water and/or oil solubility
pKa of drug
• Dissolution is directly related to solubility. Drugs that have solubility
greater than 1% (1 w/v) are generally no problem. It is applied primarily
to those drugs which have low solubility. There is rapid increase in
dissolution testing of different dosage form in official pharmacopoeias.
• Since drug absorption and physiological availability are largely
dependent upon having the drug in dissolve state suitable dissolution
characteristic are an important property of QC. Usually method of
dissolution and specifications are given in individual monographs.
DOSAGE FORMS TO BE TESTED
• Immediate release dosage forms
• Controlled release dosage forms
• Transdermal systems
• Implants
OFFICIAL DISSOLUTION MONOGRAPHS
• United States Pharmacopoeia USP XXX (30)
• European pharmacopoeia
• Ph. Eur. 5th edition supplement 5.3
• British Pharmacopoeia BP 2007
• Japanese Pharmacopoeia JP XIV (14)
13
SUBLINGUAL TABLETS
• Apply the test for uncoated tablets but omit the use of disc. Observe the
tablets within the time limit specified in individual monograph; all the
tablets have disintegrated.
• If 1 and 2 tablets fail to disintegrate completely repeat the test on 12
additional tablets; not less than 16 of the total of 18 tablets tested
disintegrate completely.
2. DISSOLUTION TEST
• It is a process by which solid enters into solution. It is one of the most
important QC test. Dissolution test represents in-vivo drug dissolution
however far from being understood properly. Therapeutic deficiency
cannot rely on dissolution test alone. In considering drug absorption one
must consider:
Total dose required
Water and/or oil solubility
pKa of drug
• Dissolution is directly related to solubility. Drugs that have solubility
greater than 1% (1 w/v) are generally no problem. It is applied primarily
to those drugs which have low solubility. There is rapid increase in
dissolution testing of different dosage form in official pharmacopoeias.
• Since drug absorption and physiological availability are largely
dependent upon having the drug in dissolve state suitable dissolution
characteristic are an important property of QC. Usually method of
dissolution and specifications are given in individual monographs.
DOSAGE FORMS TO BE TESTED
• Immediate release dosage forms
• Controlled release dosage forms
• Transdermal systems
• Implants
OFFICIAL DISSOLUTION MONOGRAPHS
• United States Pharmacopoeia USP XXX (30)
• European pharmacopoeia
• Ph. Eur. 5th edition supplement 5.3
• British Pharmacopoeia BP 2007
• Japanese Pharmacopoeia JP XIV (14)
13
GM Hamad
OFFICIAL DISSOLUTION APPARATUSES
• Rotating basket
• Paddle
• Reciprocating cylinder
• Flow through cell
• Paddle over disk
• Rotating cylinder
• Reciprocating holder
SELECTION OF APPARATUS
• The choice of the apparatus is based on one’s knowledge regarding the
formulation design, dosage form and performance. Besides the selection
of an adequate dissolution apparatus adequate test conditions are
crucial for all purposes.
• It depends upon one’s intention
QUALITY CONTROL
• Examining batch homogeneity
• Examining batch to batch conformity
• Examining stability
RESEARCH AND DEVELOPMENT
• Examining drug release behavior in preformulations
• In-vitro stimulation of the GIT passage
APPARATUS 1 – BASKET
• It is useful for capsules, bead, delayed release/ enteric
coated dosage forms, floating dosage forms,
surfactants in media. The standard volume is 900/ 1000
ml. 1, 2- and 4-liter vessels.
• It consist of following parts:
1000 ml vessel
A variable speed vessel
Cylindrical stainless-steel basket
Water bath (whole assembly is immersed in it for keeping
temperature constant at 37 ± 0.5℃ throughout the test).
Vessel: it is made up of glass or any other inert transparent
material. It is 1000 ml in volume capacity. It has slightly concave
14
OFFICIAL DISSOLUTION APPARATUSES
• Rotating basket
• Paddle
• Reciprocating cylinder
• Flow through cell
• Paddle over disk
• Rotating cylinder
• Reciprocating holder
SELECTION OF APPARATUS
• The choice of the apparatus is based on one’s knowledge regarding the
formulation design, dosage form and performance. Besides the selection
of an adequate dissolution apparatus adequate test conditions are
crucial for all purposes.
• It depends upon one’s intention
QUALITY CONTROL
• Examining batch homogeneity
• Examining batch to batch conformity
• Examining stability
RESEARCH AND DEVELOPMENT
• Examining drug release behavior in preformulations
• In-vitro stimulation of the GIT passage
APPARATUS 1 – BASKET
• It is useful for capsules, bead, delayed release/ enteric
coated dosage forms, floating dosage forms,
surfactants in media. The standard volume is 900/ 1000
ml. 1, 2- and 4-liter vessels.
• It consist of following parts:
1000 ml vessel
A variable speed vessel
Cylindrical stainless-steel basket
Water bath (whole assembly is immersed in it for keeping
temperature constant at 37 ± 0.5℃ throughout the test).
Vessel: it is made up of glass or any other inert transparent
material. It is 1000 ml in volume capacity. It has slightly concave
14
GM Hamad
bottom with 16cm height (internal height) and 10cm inside
diameter. The slides are flanged near top end to accept a fitted
cover. Cover has four ports one of which is cantered for motor
shaft. One of the other port is for thermometer. Other two ports
are for sample removal for analysis and one for addition/
replacement of dissolution medium.
Variable speed motor: the shaft of the motor is placed in central
port to facilitate the rotation of basket assembly smoothly. Shaft
in 6 mm in diameter and 30 cm in length. Motor speed is varied
between 25 rpm – 200 rpm and to be maintained as described in
individual monograph with ± 5%. Motor is suspended in such a
way that it may be raised or lowered to position the basket.
Basket: assembly basket assembly consist of two parts:
▪ Part 1: it is attached to the shaft. It is solid metal except for
2 mm vent. It is fitted with three spring clips that allows
removal of lower parts or basket proper to admit test
sample.
▪ Parts 2: it is detachable part consist of fabricated welded
seam. It has 40 mesh stainless steel cloth formed into
cylinder shaped. Its height is 3.66 cm and diameter is 2.5cm.
▪ Basket also contain metal rim sheet at top. A gold-plated
basket coating 0.0001 inch (2.5µm) thick is recommended
for tests carried out in dilute acid medium.
ADVANTAGES
• It has a breadth of experience (more than 200 monographs)
• Full pH change during the tests
• It can be easily automated which is important for routine investigations.
DISADVANTAGES
• Disintegration – dissolution interaction
• Hydrodermic dead zone under the basket degassing is particularly
important.
• Limited volume sink conditions for poorly soluble drugs.
APPARATUS 2 – PADDLE
• It is useful for tablets, capsules, beads, delayed release dosage forms,
15
bottom with 16cm height (internal height) and 10cm inside
diameter. The slides are flanged near top end to accept a fitted
cover. Cover has four ports one of which is cantered for motor
shaft. One of the other port is for thermometer. Other two ports
are for sample removal for analysis and one for addition/
replacement of dissolution medium.
Variable speed motor: the shaft of the motor is placed in central
port to facilitate the rotation of basket assembly smoothly. Shaft
in 6 mm in diameter and 30 cm in length. Motor speed is varied
between 25 rpm – 200 rpm and to be maintained as described in
individual monograph with ± 5%. Motor is suspended in such a
way that it may be raised or lowered to position the basket.
Basket: assembly basket assembly consist of two parts:
▪ Part 1: it is attached to the shaft. It is solid metal except for
2 mm vent. It is fitted with three spring clips that allows
removal of lower parts or basket proper to admit test
sample.
▪ Parts 2: it is detachable part consist of fabricated welded
seam. It has 40 mesh stainless steel cloth formed into
cylinder shaped. Its height is 3.66 cm and diameter is 2.5cm.
▪ Basket also contain metal rim sheet at top. A gold-plated
basket coating 0.0001 inch (2.5µm) thick is recommended
for tests carried out in dilute acid medium.
ADVANTAGES
• It has a breadth of experience (more than 200 monographs)
• Full pH change during the tests
• It can be easily automated which is important for routine investigations.
DISADVANTAGES
• Disintegration – dissolution interaction
• Hydrodermic dead zone under the basket degassing is particularly
important.
• Limited volume sink conditions for poorly soluble drugs.
APPARATUS 2 – PADDLE
• It is useful for tablets, capsules, beads, delayed release dosage forms,
15
GM Hamad
enteric coated dosage forms. Its standard volume is 900/
1000ml. it is normally the method of first choice.
ADVANTAGES
• It is easy to use
• It is robust
• It is easily adapted to apparatus 5
• T has breadth of experience
• pH alteration is possible
• It can be easily automated which is important for routine investigations.
DISADVANTAGES
• pH/ media change is often difficult
• Limited volume, sink conditions for poorly soluble drugs
• Hydrodynamics are complex, they vary with site of the dosage form in
the vessel (sticking, floating) and therefore may significantly affect drug
dissolution.
• Sinkers for floating dosage form.
APPARATUS 3 – RECIPROCATING CYLINDER
• It is useful for tablets, beads, and controlled release dosage forms.
Its standard volume is 200 – 250 ml per station.
ADVANTAGES
• It is easy to change pH
• Huge pH profiles
• Hydrodynamics can be directly influenced by varying the dip rate.
DISADVANTAGES
• It has small volume
• It has little experience and It provides limited data.
APPARATUS 4 – FLOW THROUGH CELL
• It is used for low solubility drugs, microparticulates, implants,
suppositories, and controlled release formulations. It has
variations; open or closed system.
ADVANTAGES
16
enteric coated dosage forms. Its standard volume is 900/
1000ml. it is normally the method of first choice.
ADVANTAGES
• It is easy to use
• It is robust
• It is easily adapted to apparatus 5
• T has breadth of experience
• pH alteration is possible
• It can be easily automated which is important for routine investigations.
DISADVANTAGES
• pH/ media change is often difficult
• Limited volume, sink conditions for poorly soluble drugs
• Hydrodynamics are complex, they vary with site of the dosage form in
the vessel (sticking, floating) and therefore may significantly affect drug
dissolution.
• Sinkers for floating dosage form.
APPARATUS 3 – RECIPROCATING CYLINDER
• It is useful for tablets, beads, and controlled release dosage forms.
Its standard volume is 200 – 250 ml per station.
ADVANTAGES
• It is easy to change pH
• Huge pH profiles
• Hydrodynamics can be directly influenced by varying the dip rate.
DISADVANTAGES
• It has small volume
• It has little experience and It provides limited data.
APPARATUS 4 – FLOW THROUGH CELL
• It is used for low solubility drugs, microparticulates, implants,
suppositories, and controlled release formulations. It has
variations; open or closed system.
ADVANTAGES
16
GM Hamad
• It is easy to change pH and media.
• pH profile is possible.
• No sink conditions.
• It has different modes; open and closed system.
DISADVANTAGES
• Deaeration is necessary
• High volume of media is required
• It is labor intensive process
APPARATUS 5 – PADDLE OVER DISK
• The method is useful for the transdermal patches. The
standard volume is 900 ml.
ADVANTAGES
• Standard apparatus (paddle) can be used, only add a stainless-
steel disk assembly.
DISADVANTAGES
• Disk assembly restricts patch size.
APPARATUS 6 – ROTATING CYLINDER
• Most probably will be removed from USP.
APPARATUS 7 – RECIPROCATING HOLDER
• Most probably will be removed from USP.
DISSOLUTION TESTING FOR VARIOUS DOSAGE FORMS
• Solid dosage forms includes:
Immediate release dosage forms (tablets and capsules)
Delayed release
Dosage forms for oral cavity:
▪ Buccal/ sublingual tablets
▪ Medicated chewing gums
• Suppositories
• Semisolid dosage forms
• Soft gelatin capsules
DISSOLUTION TESTING FOR IMMEDIATE RELEASE (IR) DOSAGE FORMS
17
• It is easy to change pH and media.
• pH profile is possible.
• No sink conditions.
• It has different modes; open and closed system.
DISADVANTAGES
• Deaeration is necessary
• High volume of media is required
• It is labor intensive process
APPARATUS 5 – PADDLE OVER DISK
• The method is useful for the transdermal patches. The
standard volume is 900 ml.
ADVANTAGES
• Standard apparatus (paddle) can be used, only add a stainless-
steel disk assembly.
DISADVANTAGES
• Disk assembly restricts patch size.
APPARATUS 6 – ROTATING CYLINDER
• Most probably will be removed from USP.
APPARATUS 7 – RECIPROCATING HOLDER
• Most probably will be removed from USP.
DISSOLUTION TESTING FOR VARIOUS DOSAGE FORMS
• Solid dosage forms includes:
Immediate release dosage forms (tablets and capsules)
Delayed release
Dosage forms for oral cavity:
▪ Buccal/ sublingual tablets
▪ Medicated chewing gums
• Suppositories
• Semisolid dosage forms
• Soft gelatin capsules
DISSOLUTION TESTING FOR IMMEDIATE RELEASE (IR) DOSAGE FORMS
17
GM Hamad
• Immediate release dosage form is designed to deliver the drug rapidly
into the systemic circulation. Therefore, the dissolution may be the rate
limiting step for the absorption. Generally, dissolution of IR dosage
forms are being conducted using apparatuses of basket, paddle,
reciprocating cylinder and flow through cell. The apparatus 1 and 2 are
most commonly used.
• USP uses basket, paddle, EP uses paddle basket and flow through cell
apparatuses for solid dosage forms of tablets, capsules. The dissolution
test is carried out at 37℃ ± 0.5℃. In general, when basket apparatus is
used rotation speed is 100 rpm with 40 mesh screen of the basket is
used.
• Paddle apparatus is used for tablets. Operating speed of 50 is used in
general.
PROCEDURE
Method I
• Unless otherwise directed in the individual monograph, place 900ml
fluid in the dissolution vessel. Vessel should previously be immersed in
water bath and allow dissolution temperature to come at 37℃ ± 0.5℃.
• Place one tablet or one capsule in the basket so that there is distance of
2.0 ± 0.2 cm between basket and bottom of vessel. Rotate the basket at
a rate specified in the monograph. Withdraw sample at the time
indicated and analyze them by procedure described in the individual
monograph. The dissolution testing is done in three stages of S1, S2, and
S3.
• In stage 1, 6 units are taken and the amount of drug from each unit
should not be less than Q + 5% where Q is the maximum amount of drug
dissolved active ingredient specified in individual monograph. Failure of
first stage (if one or two tablets fail to comply) compensates to
conductance of second stage S2 where additional 6 units are tested.
• The avg. of 12 units in two stages should be equal to or greater than Q
and no unit should be less than Q – 15%. Failure of stage 2 leads to
conductance of stage S3 where additional 12 units are tested and the
avg. of total units of three stages S1, S2 and S3 should be greater than or
equal to Q and no two units should be less than Q – 15% and none
should be less than Q – 25%.
18
• Immediate release dosage form is designed to deliver the drug rapidly
into the systemic circulation. Therefore, the dissolution may be the rate
limiting step for the absorption. Generally, dissolution of IR dosage
forms are being conducted using apparatuses of basket, paddle,
reciprocating cylinder and flow through cell. The apparatus 1 and 2 are
most commonly used.
• USP uses basket, paddle, EP uses paddle basket and flow through cell
apparatuses for solid dosage forms of tablets, capsules. The dissolution
test is carried out at 37℃ ± 0.5℃. In general, when basket apparatus is
used rotation speed is 100 rpm with 40 mesh screen of the basket is
used.
• Paddle apparatus is used for tablets. Operating speed of 50 is used in
general.
PROCEDURE
Method I
• Unless otherwise directed in the individual monograph, place 900ml
fluid in the dissolution vessel. Vessel should previously be immersed in
water bath and allow dissolution temperature to come at 37℃ ± 0.5℃.
• Place one tablet or one capsule in the basket so that there is distance of
2.0 ± 0.2 cm between basket and bottom of vessel. Rotate the basket at
a rate specified in the monograph. Withdraw sample at the time
indicated and analyze them by procedure described in the individual
monograph. The dissolution testing is done in three stages of S1, S2, and
S3.
• In stage 1, 6 units are taken and the amount of drug from each unit
should not be less than Q + 5% where Q is the maximum amount of drug
dissolved active ingredient specified in individual monograph. Failure of
first stage (if one or two tablets fail to comply) compensates to
conductance of second stage S2 where additional 6 units are tested.
• The avg. of 12 units in two stages should be equal to or greater than Q
and no unit should be less than Q – 15%. Failure of stage 2 leads to
conductance of stage S3 where additional 12 units are tested and the
avg. of total units of three stages S1, S2 and S3 should be greater than or
equal to Q and no two units should be less than Q – 15% and none
should be less than Q – 25%.
18
GM Hamad
Stage Number tested Acceptance criteria
S1 6
Amount of drug release from each unit not less
than Q + 5%.
S2 6
Avg. of S1+S2 (12 units) should be equal to or
greater than Q and no unit be less than Q -15%.
S3 12
Avg. of S1+S2+S3 (24 units) should be equal to or
greater than Q and not more than one unit should
be less than Q – 25%.
METHOD II
• Use apparatus described under tablet disintegration with some changes.
Replace 10 mesh stainless steel cloth in basket rack assembly with 40
mesh also, to the top of the assembly to provide for immersion in the
dissolution medium.
• Adjust the apparatus so that it descend to 1 ± 0.1cm from bottom of the
vessel on download stoke. Use dissolution as specified in individual
monograph.
• Apply the test firstly on 6 unit, if one or two tablets fails the specification
then perform test on 6 additional tablets. 10 out of 12 should pass the
specification.
DISSOLUTION TEST FOR DOSAGE FORMS OF THE ORAL CAVITY
• Development of dissolution method for these dosage forms possess
several challenges due to short resistance time of dosage form in the
mouth and limited volume of dissolution medium for dissolving the
dosage form.
DISSOLUTION TEST FOR CHEWABLE TABLETS
• USP insisted the use of apparatus 2 for dissolution excepting ampicillin
where apparatus 1 is recommended and carbamazepine where
apparatus 2 and 3 are used.
• The design of apparatus should consist of a mechanical breakage of
tablet prior to dissolution.
DISSOLUTION TEST FOR BUCCAL/ SUBLINGUAL TABLETS
• Initially USP stated the use of disintegration apparatus for the
ergotamine category sublingual products. Later modified USP 3
apparatus with 20 strokes/ min was used for hydrocortisone
19
Stage Number tested Acceptance criteria
S1 6
Amount of drug release from each unit not less
than Q + 5%.
S2 6
Avg. of S1+S2 (12 units) should be equal to or
greater than Q and no unit be less than Q -15%.
S3 12
Avg. of S1+S2+S3 (24 units) should be equal to or
greater than Q and not more than one unit should
be less than Q – 25%.
METHOD II
• Use apparatus described under tablet disintegration with some changes.
Replace 10 mesh stainless steel cloth in basket rack assembly with 40
mesh also, to the top of the assembly to provide for immersion in the
dissolution medium.
• Adjust the apparatus so that it descend to 1 ± 0.1cm from bottom of the
vessel on download stoke. Use dissolution as specified in individual
monograph.
• Apply the test firstly on 6 unit, if one or two tablets fails the specification
then perform test on 6 additional tablets. 10 out of 12 should pass the
specification.
DISSOLUTION TEST FOR DOSAGE FORMS OF THE ORAL CAVITY
• Development of dissolution method for these dosage forms possess
several challenges due to short resistance time of dosage form in the
mouth and limited volume of dissolution medium for dissolving the
dosage form.
DISSOLUTION TEST FOR CHEWABLE TABLETS
• USP insisted the use of apparatus 2 for dissolution excepting ampicillin
where apparatus 1 is recommended and carbamazepine where
apparatus 2 and 3 are used.
• The design of apparatus should consist of a mechanical breakage of
tablet prior to dissolution.
DISSOLUTION TEST FOR BUCCAL/ SUBLINGUAL TABLETS
• Initially USP stated the use of disintegration apparatus for the
ergotamine category sublingual products. Later modified USP 3
apparatus with 20 strokes/ min was used for hydrocortisone
19
GM Hamad
mucoadhesive tablets to mimic the low dissolution volume of in-vivo.
• Later another system continuous flow through filtration cell with dip
tube for filtration. 10 ml of fluid is pumped to give a short residence time
of 8 minutes.
DISSOLUTION TEST FOR CHEWING GUMS
• USP has not recommended any apparatus for dissolution testing of
chewing gums, but EP has emphasized on the use of 3 piston apparatus
that chews the gum at a rate of 60 cycles/ min in dissolution medium of
pH 6.0 at 37℃.
• Still controversies regarding this issue are existing and urges for
development of an appropriate apparatus.
3. CONTENT UNIFORMITY
• The content uniformity test is done to ensure that each dosage form
contains the exact stated amount of drug within a batch. Mainly it is
used for testing the consistency of:
Bulk powders before or after compression
Liquid orals before filling
During filling of powders into capsules or liquids into vials and
ampules
Amount of API within individual unit of tablet and capsule
• Only when the ingredient of the tablet granulation are homogenous,
tablet weight test as described earlier can be considered as measure of
drug content.
• Routinely the assay of the drug content in tablets involve the grinding of
tablet of large sample of tablets followed by the analysis of an aliquot.
Normally testing is confirmed by performing specific assay to determine
the content of drug material contained in particular dosage form.
Results obtained are expressed as percentage of active ingredient in the
tablet or on individual tablet basis. Different pharmacopoeias describe
the procedure of content uniformity test and giver their specifications.
CONTENT UNIFORMITY TEST USP
STAGE 1
20
mucoadhesive tablets to mimic the low dissolution volume of in-vivo.
• Later another system continuous flow through filtration cell with dip
tube for filtration. 10 ml of fluid is pumped to give a short residence time
of 8 minutes.
DISSOLUTION TEST FOR CHEWING GUMS
• USP has not recommended any apparatus for dissolution testing of
chewing gums, but EP has emphasized on the use of 3 piston apparatus
that chews the gum at a rate of 60 cycles/ min in dissolution medium of
pH 6.0 at 37℃.
• Still controversies regarding this issue are existing and urges for
development of an appropriate apparatus.
3. CONTENT UNIFORMITY
• The content uniformity test is done to ensure that each dosage form
contains the exact stated amount of drug within a batch. Mainly it is
used for testing the consistency of:
Bulk powders before or after compression
Liquid orals before filling
During filling of powders into capsules or liquids into vials and
ampules
Amount of API within individual unit of tablet and capsule
• Only when the ingredient of the tablet granulation are homogenous,
tablet weight test as described earlier can be considered as measure of
drug content.
• Routinely the assay of the drug content in tablets involve the grinding of
tablet of large sample of tablets followed by the analysis of an aliquot.
Normally testing is confirmed by performing specific assay to determine
the content of drug material contained in particular dosage form.
Results obtained are expressed as percentage of active ingredient in the
tablet or on individual tablet basis. Different pharmacopoeias describe
the procedure of content uniformity test and giver their specifications.
CONTENT UNIFORMITY TEST USP
STAGE 1
20
GM Hamad
• Take 10 units randomly and perform the assay. It passes the test if
relative standard deviation is less than 6% and no value is outside 85 –
115%. Fails the tests if one or more values are out of 75 – 125%.
STAGE 2
• Take 20 more units and perform the assay. Pass the test if RSD of all 30
tablets is less than 7.8%, not more than one value is outside 85 – 115 %
and no value is outside 75 – 125% or else, the batch fails the test.
CONTENT UNIFORMITY TEST BP
Test A
• The test is applicable for tablets, powders and parenteral use and
suspensions for injection.
• Selects 10 units at random and perform the assay. Passes the test each
individual unit is between 85 – 115% of the average content.
• Fails the test if more than one individual unit is outside these limits or if
even one unit is outsides the limit of 75 – 125% of the avg. content. But
if one unit is outside the limit of 85 – 115 % and within 75 – 125 % then
take another 10 units at random and perform the assay.
• The lot passes the test if not more than 1 unit of 30 units is outside 85 –
115% and not even one unit is outside the limit of 75 – 125% of the avg.
content.
TEST B
• The test is used for capsules, powders, other than parenteral use ,
granules, suppositories and pessaries.
• Selects 10 units at random and perform the assay. Passes the test if not
more than 1 individual unit is outside the limits of 85 – 115% and none is
outside the limits of 75 – 125%of the labelled content.
• The batch fails the test if more than 3 units are outside the limit of 85 –
115% or if one or more units are outside the limits of 75 – 125% of the
labelled content.
• If 2 or 3 units are outside the limits of 85 – 115% but within the limits of
75 – 125% then select another 20 units at random.
• The batch complies the test when not more than 3 units are out of these
30 units are outside the limits of 85 – 115% and not even one unit is
outside the limits of 75 – 125% of the labelled content.
21
• Take 10 units randomly and perform the assay. It passes the test if
relative standard deviation is less than 6% and no value is outside 85 –
115%. Fails the tests if one or more values are out of 75 – 125%.
STAGE 2
• Take 20 more units and perform the assay. Pass the test if RSD of all 30
tablets is less than 7.8%, not more than one value is outside 85 – 115 %
and no value is outside 75 – 125% or else, the batch fails the test.
CONTENT UNIFORMITY TEST BP
Test A
• The test is applicable for tablets, powders and parenteral use and
suspensions for injection.
• Selects 10 units at random and perform the assay. Passes the test each
individual unit is between 85 – 115% of the average content.
• Fails the test if more than one individual unit is outside these limits or if
even one unit is outsides the limit of 75 – 125% of the avg. content. But
if one unit is outside the limit of 85 – 115 % and within 75 – 125 % then
take another 10 units at random and perform the assay.
• The lot passes the test if not more than 1 unit of 30 units is outside 85 –
115% and not even one unit is outside the limit of 75 – 125% of the avg.
content.
TEST B
• The test is used for capsules, powders, other than parenteral use ,
granules, suppositories and pessaries.
• Selects 10 units at random and perform the assay. Passes the test if not
more than 1 individual unit is outside the limits of 85 – 115% and none is
outside the limits of 75 – 125%of the labelled content.
• The batch fails the test if more than 3 units are outside the limit of 85 –
115% or if one or more units are outside the limits of 75 – 125% of the
labelled content.
• If 2 or 3 units are outside the limits of 85 – 115% but within the limits of
75 – 125% then select another 20 units at random.
• The batch complies the test when not more than 3 units are out of these
30 units are outside the limits of 85 – 115% and not even one unit is
outside the limits of 75 – 125% of the labelled content.
21
GM Hamad
TEST C
• The test is applicable to only transdermal patches.
• The preparation passes test only if the avg. content of 10 units is
between 90 – 110% and if the content of each unit is between 75 –
125% of the avg. content.
4. CHEMICAL ASSAY OF TABLETS
• Routinely the assay of the drug content in tablets involve the
grinding of tablet of large sample of tablets followed by the
analysis of an aliquot. Representing the certain amount of drug
normally in a single unit.
• Analysis is performed by the methods prescribed in the
individual monographs. Results obtained are expressed in
percentage of the active ingredient in the tablet or unit dose
compared with limits in the monograph of the drug.
• Common assay procedure involves:
Titrimetric analysis
Spectrophotometric methods
UV spectroscopy
HPLC
Biological assay
Microbial assay
TESTS FOR COATED TABLETS
• Water vapor permeability
• Film tensile strength
• Coted tablets evaluations
ADHESION TEST WITH TENSILE STRENGTH TESTER
• It measure the force required to peel the film of from the tablet
surface.
DIAMETRAL CRUSHING STRENGTH OF COATED TABLETS
• Tablets hardness testers are used. This test gives information
22
TEST C
• The test is applicable to only transdermal patches.
• The preparation passes test only if the avg. content of 10 units is
between 90 – 110% and if the content of each unit is between 75 –
125% of the avg. content.
4. CHEMICAL ASSAY OF TABLETS
• Routinely the assay of the drug content in tablets involve the
grinding of tablet of large sample of tablets followed by the
analysis of an aliquot. Representing the certain amount of drug
normally in a single unit.
• Analysis is performed by the methods prescribed in the
individual monographs. Results obtained are expressed in
percentage of the active ingredient in the tablet or unit dose
compared with limits in the monograph of the drug.
• Common assay procedure involves:
Titrimetric analysis
Spectrophotometric methods
UV spectroscopy
HPLC
Biological assay
Microbial assay
TESTS FOR COATED TABLETS
• Water vapor permeability
• Film tensile strength
• Coted tablets evaluations
ADHESION TEST WITH TENSILE STRENGTH TESTER
• It measure the force required to peel the film of from the tablet
surface.
DIAMETRAL CRUSHING STRENGTH OF COATED TABLETS
• Tablets hardness testers are used. This test gives information
22
GM Hamad
on the relative increase in crushing strength provided by the
film and the contribution made by changes in the film
composition.
• Temperature and humidity may cause film defects, hence
studies are to be carried out.
• Quantification of film roughness, hardness and color
uniformity.
• Visual inspection or instruments are used. Resistance of coated
tablets on a white sheet of paper. Resilient films remain intact
and no color is transferred to the paper, very softy coating are
readily “erased” from the tablet surface to the paper.
QUALITY CONTROL OF CAPSULES
• Quality control tests for capsules includes:
Weight variation
Content uniformity
Disintegration
Dissolution
Chemical or biological
assay
1. DISSOLUTION TEST FOR CAPSULES
• Special type of basket rack assembly is used. The apparatus consisting
of:
A glass tube 80 – 100 mm long with internal diameter of about
28mm and external diameter of 30-31 mm. At the bottom of the
tube rust proof wire gauze (sieve # 1.7mm or 10 mesh) is attached
to form a basket. Wire gauze is fitted to the tube in such a manner
that the overall diameter of the basket in not materially increased.
A glass cylinder with a flat base and an internal diameter of about
45mm used for disintegration media. Cylinder contains water
media not less than 15 cm deep maintained at the temperature
37±2℃ by suitable means.
Basket is raised and lowered repeatedly in a uniform manner so
that at highest position the gauze breaks the surface of water and
at lowest position the upper rim of basket cylinder just remains
clear of water. Guiding disk made up of suitable material, lowering
23
on the relative increase in crushing strength provided by the
film and the contribution made by changes in the film
composition.
• Temperature and humidity may cause film defects, hence
studies are to be carried out.
• Quantification of film roughness, hardness and color
uniformity.
• Visual inspection or instruments are used. Resistance of coated
tablets on a white sheet of paper. Resilient films remain intact
and no color is transferred to the paper, very softy coating are
readily “erased” from the tablet surface to the paper.
QUALITY CONTROL OF CAPSULES
• Quality control tests for capsules includes:
Weight variation
Content uniformity
Disintegration
Dissolution
Chemical or biological
assay
1. DISSOLUTION TEST FOR CAPSULES
• Special type of basket rack assembly is used. The apparatus consisting
of:
A glass tube 80 – 100 mm long with internal diameter of about
28mm and external diameter of 30-31 mm. At the bottom of the
tube rust proof wire gauze (sieve # 1.7mm or 10 mesh) is attached
to form a basket. Wire gauze is fitted to the tube in such a manner
that the overall diameter of the basket in not materially increased.
A glass cylinder with a flat base and an internal diameter of about
45mm used for disintegration media. Cylinder contains water
media not less than 15 cm deep maintained at the temperature
37±2℃ by suitable means.
Basket is raised and lowered repeatedly in a uniform manner so
that at highest position the gauze breaks the surface of water and
at lowest position the upper rim of basket cylinder just remains
clear of water. Guiding disk made up of suitable material, lowering
23
GM Hamad
and raising device.
BP METHOD
• Place five capsules in the basket. Raise and lower the basket in such a
manner that complete up and down movement is repeated thirty times
per minute.
• Capsules are disintegrated when no particle of any solid content remains
above the gauze which would not readily pass through it.
• Time required for capsules to disintegrate not more than 15 minutes
unless otherwise stated in individual monograph. If capsules fail the
disintegration test because of aggregation, further five capsules may be
tested individually.
• The longest time taken by one of the five capsules is the disintegration
time.
USP METHOD
• According to USP disintegration test is usually not require for capsules
unless have been treated to resist solution in gastric fluid (enteric
coated). In this case they must meet the requirements of disintegration
test of enteric coated tablet i.e.
• Assemble the apparatus as described using 2.5 L of simulated gastric
fluid in place of water. Remove the basket rack assembly from the
simulated fluid and disassemble.
• Select at random six capsule from the sample and place one in each of
the tubes of the basket rack assembly. Place a guided disc. Insert the
assembly in the simulated gastric fluid and set the machine in motion.
• At the end of 60 minutes of operation, remove the basket rack assembly
from the fluid and gently rinse with water. Enteric coated capsule fail the
test if any of tablet show distant evidence of disintegration. Replace the
simulated gastric fluid in the jar with 2.5 L of simulated intestinal fluid.
• Reinsert the guided disc. Continue the test by setting the machine in
motion for 60 minutes. After 60 minutes remove the basket rack
assembly from the fluid. Enteric coated tablets pass the test each of the
six tablets disintegrates in not more than 30 minutes in the simulated
intestinal fluid.
2. WEIGHT VARIATION TEST FOR CAPSULES
• There are two methods for testing uniformity of weight of capsules:
24
and raising device.
BP METHOD
• Place five capsules in the basket. Raise and lower the basket in such a
manner that complete up and down movement is repeated thirty times
per minute.
• Capsules are disintegrated when no particle of any solid content remains
above the gauze which would not readily pass through it.
• Time required for capsules to disintegrate not more than 15 minutes
unless otherwise stated in individual monograph. If capsules fail the
disintegration test because of aggregation, further five capsules may be
tested individually.
• The longest time taken by one of the five capsules is the disintegration
time.
USP METHOD
• According to USP disintegration test is usually not require for capsules
unless have been treated to resist solution in gastric fluid (enteric
coated). In this case they must meet the requirements of disintegration
test of enteric coated tablet i.e.
• Assemble the apparatus as described using 2.5 L of simulated gastric
fluid in place of water. Remove the basket rack assembly from the
simulated fluid and disassemble.
• Select at random six capsule from the sample and place one in each of
the tubes of the basket rack assembly. Place a guided disc. Insert the
assembly in the simulated gastric fluid and set the machine in motion.
• At the end of 60 minutes of operation, remove the basket rack assembly
from the fluid and gently rinse with water. Enteric coated capsule fail the
test if any of tablet show distant evidence of disintegration. Replace the
simulated gastric fluid in the jar with 2.5 L of simulated intestinal fluid.
• Reinsert the guided disc. Continue the test by setting the machine in
motion for 60 minutes. After 60 minutes remove the basket rack
assembly from the fluid. Enteric coated tablets pass the test each of the
six tablets disintegrates in not more than 30 minutes in the simulated
intestinal fluid.
2. WEIGHT VARIATION TEST FOR CAPSULES
• There are two methods for testing uniformity of weight of capsules:
24
GM Hamad
METHOD A
• Method A is for capsules with dry content. Weigh a capsule, open it
without loss of shell material, remove the contents and weigh all parts of
shell.
• The difference between the weights represents the weight of contents
of capsule. Repeat the operation with further 19 capsules (total 20).
• Capsules pass the test if not more than 2 capsules deviate from the
mean weight by more than percentage given in table.
• For one or two capsules (which are outside above given range) the
weight of the content should not be more than percentage given in the
table below.
Average weight
Percentage deviation
A B
0.120g or less ± 10% (18 out of 20) ± 20% (2 out of 20)
More than 0.120g ± 7.5% (18 out of 20) ± 15%(2 out of 20)
METHOD B
• The method B is for capsules containing liquid or base. Weigh a capsule,
open it without loss of shell material express as much of the contents is
possible. Wash the shell with solvent ether, reject the washing. Allow
the shell to stand until all the odor of ether is no longer perceptible and
weigh.
• The difference between the whole weight and shell weight represents in
weigh of contents. Repeat the operation with further 9 capsules (total
10) and calculate the average weight content of 10 capsules. The weight
of each capsule does not differ from the average weight by more than
7.5%. Except the one capsule the weight of content may differ by not
more than 15%.
• Regardless of the weight of content of this type of capsules the
percentage deviation range should be between ±7.5 – ±15%.
DISSOLUTION TEST
• The dissolution may be the rate limiting step in capsules absorption.
Generally, the dissolution test of capsules is conducted in paddle or
basket assembly. USP uses basket, paddle, EP uses paddle, basket, and
flow through cell apparatuses for solid dosage forms of tablets and
capsules. The choice of apparatus is based on the knowledge regarding
25
METHOD A
• Method A is for capsules with dry content. Weigh a capsule, open it
without loss of shell material, remove the contents and weigh all parts of
shell.
• The difference between the weights represents the weight of contents
of capsule. Repeat the operation with further 19 capsules (total 20).
• Capsules pass the test if not more than 2 capsules deviate from the
mean weight by more than percentage given in table.
• For one or two capsules (which are outside above given range) the
weight of the content should not be more than percentage given in the
table below.
Average weight
Percentage deviation
A B
0.120g or less ± 10% (18 out of 20) ± 20% (2 out of 20)
More than 0.120g ± 7.5% (18 out of 20) ± 15%(2 out of 20)
METHOD B
• The method B is for capsules containing liquid or base. Weigh a capsule,
open it without loss of shell material express as much of the contents is
possible. Wash the shell with solvent ether, reject the washing. Allow
the shell to stand until all the odor of ether is no longer perceptible and
weigh.
• The difference between the whole weight and shell weight represents in
weigh of contents. Repeat the operation with further 9 capsules (total
10) and calculate the average weight content of 10 capsules. The weight
of each capsule does not differ from the average weight by more than
7.5%. Except the one capsule the weight of content may differ by not
more than 15%.
• Regardless of the weight of content of this type of capsules the
percentage deviation range should be between ±7.5 – ±15%.
DISSOLUTION TEST
• The dissolution may be the rate limiting step in capsules absorption.
Generally, the dissolution test of capsules is conducted in paddle or
basket assembly. USP uses basket, paddle, EP uses paddle, basket, and
flow through cell apparatuses for solid dosage forms of tablets and
capsules. The choice of apparatus is based on the knowledge regarding
25
GM Hamad
the size and type of capsules and selected according to individual
monograph.
• The dissolution test is carried out at 37℃ ± 0.5℃. In general, when
basket apparatus is used rotation speed is 100 rpm with 40 mesh screen
of the basket is used. Other mesh sizes may also be used if supported by
necessary date documentation. It is generally used for capsules and
floating type dosage forms or to those which tend to disintegrate slowly.
For floating type of dosage forms sinker may be used to prevent the
floating of capsules.
• Samples are withdrawn according to specifications with tolerance of ±
5%. The test is conducted on the equipment which was pre-calibrated
with USP salicylic acid and prednisone calibrator tablets (according to
USP).
• The dissolution medium used should be deaerated and may be water,
buffered aq. Solution of pH 4 – 8 and dilute acid of 0.001N to 0.1N HCl
are used. The test time is 30 – 60 minutes and with a single point
specification or as specified in individual monographs.
PROCEDURE
• Unless otherwise directed in the individual monograph, place 900ml
fluid in the dissolution vessel. Vessel should previously be immersed in
water bath and allow dissolution temperature to come at 37℃ ± 0.5℃.
• Place one tablet or one capsule in the basket so that there is distance of
2.0 ± 0.2 cm between basket and bottom of vessel. Rotate the basket at
a rate specified in the monograph. Withdraw sample at the time
indicated and analyze them by procedure described in the individual
monograph.
• The dissolution testing is done in three stages of S1, S2, and S3. In stage
1, 6 units are taken and the amount of drug from each unit should not
be less than Q + 5% where Q is the maximum amount of drug dissolved
active ingredient specified in individual monograph.
• Failure of first stage (if one or two tablets fail to comply) compensates to
conductance of second stage S2 where additional 6 units are tested. The
avg. of 12 units in two stages should be equal to or greater than Q and
no unit should be less than Q – 15%.
• Failure of stage 2 leads to conductance of stage S3 where additional 12
units are tested and the avg. of total units of three stages S1, S2 and S3
26
the size and type of capsules and selected according to individual
monograph.
• The dissolution test is carried out at 37℃ ± 0.5℃. In general, when
basket apparatus is used rotation speed is 100 rpm with 40 mesh screen
of the basket is used. Other mesh sizes may also be used if supported by
necessary date documentation. It is generally used for capsules and
floating type dosage forms or to those which tend to disintegrate slowly.
For floating type of dosage forms sinker may be used to prevent the
floating of capsules.
• Samples are withdrawn according to specifications with tolerance of ±
5%. The test is conducted on the equipment which was pre-calibrated
with USP salicylic acid and prednisone calibrator tablets (according to
USP).
• The dissolution medium used should be deaerated and may be water,
buffered aq. Solution of pH 4 – 8 and dilute acid of 0.001N to 0.1N HCl
are used. The test time is 30 – 60 minutes and with a single point
specification or as specified in individual monographs.
PROCEDURE
• Unless otherwise directed in the individual monograph, place 900ml
fluid in the dissolution vessel. Vessel should previously be immersed in
water bath and allow dissolution temperature to come at 37℃ ± 0.5℃.
• Place one tablet or one capsule in the basket so that there is distance of
2.0 ± 0.2 cm between basket and bottom of vessel. Rotate the basket at
a rate specified in the monograph. Withdraw sample at the time
indicated and analyze them by procedure described in the individual
monograph.
• The dissolution testing is done in three stages of S1, S2, and S3. In stage
1, 6 units are taken and the amount of drug from each unit should not
be less than Q + 5% where Q is the maximum amount of drug dissolved
active ingredient specified in individual monograph.
• Failure of first stage (if one or two tablets fail to comply) compensates to
conductance of second stage S2 where additional 6 units are tested. The
avg. of 12 units in two stages should be equal to or greater than Q and
no unit should be less than Q – 15%.
• Failure of stage 2 leads to conductance of stage S3 where additional 12
units are tested and the avg. of total units of three stages S1, S2 and S3
26
GM Hamad
should be greater than or equal to Q and no two units should be less
than Q – 15% and none should be less than Q – 25%.
Stage Number tested Acceptance criteria
S1 6
Amount of drug release from each unit not less
than Q + 5%.
S2 6
Avg. of S1+S2 (12 units) should be equal to or
greater than Q and no unit be less than Q -15%.
S3 12
Avg. of S1+S2+S3 (24 units) should be equal to or
greater than Q and not more than one unit should
be less than Q – 25%.
DISSOLUTION TESTING OF SOFT GELATIN CAPSULES
• USP has recommended the use of apparatus 1 and 2, but since there had
been serious disadvantages related, attempts had been made in
literature to develop new methods for lipid filled soft gelatin capsules.
ASSAY OF ACTIVE INGREDIENTS IN CAPSULES
• Determine the amount of active ingredient by the method described in
the assay.
• Calculate the amount of API in the mixed contents of the capsules taken
and divide by the number of capsules taken. The result should lie within
the range specified in individual monograph.
• Sometime biological assay is described in the mono graph e.g. drugs of
natural origin (vitamins, antibiotics, insulin etc.).
QUALITY CONTROL OF POWDERS
POWDER FLOW
• The widespread use of powder in the pharmaceutical industries has
generalized a variety of methods for characterizing powder flow. Several
references appear in the pharmaceutical literature attempting to
correlate the various measure of powder flow to manufacturing
properties. The development of such a variety of test methods was
inevitable; powder behavior is multifaceted and thus complicates the
efforts to characterize the flow properties of the pharmaceutical
powders.
27
should be greater than or equal to Q and no two units should be less
than Q – 15% and none should be less than Q – 25%.
Stage Number tested Acceptance criteria
S1 6
Amount of drug release from each unit not less
than Q + 5%.
S2 6
Avg. of S1+S2 (12 units) should be equal to or
greater than Q and no unit be less than Q -15%.
S3 12
Avg. of S1+S2+S3 (24 units) should be equal to or
greater than Q and not more than one unit should
be less than Q – 25%.
DISSOLUTION TESTING OF SOFT GELATIN CAPSULES
• USP has recommended the use of apparatus 1 and 2, but since there had
been serious disadvantages related, attempts had been made in
literature to develop new methods for lipid filled soft gelatin capsules.
ASSAY OF ACTIVE INGREDIENTS IN CAPSULES
• Determine the amount of active ingredient by the method described in
the assay.
• Calculate the amount of API in the mixed contents of the capsules taken
and divide by the number of capsules taken. The result should lie within
the range specified in individual monograph.
• Sometime biological assay is described in the mono graph e.g. drugs of
natural origin (vitamins, antibiotics, insulin etc.).
QUALITY CONTROL OF POWDERS
POWDER FLOW
• The widespread use of powder in the pharmaceutical industries has
generalized a variety of methods for characterizing powder flow. Several
references appear in the pharmaceutical literature attempting to
correlate the various measure of powder flow to manufacturing
properties. The development of such a variety of test methods was
inevitable; powder behavior is multifaceted and thus complicates the
efforts to characterize the flow properties of the pharmaceutical
powders.
27
GM Hamad
• In addition, no single or simple method can adequately characterize the
flow properties of pharmaceutical powders.
• Four common reported methods for testing powder flow rate are:
Angle of repose
Compressibility index or Hausner ratio
Flow rate through an orifice
Shear cell
1. ANGLE OF REPOSE
• The angle of repose have been used in several branches of solid-state
science to characterize the flow properties of solid. Angle of repose is a
characteristic related to interparticulate resistance or friction to
movement between particles.
• Angle of repose test result are reported to be very dependent upon the
method used. Experimental difficulties arise as a result of segregation of
material and consolidation or aeration of the powder as the cone is
formed.
• It is the constant three-dimensional angle (relative to horizontal base)
assumed by a cone like pile of material formed by any of several
different methods. Basic methods of angle of repose are as follows:
Static angle of repose
Drained angle of repose
Dynamic angle of repose
STATIC ANGLE OF REPOSE
• Static angle of repose is calculated by the use of funnel. The most
common method of determining the static angle of repose can be
classified on the bases of following two important experimental
variables:
The height of the funnel through which the powder passes may be
fixed relative to the base or the height may be varied as the pile
forms.
The base upon which the pile forms may be of fixed diameter or
the diameter of the powder cone may be followed to vary as the
pile forms.
DRAINED ANGLE OF REPOSE
• Drained angle of repose is determined by allowing an excess quantity of
28
• In addition, no single or simple method can adequately characterize the
flow properties of pharmaceutical powders.
• Four common reported methods for testing powder flow rate are:
Angle of repose
Compressibility index or Hausner ratio
Flow rate through an orifice
Shear cell
1. ANGLE OF REPOSE
• The angle of repose have been used in several branches of solid-state
science to characterize the flow properties of solid. Angle of repose is a
characteristic related to interparticulate resistance or friction to
movement between particles.
• Angle of repose test result are reported to be very dependent upon the
method used. Experimental difficulties arise as a result of segregation of
material and consolidation or aeration of the powder as the cone is
formed.
• It is the constant three-dimensional angle (relative to horizontal base)
assumed by a cone like pile of material formed by any of several
different methods. Basic methods of angle of repose are as follows:
Static angle of repose
Drained angle of repose
Dynamic angle of repose
STATIC ANGLE OF REPOSE
• Static angle of repose is calculated by the use of funnel. The most
common method of determining the static angle of repose can be
classified on the bases of following two important experimental
variables:
The height of the funnel through which the powder passes may be
fixed relative to the base or the height may be varied as the pile
forms.
The base upon which the pile forms may be of fixed diameter or
the diameter of the powder cone may be followed to vary as the
pile forms.
DRAINED ANGLE OF REPOSE
• Drained angle of repose is determined by allowing an excess quantity of
28
GM Hamad
material positioned above a fixed container base to drain from the
container.
• Formulation of a cone of a powder on a fixed diameter base allows
determination of the drained angle of repose.
DYNAMIC ANGLE OF REPOSE
• Dynamic angle of repose is calculated by filling a cylinder (with a clear
flat cover at the end) and rotating at a specific speed.
• The dynamic angle of a repose is an angle (relative to the horizontal)
formed by the flowing powder. The internal angle of kinetic friction is
defined by the plane separating those particles sliding down the top
layer of the powder and those particles that are rotating with the drum
(with roughened surface).
SPECIFICATIONS
• Although there is some variation in the qualitative description of powder
flow using the angle of repose, much of the pharmaceutical literature
appears to be consistent with the classification shown in the table.
Flow property Angle of repose (degrees)
Excellent 25 – 30
Good 31 -35
Fair – aid not needed 36 – 40
Passable – may hang up 41 – 45
Poor – must agitate, vibrate 46 – 55
Very poor 56 – 65
Very, very poor > 66
PROCEDURE FOR ANGLE OF REPOSE
• The base should be free from vibration. Vary the height of the funnel to
carefully build up a symmetrical cone of powder. Care should be taken
to prevent vibration as the funnel moved.
• The funnel height should be maintained approximately 2 – 4 cm from
the top of powder pile as it being formed in order to minimize the
impact of falling powder on the tip of the cone.
• If a symmetrical cone of powder cannot be successfully or reproducibly
prepared, this method is not appropriate. Determine the angle of repose
by measuring the height of the cone of powder and calculating the angle
of repose from the following equation:
29
material positioned above a fixed container base to drain from the
container.
• Formulation of a cone of a powder on a fixed diameter base allows
determination of the drained angle of repose.
DYNAMIC ANGLE OF REPOSE
• Dynamic angle of repose is calculated by filling a cylinder (with a clear
flat cover at the end) and rotating at a specific speed.
• The dynamic angle of a repose is an angle (relative to the horizontal)
formed by the flowing powder. The internal angle of kinetic friction is
defined by the plane separating those particles sliding down the top
layer of the powder and those particles that are rotating with the drum
(with roughened surface).
SPECIFICATIONS
• Although there is some variation in the qualitative description of powder
flow using the angle of repose, much of the pharmaceutical literature
appears to be consistent with the classification shown in the table.
Flow property Angle of repose (degrees)
Excellent 25 – 30
Good 31 -35
Fair – aid not needed 36 – 40
Passable – may hang up 41 – 45
Poor – must agitate, vibrate 46 – 55
Very poor 56 – 65
Very, very poor > 66
PROCEDURE FOR ANGLE OF REPOSE
• The base should be free from vibration. Vary the height of the funnel to
carefully build up a symmetrical cone of powder. Care should be taken
to prevent vibration as the funnel moved.
• The funnel height should be maintained approximately 2 – 4 cm from
the top of powder pile as it being formed in order to minimize the
impact of falling powder on the tip of the cone.
• If a symmetrical cone of powder cannot be successfully or reproducibly
prepared, this method is not appropriate. Determine the angle of repose
by measuring the height of the cone of powder and calculating the angle
of repose from the following equation:
29
GM Hamad
tanθ=
height
0.5 base
OR
θ=tan
−1
h
R
2. COMPRESSIBILITY INDEX & HAUSNER RATIO
• It is simple, fast and popular method of determining powder flow
characteristics. The compressibility index has been proposed as an
indirect measure of bulk density, size and shape, surface area, moisture
content, and cohesiveness of materials because all of these can
influence the observed compressibility index.
• The compressibility index and the Hausner ratio are determined by
measuring both the bulk volume and the tapped volume of powder.
• Although there are some variations in the method of determining the
compressibility index and Hausner ratio, the basic procedure is to
measure:
The unsettled apparent volume V0
The final tapped volume V1 of the powder then tapping the
material until no further volume changes occur.
• The compressibility and Hausner ratio are calculated as follows:
Compressibilty index=100×(
??????
0−??????
1
??????
0
)
Hausner ratio=
??????
1
??????
0
• Alternatively, the compressibility index and Hausner ratio may be
calculated using measured values for bilk density and tapped density as
fallows
Compressibility index= 100×(
ρ
bulk − ρ
tap
ρ
bulk
)
Hausner ratio =
ρ
tap
ρ
bulk
• For compressibility and Hausner ratio the generally accepted scale of
flow ability is given in the table:
30
tanθ=
height
0.5 base
OR
θ=tan
−1
h
R
2. COMPRESSIBILITY INDEX & HAUSNER RATIO
• It is simple, fast and popular method of determining powder flow
characteristics. The compressibility index has been proposed as an
indirect measure of bulk density, size and shape, surface area, moisture
content, and cohesiveness of materials because all of these can
influence the observed compressibility index.
• The compressibility index and the Hausner ratio are determined by
measuring both the bulk volume and the tapped volume of powder.
• Although there are some variations in the method of determining the
compressibility index and Hausner ratio, the basic procedure is to
measure:
The unsettled apparent volume V0
The final tapped volume V1 of the powder then tapping the
material until no further volume changes occur.
• The compressibility and Hausner ratio are calculated as follows:
Compressibilty index=100×(
??????
0−??????
1
??????
0
)
Hausner ratio=
??????
1
??????
0
• Alternatively, the compressibility index and Hausner ratio may be
calculated using measured values for bilk density and tapped density as
fallows
Compressibility index= 100×(
ρ
bulk − ρ
tap
ρ
bulk
)
Hausner ratio =
ρ
tap
ρ
bulk
• For compressibility and Hausner ratio the generally accepted scale of
flow ability is given in the table:
30
GM Hamad
Compressibility index % Flow character Hausner ratio
< 10 Excellent 1.35 – 1.45
11 – 15 Good 1.46 – 1.59
16 – 20 Fair > 1.60
21 – 25 Passable 1.00 – 1.11
26 -31 Poor 1.12 – 1.18
32 – 37 Very poor 1.19 – 1.25
> 38 Very very poor 1.26 – 1.34
• Compressibility index and Hausner ratio are not intrinsic properties of
the powder. They are very much dependent upon the methodology
used.
• Recommended procedure for Compressibility index and Hausner ratio:
Use a 250 ml volumetric flask with a test sample weight of 100g.
Smaller weights and volume may be used but variations in the
method should be described with the results. An average of three
determinations are recommended.
3. FLOW THROUGH AN ORIFICE
• The flow rate of a substance depends upon many factors some of which
are particle related and some related to the process.
• Monitoring the rate of flow of material through an orifice has been
proposed as a better measure of powder flowability. Of particular
significance is the utility of monitoring flow continuously because
pulsating flow patterns have been observed even for free floating
materials. Changes in the flow rate as the container empties can also be
observed.
RECOMMENDED PROCEDURE FOR FLOW THROUGH AN ORIFICE
• Flow through an orifice is generally measured as the mass per time
flowing from any of a number of types of containers (cylinders, funnels,
hoppers). It can be used only for that materials having some capacity to
flow, it is not useful for cohesive materials.
• Provided that the height of the powdered bed is much greater than the
diameter of the orifice, the flow rate is virtually independent of the
powder head.
Use the cylinder as a container because the cylinder material
should have little effect on flow.
31
Compressibility index % Flow character Hausner ratio
< 10 Excellent 1.35 – 1.45
11 – 15 Good 1.46 – 1.59
16 – 20 Fair > 1.60
21 – 25 Passable 1.00 – 1.11
26 -31 Poor 1.12 – 1.18
32 – 37 Very poor 1.19 – 1.25
> 38 Very very poor 1.26 – 1.34
• Compressibility index and Hausner ratio are not intrinsic properties of
the powder. They are very much dependent upon the methodology
used.
• Recommended procedure for Compressibility index and Hausner ratio:
Use a 250 ml volumetric flask with a test sample weight of 100g.
Smaller weights and volume may be used but variations in the
method should be described with the results. An average of three
determinations are recommended.
3. FLOW THROUGH AN ORIFICE
• The flow rate of a substance depends upon many factors some of which
are particle related and some related to the process.
• Monitoring the rate of flow of material through an orifice has been
proposed as a better measure of powder flowability. Of particular
significance is the utility of monitoring flow continuously because
pulsating flow patterns have been observed even for free floating
materials. Changes in the flow rate as the container empties can also be
observed.
RECOMMENDED PROCEDURE FOR FLOW THROUGH AN ORIFICE
• Flow through an orifice is generally measured as the mass per time
flowing from any of a number of types of containers (cylinders, funnels,
hoppers). It can be used only for that materials having some capacity to
flow, it is not useful for cohesive materials.
• Provided that the height of the powdered bed is much greater than the
diameter of the orifice, the flow rate is virtually independent of the
powder head.
Use the cylinder as a container because the cylinder material
should have little effect on flow.
31
GM Hamad
The configuration results in flow rate being determined by the
movement of powder over powder rather than powder along the
wall of the container.
Powder flow rate often increases when the height of the powder
column is less than two times of the diameter of the column.
The orifice should be circular and the cylinder should be free of
vibration.
• General guidelines for dimensions of the cylinder are as follows:
Diameter of opening > 6 times of the diameter of the particles
Diameter of the cylinder > 2 times the diameter of the opening
• For the opening in the cylinder use a flat faced bottom plate with an
option to vary orifice diameter to provide maximum flexibility and to
better ensure the powder over powder flow pattern.
• Rate measurement can either be discrete or continuous. Continuous
measurement using an electronic balance can more effectively detect
momentary flow rate variations.
GENERAL SCALE FOR FLOWABILITY FOR FLOW THROUGH AND ORIFICE
• No general scale is available because flow rate is critically dependent on
the method used to measure it. Comparison between the published
results is difficult.
4. SHEAR CELL METHODS
• In an effort to put powder flow studies and hopper design on a more
fundamental basis, a verity of powder shear testers and methods that
permit more thorough and precisely, defined assessment of powder flow
properties have been developed.
• Shear cell methodology has been used extensively in the study of
pharmaceutical materials.
• From these methods a wide verity of parameters can be obtained
including the yield loci representing the shear stress and shear stain
relationship. The angle of internal friction, the unconfirmed yield
strength, the tensile strength and the verity of divided parameters such
as the flow factor and other flowability indices.
• Because of the ability to move precisely control experimental
parameters flow properties can also be determined as a function of
consolidation load, time and other environmental conditions.
BASIC METHODS FOR SHEAR CELL
32
The configuration results in flow rate being determined by the
movement of powder over powder rather than powder along the
wall of the container.
Powder flow rate often increases when the height of the powder
column is less than two times of the diameter of the column.
The orifice should be circular and the cylinder should be free of
vibration.
• General guidelines for dimensions of the cylinder are as follows:
Diameter of opening > 6 times of the diameter of the particles
Diameter of the cylinder > 2 times the diameter of the opening
• For the opening in the cylinder use a flat faced bottom plate with an
option to vary orifice diameter to provide maximum flexibility and to
better ensure the powder over powder flow pattern.
• Rate measurement can either be discrete or continuous. Continuous
measurement using an electronic balance can more effectively detect
momentary flow rate variations.
GENERAL SCALE FOR FLOWABILITY FOR FLOW THROUGH AND ORIFICE
• No general scale is available because flow rate is critically dependent on
the method used to measure it. Comparison between the published
results is difficult.
4. SHEAR CELL METHODS
• In an effort to put powder flow studies and hopper design on a more
fundamental basis, a verity of powder shear testers and methods that
permit more thorough and precisely, defined assessment of powder flow
properties have been developed.
• Shear cell methodology has been used extensively in the study of
pharmaceutical materials.
• From these methods a wide verity of parameters can be obtained
including the yield loci representing the shear stress and shear stain
relationship. The angle of internal friction, the unconfirmed yield
strength, the tensile strength and the verity of divided parameters such
as the flow factor and other flowability indices.
• Because of the ability to move precisely control experimental
parameters flow properties can also be determined as a function of
consolidation load, time and other environmental conditions.
BASIC METHODS FOR SHEAR CELL
32
GM Hamad
• One type of the cell is the cylindrical shear cell that is split horizontally
forming a shear place between the lower stationary base and the upper
movable portion of the shear cell ring. After powder bed consolidation in
the shear cell (using a well-defined procedure) the force necessary shear
the powder bed by moving the upper ring is determined.
• Annular shear cell designs offer some advantages over the cylindrical
shear cell design including the need for less material. A disadvantage,
however, is that because of its design the powder bed is not sheared as
uniformly i.e. material on the outside of the annulus is sheared more
than material in the inner region.
• A third type of shear cell, plate shear cell consist of a thin sandwich
powder between a lower stationary rough surface and an upper rough
surface that is moveable.
• All of the shear cells have their merits and demerits. As with the other
methods of characterizing powder flow many variations are described in
the literature.
• A significant advantage of shear cell methodology in general is a greater
degree of experimental control.
33
• One type of the cell is the cylindrical shear cell that is split horizontally
forming a shear place between the lower stationary base and the upper
movable portion of the shear cell ring. After powder bed consolidation in
the shear cell (using a well-defined procedure) the force necessary shear
the powder bed by moving the upper ring is determined.
• Annular shear cell designs offer some advantages over the cylindrical
shear cell design including the need for less material. A disadvantage,
however, is that because of its design the powder bed is not sheared as
uniformly i.e. material on the outside of the annulus is sheared more
than material in the inner region.
• A third type of shear cell, plate shear cell consist of a thin sandwich
powder between a lower stationary rough surface and an upper rough
surface that is moveable.
• All of the shear cells have their merits and demerits. As with the other
methods of characterizing powder flow many variations are described in
the literature.
• A significant advantage of shear cell methodology in general is a greater
degree of experimental control.
33
GM Hamad
QUALITY CONTROL OF SYRUPS AND ELIXIRS
SYRUPS
• “Syrups are concentrated, viscous aqueous solutions of sugar or sugar
substitutes with or without flavor and medical substances.”
• When purified water alone is used in making the solution of sucrose, the
preparation is known as syrup, or simple syrup if the sucrose
concentration is 85%.
PROPERTIES
• Syrups are aqueous preparations characterized by a sweet taste and
viscous consistency.
• They may contain sucrose at a concentration of at least 45% w/w.
• The sweet taste can also be obtained by using other polyols or
sweetening agents.
• Syrups usually contain aromatic or other flavoring agents.
ELIXIRS
• “Elixirs are clear, pleasantly flavored, sweetened hydroalcoholic liquids
intended for oral use.” The main ingredients in elixirs is ethanol and
water but glycine, sorbitol, PEG, flavoring agent, preservative and syrups
often are used in the preparation of final product.
PROPERTIES
• The solvents are often used to increase the solubility of the drug
substance in the dosage form.
• Elixirs are more fluid then syrups, due to use of less viscous ingredients
such as alcohol and the minimal use of viscosity improving agents such
as sucrose.
• They are used as flavors and vehicles such as aromatic elixir USP for drug
substances, with drug substances they are called medicated elixirs. For
example: Dexamethasone elixir.
QUALITY CONTROL TESTS
• The quality control tests for syrups and elixirs are as follows:
1. Viscosity 2. Weight per ml
34
QUALITY CONTROL OF SYRUPS AND ELIXIRS
SYRUPS
• “Syrups are concentrated, viscous aqueous solutions of sugar or sugar
substitutes with or without flavor and medical substances.”
• When purified water alone is used in making the solution of sucrose, the
preparation is known as syrup, or simple syrup if the sucrose
concentration is 85%.
PROPERTIES
• Syrups are aqueous preparations characterized by a sweet taste and
viscous consistency.
• They may contain sucrose at a concentration of at least 45% w/w.
• The sweet taste can also be obtained by using other polyols or
sweetening agents.
• Syrups usually contain aromatic or other flavoring agents.
ELIXIRS
• “Elixirs are clear, pleasantly flavored, sweetened hydroalcoholic liquids
intended for oral use.” The main ingredients in elixirs is ethanol and
water but glycine, sorbitol, PEG, flavoring agent, preservative and syrups
often are used in the preparation of final product.
PROPERTIES
• The solvents are often used to increase the solubility of the drug
substance in the dosage form.
• Elixirs are more fluid then syrups, due to use of less viscous ingredients
such as alcohol and the minimal use of viscosity improving agents such
as sucrose.
• They are used as flavors and vehicles such as aromatic elixir USP for drug
substances, with drug substances they are called medicated elixirs. For
example: Dexamethasone elixir.
QUALITY CONTROL TESTS
• The quality control tests for syrups and elixirs are as follows:
1. Viscosity 2. Weight per ml
34
GM Hamad
3. Content uniformity 4. Drug assay
VISCOSITY
INTRODUCTION
• Viscosity is the property of liquid that is closely related to resistance to
flow. It is defined as, “the force required to move one plane surface
continuously past another under specified steady state conditions when
the space between is filled by the liquid in question.
• It is defined as the shear stress divided by rate of shear strain.
• There are two types of viscosity:
Dynamic viscosity
Kinematic viscosity
DYNAMIC VISCOSITY
• The dynamic viscosity or the viscosity coefficient ‘h’ is the tangential
force per unit surface, known as shearing stress ‘t’ and expressed in
Pascal, necessary to move parallel to the sliding plane a layer of liquid of
1 square meter at a rate ‘v’ of 1ms
-1
relative to parallel layer at a
distance ‘x’ of 1 meter.
• The ratio dv/dx is speed gradient giving the rate of shear D expressed in
reciprocal seconds that:
h =
t
D
• The unit of dynamic viscosity is the Pascal second (Pa.s). The most
commonly used submultiple is the milliPascal second (mPa.s)
KINEMATIC VISCOSITY
• The kinematic viscosity ‘V’ is expressed in square meter per second is
obtained by diving the dynamic viscosity ‘h’ by the density ‘d’ expressed
in kilograms per cubic meter of the liquid measured at same
temperature:
V =
h
d
• The kinematic viscosity is expressed in square millimeters per second.
35
3. Content uniformity 4. Drug assay
VISCOSITY
INTRODUCTION
• Viscosity is the property of liquid that is closely related to resistance to
flow. It is defined as, “the force required to move one plane surface
continuously past another under specified steady state conditions when
the space between is filled by the liquid in question.
• It is defined as the shear stress divided by rate of shear strain.
• There are two types of viscosity:
Dynamic viscosity
Kinematic viscosity
DYNAMIC VISCOSITY
• The dynamic viscosity or the viscosity coefficient ‘h’ is the tangential
force per unit surface, known as shearing stress ‘t’ and expressed in
Pascal, necessary to move parallel to the sliding plane a layer of liquid of
1 square meter at a rate ‘v’ of 1ms
-1
relative to parallel layer at a
distance ‘x’ of 1 meter.
• The ratio dv/dx is speed gradient giving the rate of shear D expressed in
reciprocal seconds that:
h =
t
D
• The unit of dynamic viscosity is the Pascal second (Pa.s). The most
commonly used submultiple is the milliPascal second (mPa.s)
KINEMATIC VISCOSITY
• The kinematic viscosity ‘V’ is expressed in square meter per second is
obtained by diving the dynamic viscosity ‘h’ by the density ‘d’ expressed
in kilograms per cubic meter of the liquid measured at same
temperature:
V =
h
d
• The kinematic viscosity is expressed in square millimeters per second.
35
GM Hamad
UNITS OF VISCOSITY
• The basic unit is the poise (according to USP). However, viscosities
commonly encountered represent fractions of the poise, so that the
centipoise proves to be an easier unit.
• When the absolute scale viscosity is measured in poises or centipoises
for convenience the kinematic scale in which the units are strokes and
centi-strokes is commonly used.
• To obtain the kinematic viscosity from absolute viscosity the latter is
divided by the density of the liquid at the same temperature:
????????????�����??????� �??????����??????�?????? = �������� �??????����??????�?????? / ����??????�??????
MEASUREMENT OF VISCOSITY
• A capillary viscometer is used for the determination of viscosity of
Newtonian liquid and a rotating viscometer is used for the
determination of viscosity of both Newtonian and Non – Newtonian
liquids. Other viscometers may be used provided that the accuracy and
precision is not less than that obtained with the viscometers described
above.
• The absolute viscosity can be measured directly if accurate dimensions
of the measuring instrument are known, but it is more common practice
to calibrate the instrument with a liquid of known viscosity and to
determine the viscosity of the unknown fluid by comparison with that of
the known.
CAPILLARY VISCOMETER
• The usual method for the measurement of viscosity involves the
determination of the liquid required for a given volume of the liquid to
flow through a capillary.
EXAMPLE
• Many capillary tube viscometers have been devised, Ostwald and
Ubbelohde viscometers are among the most frequently used.
ROTATIONAL VISCOMETER
• A particularly easier and rapid type of instrument is a Rotational
viscometer, which utilizes the bob or a spindle immersed in the test
specimen and measures the resistance to movement of the rotating
part.
36
UNITS OF VISCOSITY
• The basic unit is the poise (according to USP). However, viscosities
commonly encountered represent fractions of the poise, so that the
centipoise proves to be an easier unit.
• When the absolute scale viscosity is measured in poises or centipoises
for convenience the kinematic scale in which the units are strokes and
centi-strokes is commonly used.
• To obtain the kinematic viscosity from absolute viscosity the latter is
divided by the density of the liquid at the same temperature:
????????????�����??????� �??????����??????�?????? = �������� �??????����??????�?????? / ����??????�??????
MEASUREMENT OF VISCOSITY
• A capillary viscometer is used for the determination of viscosity of
Newtonian liquid and a rotating viscometer is used for the
determination of viscosity of both Newtonian and Non – Newtonian
liquids. Other viscometers may be used provided that the accuracy and
precision is not less than that obtained with the viscometers described
above.
• The absolute viscosity can be measured directly if accurate dimensions
of the measuring instrument are known, but it is more common practice
to calibrate the instrument with a liquid of known viscosity and to
determine the viscosity of the unknown fluid by comparison with that of
the known.
CAPILLARY VISCOMETER
• The usual method for the measurement of viscosity involves the
determination of the liquid required for a given volume of the liquid to
flow through a capillary.
EXAMPLE
• Many capillary tube viscometers have been devised, Ostwald and
Ubbelohde viscometers are among the most frequently used.
ROTATIONAL VISCOMETER
• A particularly easier and rapid type of instrument is a Rotational
viscometer, which utilizes the bob or a spindle immersed in the test
specimen and measures the resistance to movement of the rotating
part.
36
GM Hamad
• Different spindles are available for given viscosity ranges and several
rotational speeds are generally available. Other rotational instruments
may have a stationary bob or rotating cup.
EXAMPLE
• Brookfield, Rotouisco, and Stormer viscometers are the examples of
rotating viscometers. MacMichael is an example of rotating cup
instrument.
TEMPERATURE SPECIFICATION
• Specifying the temperature is important because viscosity changes with
the temperature. In general viscosity decreases as the temperature is
raised.
• For the measurement of viscosity or apparent viscosity the temperature
of the substances must be accurately controlled, since small changes in
temperature may lead to marked changes in the viscosity. For usual
pharmaceutical purposes, temperature should be held to within ± 0.1.
COMMON METHODS FOR THE DETERMINATION OF VISCOSITY
METHOD I (U-TUBE VISCOMETER)
• The monograph states the size of the viscometer to be used.
APPARATUS
• The apparatus consist of a glass U – tube viscometer made up of clear
borosilicate and constructed in accordance with the dimensions given in
official books.
PROCEDURE
• Fill the viscometer with the liquid being in question through tube
L to slightly above the mark G using a long pipette to minimize
wetting the tube above the mark.
• Place the tube vertically in the water bath when it has attained
the specific temperature adjust the volume of the liquid so that
the bottom of the meniscus settles at the mark G.
• Adjust the liquid to a point about 5 mm above the mark E.
• After releasing the pressure or suction measure the time taken
for the bottom of the meniscus to fall from the top edge of mark
E to top edge of mark F.
37
• Different spindles are available for given viscosity ranges and several
rotational speeds are generally available. Other rotational instruments
may have a stationary bob or rotating cup.
EXAMPLE
• Brookfield, Rotouisco, and Stormer viscometers are the examples of
rotating viscometers. MacMichael is an example of rotating cup
instrument.
TEMPERATURE SPECIFICATION
• Specifying the temperature is important because viscosity changes with
the temperature. In general viscosity decreases as the temperature is
raised.
• For the measurement of viscosity or apparent viscosity the temperature
of the substances must be accurately controlled, since small changes in
temperature may lead to marked changes in the viscosity. For usual
pharmaceutical purposes, temperature should be held to within ± 0.1.
COMMON METHODS FOR THE DETERMINATION OF VISCOSITY
METHOD I (U-TUBE VISCOMETER)
• The monograph states the size of the viscometer to be used.
APPARATUS
• The apparatus consist of a glass U – tube viscometer made up of clear
borosilicate and constructed in accordance with the dimensions given in
official books.
PROCEDURE
• Fill the viscometer with the liquid being in question through tube
L to slightly above the mark G using a long pipette to minimize
wetting the tube above the mark.
• Place the tube vertically in the water bath when it has attained
the specific temperature adjust the volume of the liquid so that
the bottom of the meniscus settles at the mark G.
• Adjust the liquid to a point about 5 mm above the mark E.
• After releasing the pressure or suction measure the time taken
for the bottom of the meniscus to fall from the top edge of mark
E to top edge of mark F.
37
GM Hamad
• Calculate as either the kinematic viscosity ‘V’ in sq. millimeters per
second (mm
2
s
-1
) from the expression:
??????=?????? �
• Or the dynamic viscosity in milli Pascal seconds from the expression:
ɳ=k ρ t
• Where, t = time in seconds for the meniscus to fall from E to F, � = mass/
volume (gcm
-3
) obtained by multiplying the relative density (Appendix V)
the G is being examined by 0.9982, K = the constant of the instrument is
determined by using the appropriate European pharmacopoeia
reference liquid for viscometers.
METHOD II (CAPILLARY VISCOMETERS METHOD)
• The determination of viscosity by using a suitable capillary viscometer is
carried out a temperature of 20 ± 0.1℃ unless otherwise prescribed. The
time required for the level of the liquid to drop form one mark to the
other is measured with a stopwatch to the nearest one-fifth of a second.
• The result is valid only if two consecutive readings do not differ by more
than 1%. The average of not fewer than 3 readings gives the flow time of
the liquid to be examined.
• The determination may be carried out with an apparatus having the
specifications described in official books. The minimum flow time should
be 350s for size no. 1 and 200s for all other sizes.
PROCEDURE
•
• Fill the viscometer through tube L with sufficient quantity of
the liquid in question, previously brought to 20℃ unless
otherwise prescribed to fill blub A but ensuring that the level of
liquid in bulb B is below the exit to ventilation tube M.
• Immerse the viscometer in the water bath of water at 20 ±
0.1℃ unless otherwise prescribed, maintain it in upright
position and allow to stand not less than 30 minutes to allow
the temperature to reach equilibrium.
• Close the tube M and raise the level of the liquid in tube N up
to a level about 8 mm above mark E.
38
• Calculate as either the kinematic viscosity ‘V’ in sq. millimeters per
second (mm
2
s
-1
) from the expression:
??????=?????? �
• Or the dynamic viscosity in milli Pascal seconds from the expression:
ɳ=k ρ t
• Where, t = time in seconds for the meniscus to fall from E to F, � = mass/
volume (gcm
-3
) obtained by multiplying the relative density (Appendix V)
the G is being examined by 0.9982, K = the constant of the instrument is
determined by using the appropriate European pharmacopoeia
reference liquid for viscometers.
METHOD II (CAPILLARY VISCOMETERS METHOD)
• The determination of viscosity by using a suitable capillary viscometer is
carried out a temperature of 20 ± 0.1℃ unless otherwise prescribed. The
time required for the level of the liquid to drop form one mark to the
other is measured with a stopwatch to the nearest one-fifth of a second.
• The result is valid only if two consecutive readings do not differ by more
than 1%. The average of not fewer than 3 readings gives the flow time of
the liquid to be examined.
• The determination may be carried out with an apparatus having the
specifications described in official books. The minimum flow time should
be 350s for size no. 1 and 200s for all other sizes.
PROCEDURE
•
• Fill the viscometer through tube L with sufficient quantity of
the liquid in question, previously brought to 20℃ unless
otherwise prescribed to fill blub A but ensuring that the level of
liquid in bulb B is below the exit to ventilation tube M.
• Immerse the viscometer in the water bath of water at 20 ±
0.1℃ unless otherwise prescribed, maintain it in upright
position and allow to stand not less than 30 minutes to allow
the temperature to reach equilibrium.
• Close the tube M and raise the level of the liquid in tube N up
to a level about 8 mm above mark E.
38
GM Hamad
• Keep the liquid at this level by closing tube N and opening tube M. Open
the tube N and measure with a stopwatch to the nearest 1/5
th
of a
second, the time required for the level of the liquid to drop from mark E
to mark F.
• Calculate kinematic viscosity ‘V’ in sq. millimeters per second (mm
2
s
-1
)
from the expression:
??????=� �
• Calculate the dynamic viscosity in milli Pascal seconds from the
expression:
ɳ=k ρ t
METHOD III (ROTATING VISCOMETER METHOD)
• The principle of this method is to measure the force acting on a rotor
(torque) when it rotates at a constant angular velocity (rotational speed)
in the liquid.
• A rotating viscometer is used for the determination of viscosity of both
Non – Newtonian liquids (shear dependent viscosity or apparent
viscosity) and Newtonian (shear independent viscosity).
• The rotating viscometer can be divided into two groups:
Absolute viscometers
Relative viscometers
• In the relative viscometers the flow in the measuring geometry is not
defined. The measurements result in relative viscosity values, which
cannot be compared with absolute values or other relative values if not
determined by the same relative viscometer method.
• In absolute viscometers the flow in the measuring geometry is well
defined. The measurements result in absolute viscosity values, which
can be compared with any other absolute values.
• Different measuring systems are available for given viscosity ranges as
well as several rotational speed.
APPARATUS
• Following viscometers are used:
Concentric cylinder viscometers
Cone plate viscometers
Spindle viscometers
I. CONCENTRIC CYLINDER VISCOMETERS (ABSOLUTE VISCOMETER)
39
• Keep the liquid at this level by closing tube N and opening tube M. Open
the tube N and measure with a stopwatch to the nearest 1/5
th
of a
second, the time required for the level of the liquid to drop from mark E
to mark F.
• Calculate kinematic viscosity ‘V’ in sq. millimeters per second (mm
2
s
-1
)
from the expression:
??????=� �
• Calculate the dynamic viscosity in milli Pascal seconds from the
expression:
ɳ=k ρ t
METHOD III (ROTATING VISCOMETER METHOD)
• The principle of this method is to measure the force acting on a rotor
(torque) when it rotates at a constant angular velocity (rotational speed)
in the liquid.
• A rotating viscometer is used for the determination of viscosity of both
Non – Newtonian liquids (shear dependent viscosity or apparent
viscosity) and Newtonian (shear independent viscosity).
• The rotating viscometer can be divided into two groups:
Absolute viscometers
Relative viscometers
• In the relative viscometers the flow in the measuring geometry is not
defined. The measurements result in relative viscosity values, which
cannot be compared with absolute values or other relative values if not
determined by the same relative viscometer method.
• In absolute viscometers the flow in the measuring geometry is well
defined. The measurements result in absolute viscosity values, which
can be compared with any other absolute values.
• Different measuring systems are available for given viscosity ranges as
well as several rotational speed.
APPARATUS
• Following viscometers are used:
Concentric cylinder viscometers
Cone plate viscometers
Spindle viscometers
I. CONCENTRIC CYLINDER VISCOMETERS (ABSOLUTE VISCOMETER)
39
GM Hamad
• In this type of viscometer (coaxial double cylinder viscometer or simply
coaxial cylinder viscometer) the viscosity is measured by placing the
liquid in the gap between the inner cylinder and the outer cylinder.
• Viscosity measurement can be performed by rotating the inner cylinder
(Searle type viscometer) or the outer cylinder (Couette viscometer).
• For laminar flow, the viscosity ‘h’ expressed in Pascal seconds is given by
the following:
ɳ=
1
ω
(
M
4πh
)(
1
R
i
−
1
R
o
)=k
M
ω
• For Non – Newtonian liquids it is indispensable to specify the shear
stress T or the shear rate γ at which the viscosity is measured. Under
narrow gap conditions (conditions satisfied in absolute viscometers)
there is a proportional relationship between M and t and also between
?????? and γ:
t=A M γ=Bω
Where, A and B are the constants for the instruments and are
calculated by following expressions:
• For concentric surfaces:
A=
1R
i
2
+R
o
2
4πh R
i
2
R
o
2
�=
??????
??????
2
+??????
??????
2
??????
??????
2
−??????
??????
2
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, h = height of immersion in meters
of the inner cylinder in the liquid medium, Ri = radius in meter of inner
cylinder, Ro = radius in meter of outer cylinder, R = radius in meters of
the cone.
40
• In this type of viscometer (coaxial double cylinder viscometer or simply
coaxial cylinder viscometer) the viscosity is measured by placing the
liquid in the gap between the inner cylinder and the outer cylinder.
• Viscosity measurement can be performed by rotating the inner cylinder
(Searle type viscometer) or the outer cylinder (Couette viscometer).
• For laminar flow, the viscosity ‘h’ expressed in Pascal seconds is given by
the following:
ɳ=
1
ω
(
M
4πh
)(
1
R
i
−
1
R
o
)=k
M
ω
• For Non – Newtonian liquids it is indispensable to specify the shear
stress T or the shear rate γ at which the viscosity is measured. Under
narrow gap conditions (conditions satisfied in absolute viscometers)
there is a proportional relationship between M and t and also between
?????? and γ:
t=A M γ=Bω
Where, A and B are the constants for the instruments and are
calculated by following expressions:
• For concentric surfaces:
A=
1R
i
2
+R
o
2
4πh R
i
2
R
o
2
�=
??????
??????
2
+??????
??????
2
??????
??????
2
−??????
??????
2
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, h = height of immersion in meters
of the inner cylinder in the liquid medium, Ri = radius in meter of inner
cylinder, Ro = radius in meter of outer cylinder, R = radius in meters of
the cone.
40
GM Hamad
II. CONE PLATE VISCOMETERS (ABSOLUTE VISCOMETERS)
• In the cone plate viscometer, the liquid is introduced into the gap
between the flat disc and a cone forming a define angle. Viscosity
measurement can be performed by rotating the cone or the flat disc. For
laminar flow, the viscosity ‘h’ expressed in Pascal seconds is given by the
following:
ɳ=(
M
ω
)(
3α
2πR
3
)=k
M
ω
• For Non – Newtonian liquids it is indispensable to specify the shear
stress T or the shear rate γ at which the viscosity is measured. Under
narrow gap conditions (conditions satisfied in absolute viscometers)
there is a proportional relationship between M and t and also between
?????? and γ
t=A M γ=Bω
• Where, A and B are the constants for the instruments and are calculated
by following expressions; For cone plates:
A=
3
2πR
3
B=
1
α
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, h = height of immersion in meters
of the inner cylinder in the liquid medium, R = radius in meters of the
cone, α = angle in radians between the flat disc and the cone, K =
constant of the apparatus expressed in radians per cubic meter
41
II. CONE PLATE VISCOMETERS (ABSOLUTE VISCOMETERS)
• In the cone plate viscometer, the liquid is introduced into the gap
between the flat disc and a cone forming a define angle. Viscosity
measurement can be performed by rotating the cone or the flat disc. For
laminar flow, the viscosity ‘h’ expressed in Pascal seconds is given by the
following:
ɳ=(
M
ω
)(
3α
2πR
3
)=k
M
ω
• For Non – Newtonian liquids it is indispensable to specify the shear
stress T or the shear rate γ at which the viscosity is measured. Under
narrow gap conditions (conditions satisfied in absolute viscometers)
there is a proportional relationship between M and t and also between
?????? and γ
t=A M γ=Bω
• Where, A and B are the constants for the instruments and are calculated
by following expressions; For cone plates:
A=
3
2πR
3
B=
1
α
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, h = height of immersion in meters
of the inner cylinder in the liquid medium, R = radius in meters of the
cone, α = angle in radians between the flat disc and the cone, K =
constant of the apparatus expressed in radians per cubic meter
41
GM Hamad
III. SPINDLE VISCOMETERS (RELATIVE VISCOMETERS)
• In the spindle viscometers the viscosity is determined by rotating a
spindle (cylinder or disc shaped) immersed in the liquid. Relative values
of viscosity can be directly calculated using conversion factors from the
scale reading at a given rotational speed.
• In general way, the constant K of the apparatus may be determined at
various speeds of rotation using a certified viscometer calibration liquid.
The viscosity then corresponds to the expression:
η=K
M
ω
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, K = constant of the apparatus
expressed in radians per cubic meter.
PROCEDURE
• Measuring the viscosity according to the instructions for the operation
of the rotating viscometer.
• The temperature of measuring the viscosity is indicated in the
monograph.
• For non – Newtonian systems the monograph indicates the type of
viscometer to be used and if absolute viscometers are used the angular
velocity or the shear rate at which the
measurement is made.
• If it is impossible to obtain the indicated shear rate use a shear rate
slightly higher and a shear rate slightly lower and interpolate.
• With relative viscometers the shear rate is not the same throughout the
sample and therefore it cannot be defined.
• Under these conditions the viscosity of non – Newtonian liquids
determined from the previous formula has a relative character, which
depends upon the type of the spindle and the angular velocity as well as
the dimensions of the sample container and the depth of the immersion
of the spindle.
• The values obtained are comparable only if the method is carried out
under experimental conditions that are rigorously the same.
IV. METHOD IV (FALLING BALL VISCOMETER METHOD)
42
III. SPINDLE VISCOMETERS (RELATIVE VISCOMETERS)
• In the spindle viscometers the viscosity is determined by rotating a
spindle (cylinder or disc shaped) immersed in the liquid. Relative values
of viscosity can be directly calculated using conversion factors from the
scale reading at a given rotational speed.
• In general way, the constant K of the apparatus may be determined at
various speeds of rotation using a certified viscometer calibration liquid.
The viscosity then corresponds to the expression:
η=K
M
ω
• Where, M = torque in newton meters acting on cylinder surface, ?????? =
angular velocity in radians per second, K = constant of the apparatus
expressed in radians per cubic meter.
PROCEDURE
• Measuring the viscosity according to the instructions for the operation
of the rotating viscometer.
• The temperature of measuring the viscosity is indicated in the
monograph.
• For non – Newtonian systems the monograph indicates the type of
viscometer to be used and if absolute viscometers are used the angular
velocity or the shear rate at which the
measurement is made.
• If it is impossible to obtain the indicated shear rate use a shear rate
slightly higher and a shear rate slightly lower and interpolate.
• With relative viscometers the shear rate is not the same throughout the
sample and therefore it cannot be defined.
• Under these conditions the viscosity of non – Newtonian liquids
determined from the previous formula has a relative character, which
depends upon the type of the spindle and the angular velocity as well as
the dimensions of the sample container and the depth of the immersion
of the spindle.
• The values obtained are comparable only if the method is carried out
under experimental conditions that are rigorously the same.
IV. METHOD IV (FALLING BALL VISCOMETER METHOD)
42
GM Hamad
• The determination of dynamic viscosity of Newtonian liquids using a
suitable falling ball viscometer is performed at 20 ± 0.1℃ unless
otherwise prescribed, in the monograph.
• The time required for the test ball to fall in the liquid to be examined
from one ring mark to the other is determined. If no striker limit is not
defined for the equipment used the results is valid only if two
consecutive measurements do not differ by more than 1.5%.
APPARATUS
• The falling ball viscometer consist of a glass tube enclosed in a mantle
that allows direct control of temperature. Six balls made of glass, nickel,
iron or steel with different densities and diameters. The tube is fixed in
such a way that the axis is inclined by 10 ± 0.1
o
with regard to the
vertical. The tube has two ring marks which defines the distance the ball
has to roll.
• Commercially available apparatus is supplied with table giving the
constants, the density of the balls and the suitability of the different
balls for the expected range of viscosity.
PROCEDURE
• Fill the clean dry tube of the viscometer previously brought to 20 ± 0.1
o
C
with the liquid to be examined avoiding bubbles.
• Add the ball suitable for the range of viscosity of the liquid so as to
obtain a falling time not less than 30s.
• Close the tube and maintain the solution at 20 ± 0.1
o
C for at least 15
• minutes. Let the ball run through the liquid between the 2 ring marks
once without measurement.
• Let it run again and measure with a stopwatch to the nearest 1/5
th
of a
second the time required for the ball to roll from the upper to the lower
ring mark.
• Repeat the test run at least 3 times.
• Calculate the dynamic viscosity in the milli Pascal using the formula:
ɳ=k(ρ
1−ρ
2)×t
• Where, K = constant expressed in millimeter sq. per second square, t =
falling time of the ball in seconds, �1 = density of the ball used,
expressed in grams per cubic centimeter, �2 = density of the liquid to be
43
• The determination of dynamic viscosity of Newtonian liquids using a
suitable falling ball viscometer is performed at 20 ± 0.1℃ unless
otherwise prescribed, in the monograph.
• The time required for the test ball to fall in the liquid to be examined
from one ring mark to the other is determined. If no striker limit is not
defined for the equipment used the results is valid only if two
consecutive measurements do not differ by more than 1.5%.
APPARATUS
• The falling ball viscometer consist of a glass tube enclosed in a mantle
that allows direct control of temperature. Six balls made of glass, nickel,
iron or steel with different densities and diameters. The tube is fixed in
such a way that the axis is inclined by 10 ± 0.1
o
with regard to the
vertical. The tube has two ring marks which defines the distance the ball
has to roll.
• Commercially available apparatus is supplied with table giving the
constants, the density of the balls and the suitability of the different
balls for the expected range of viscosity.
PROCEDURE
• Fill the clean dry tube of the viscometer previously brought to 20 ± 0.1
o
C
with the liquid to be examined avoiding bubbles.
• Add the ball suitable for the range of viscosity of the liquid so as to
obtain a falling time not less than 30s.
• Close the tube and maintain the solution at 20 ± 0.1
o
C for at least 15
• minutes. Let the ball run through the liquid between the 2 ring marks
once without measurement.
• Let it run again and measure with a stopwatch to the nearest 1/5
th
of a
second the time required for the ball to roll from the upper to the lower
ring mark.
• Repeat the test run at least 3 times.
• Calculate the dynamic viscosity in the milli Pascal using the formula:
ɳ=k(ρ
1−ρ
2)×t
• Where, K = constant expressed in millimeter sq. per second square, t =
falling time of the ball in seconds, �1 = density of the ball used,
expressed in grams per cubic centimeter, �2 = density of the liquid to be
43
GM Hamad
examined, g=expressed in gcm
-3
obtained by multiplying its relative
density by 0.9982
WEIGHT PER MILLILITER
• It is defined as, “The weight per ml of a liquid in the weight in gram of 1
ml of a liquid when weighed in the air at 20℃ unless otherwise specified
in the monograph.”
DETERMINATION
• The weight per ml is determined by dividing the weight in air expressed
in grams of the quantity of the liquid that falls on pycnometer at the
specified temperature by the capacity expressed in ml of the
pycnometer at the same temperature.
• The capacity of the pycnometer is ascertained from the weight in air
expressed in grams of the quantity of the water required to fill the
pycnometer at same temperature.
• The weight of a liter of water at specified temperatures when weighed
against brass weights in air of density 0.0012 g per ml is given in the
following table:
Temperature (
o
C) Weight of water (g)
20 997.91
25 997.07
30 995.67
DENSITY
• The density can be defined as, “the density of a substance is the ratio of
its mass to its volume at 20
o
C. It is expressed in Kgm
-3
.
DETERMINATION
• The density is determined by dividing the weight in air of the quantity of
liquid being examined that fills a pycnometer at 20
o
C by the weight of air
of water required to fill the pycnometer after making allowance for the
thrust of the air.
• The density is calculated from the expression:
ρ
20=
998.2 (??????
1+A)
(??????
2+A)
44
examined, g=expressed in gcm
-3
obtained by multiplying its relative
density by 0.9982
WEIGHT PER MILLILITER
• It is defined as, “The weight per ml of a liquid in the weight in gram of 1
ml of a liquid when weighed in the air at 20℃ unless otherwise specified
in the monograph.”
DETERMINATION
• The weight per ml is determined by dividing the weight in air expressed
in grams of the quantity of the liquid that falls on pycnometer at the
specified temperature by the capacity expressed in ml of the
pycnometer at the same temperature.
• The capacity of the pycnometer is ascertained from the weight in air
expressed in grams of the quantity of the water required to fill the
pycnometer at same temperature.
• The weight of a liter of water at specified temperatures when weighed
against brass weights in air of density 0.0012 g per ml is given in the
following table:
Temperature (
o
C) Weight of water (g)
20 997.91
25 997.07
30 995.67
DENSITY
• The density can be defined as, “the density of a substance is the ratio of
its mass to its volume at 20
o
C. It is expressed in Kgm
-3
.
DETERMINATION
• The density is determined by dividing the weight in air of the quantity of
liquid being examined that fills a pycnometer at 20
o
C by the weight of air
of water required to fill the pycnometer after making allowance for the
thrust of the air.
• The density is calculated from the expression:
ρ
20=
998.2 (??????
1+A)
(??????
2+A)
44
GM Hamad
• Where, M1 = weight in air, in the grams of the substance being
examined, M2 = wright in air in grams of water, A = the correction factor
for the thrust of the air 0.0012 M2, 998.2 = the density of water at 20
o
C
in Kgm
-3
. In most cases the correction for the thrust of the air may be
disregarded.
RELATIVE DENSITY
• The relative density can be defined as, “the relative density is the ratio
of the mass of a certain volume of a substance at a temperature t1 to the
mass of an equal volume of water at temperature t2 unless otherwise
indicated the relative density is used. Relative density is also commonly
expressed as �
4
20
• Density, defined as the mass of a unit volume of the substance at 20
o
C
may also be used, expressed in kg/m
3
or g/cm
3
.
• The quantities are related by the following equations, where the density
is expressed in g/cm
3
.
ρ
20=0.998203×d
20
20
�� d
20
20
=1.00180×ρ
20
ρ
20=0.999972×d
4
20
or d
4
20
=1.00003×ρ
20
d
4
20
=0.998203×d
20
20
• Relative density are measured with the precision to the number of
decimals prescribed in the monograph using a density bottle (solid or
liquids), a hydrostatic balance (solids), a hydrometer (liquids) or a digital
density meter with an oscillating transducer (liquids or gases).
• When the determination is made by weighing the buoyancy of air is
disregarded which may introduce an error of 1 unit in the third decimal
place. When using a density meter, the buoyancy of air has no influence.
OSCILLATING TRANSDUCER DENSITY METER
• The apparatus consist of:
A U – shaped tube made up of borosilicate glass which contains
the liquid to be examined.
A magneto electrical or piezo-electrical excitation system that
causes the tube to oscillate as a cantilever oscillator at a
characteristic frequency depending upon the density of the liquid
to be examined.
A means of measuring the oscillation period T, which may be
45
• Where, M1 = weight in air, in the grams of the substance being
examined, M2 = wright in air in grams of water, A = the correction factor
for the thrust of the air 0.0012 M2, 998.2 = the density of water at 20
o
C
in Kgm
-3
. In most cases the correction for the thrust of the air may be
disregarded.
RELATIVE DENSITY
• The relative density can be defined as, “the relative density is the ratio
of the mass of a certain volume of a substance at a temperature t1 to the
mass of an equal volume of water at temperature t2 unless otherwise
indicated the relative density is used. Relative density is also commonly
expressed as �
4
20
• Density, defined as the mass of a unit volume of the substance at 20
o
C
may also be used, expressed in kg/m
3
or g/cm
3
.
• The quantities are related by the following equations, where the density
is expressed in g/cm
3
.
ρ
20=0.998203×d
20
20
�� d
20
20
=1.00180×ρ
20
ρ
20=0.999972×d
4
20
or d
4
20
=1.00003×ρ
20
d
4
20
=0.998203×d
20
20
• Relative density are measured with the precision to the number of
decimals prescribed in the monograph using a density bottle (solid or
liquids), a hydrostatic balance (solids), a hydrometer (liquids) or a digital
density meter with an oscillating transducer (liquids or gases).
• When the determination is made by weighing the buoyancy of air is
disregarded which may introduce an error of 1 unit in the third decimal
place. When using a density meter, the buoyancy of air has no influence.
OSCILLATING TRANSDUCER DENSITY METER
• The apparatus consist of:
A U – shaped tube made up of borosilicate glass which contains
the liquid to be examined.
A magneto electrical or piezo-electrical excitation system that
causes the tube to oscillate as a cantilever oscillator at a
characteristic frequency depending upon the density of the liquid
to be examined.
A means of measuring the oscillation period T, which may be
45
GM Hamad
converted by the apparatus to give a direct reading of density or
used to calculate density using the constants A and B.
• The resonant frequency ‘f’ is a function of the spring constant ‘c’ and the
mass ‘m’ of the system.
�
2
=
1
??????
2
=
�
�
×
1
4�
2
• Hence,
??????
2
=(
??????
�
+
� × ??????
�
)×4�
2
• Where, M = mass of the tube, V = inner volume of the tube
• Introduction of 2 constants � = �/ (4�2 × ??????) and � = M/?????? leads to the
classical equation for the oscillating transducer.
ρ=A×T
2
−B
• The constants A and B are determined by operating the instrument by
the U- Tube filled with two different samples of known density. For
example; degassed water R and air. Control measurements are made
daily using degassed water R.
• The results displayed for the control measurement using degassed water
R shall not deviate from the reference value (�20 = 0.998203 gcm
-3
=
1.000000) by more than its specified error.
FACTORS AFFECTING ACCURACY
• Temperature uniformity throughout the tube
• Non – linearity over a range of density
• Parasitic resonant effect
• Viscosity, whereby solutions with a higher viscosity the calibrant have
density that is apparently higher than the true value.
APPARENT DENSITY
• The term apparent density is used in monographs for dilute Ethanol,
Industrial methylated spirit and industrial methylate spirit (ketone free).
• It is defined as, “weight in air per unit volume.” It is expressed in kgm
-3
.
It named density in the laboratory alcohol table for laboratory use.
• The apparent density is calculated by the following expression:
�������� ����??????�??????=997.0�
20
46
converted by the apparatus to give a direct reading of density or
used to calculate density using the constants A and B.
• The resonant frequency ‘f’ is a function of the spring constant ‘c’ and the
mass ‘m’ of the system.
�
2
=
1
??????
2
=
�
�
×
1
4�
2
• Hence,
??????
2
=(
??????
�
+
� × ??????
�
)×4�
2
• Where, M = mass of the tube, V = inner volume of the tube
• Introduction of 2 constants � = �/ (4�2 × ??????) and � = M/?????? leads to the
classical equation for the oscillating transducer.
ρ=A×T
2
−B
• The constants A and B are determined by operating the instrument by
the U- Tube filled with two different samples of known density. For
example; degassed water R and air. Control measurements are made
daily using degassed water R.
• The results displayed for the control measurement using degassed water
R shall not deviate from the reference value (�20 = 0.998203 gcm
-3
=
1.000000) by more than its specified error.
FACTORS AFFECTING ACCURACY
• Temperature uniformity throughout the tube
• Non – linearity over a range of density
• Parasitic resonant effect
• Viscosity, whereby solutions with a higher viscosity the calibrant have
density that is apparently higher than the true value.
APPARENT DENSITY
• The term apparent density is used in monographs for dilute Ethanol,
Industrial methylated spirit and industrial methylate spirit (ketone free).
• It is defined as, “weight in air per unit volume.” It is expressed in kgm
-3
.
It named density in the laboratory alcohol table for laboratory use.
• The apparent density is calculated by the following expression:
�������� ����??????�??????=997.0�
20
46
GM Hamad
• Where, �20 = Relative density of the substance being examined, 997.2 =
Weight in air in kg of 1 cubic meter of water.
CHEMICAL ASSAY OF SYRUP AND ELIXIR
• Routinely the assay of content in syrup and elixirs is done by the amount
of liquid representing the certain amount of drug normally in a single
unit. It is then diluted according to the procedure and instrument use.
• Analysis is performed by the method prescribed in the individual
monographs.
• Results obtained are expressed as percentage of the active ingredient in
the table or unit dose and compared with limits in the monograph of
drug.
• Common assay procedure involves:
Titrimetric method
Spectroscopic method
UV spectroscopy
HPLC
Biological assay
Microbial assay
47
• Where, �20 = Relative density of the substance being examined, 997.2 =
Weight in air in kg of 1 cubic meter of water.
CHEMICAL ASSAY OF SYRUP AND ELIXIR
• Routinely the assay of content in syrup and elixirs is done by the amount
of liquid representing the certain amount of drug normally in a single
unit. It is then diluted according to the procedure and instrument use.
• Analysis is performed by the method prescribed in the individual
monographs.
• Results obtained are expressed as percentage of the active ingredient in
the table or unit dose and compared with limits in the monograph of
drug.
• Common assay procedure involves:
Titrimetric method
Spectroscopic method
UV spectroscopy
HPLC
Biological assay
Microbial assay
47
GM Hamad
QUALITY CONTROL OF SUPPOSITORIES
SUPPOSITORIES
• Suppositories are medicated solid bodies of various sizes and shapes
suitable for introduction into body cavities for their local and systemic
effect.
• The medicament is incorporated into the base such as coca butter that
melts at body temperature or into one such as glycerol gelatin or PEG
which slowly dissolve into the mucous secretions.
• Suppositories are suited particularly for producing local action but may
also be used to produce a systemic effect or exert a mechanical effect to
facilitate emptying the lower bowel.
CLASSIFICATION OF SUPPOSITORIES
• Rectal suppositories for adults weigh 2 gm and are torpedo shape.
Children's suppositories weigh about 1 gm.
• Vaginal suppositories or Pessaries weigh about 3-5gm and are molded
in globular or oviform shape or compressed on a tablet press into conical
shapes.
• Urethral suppositories called bogies are pencil shape. Those intended
for males weigh 4 gm each and are 100-150 mm long while those for
females are 2 gm each and 60-75 mm in length.
QUALITY CONTROL TESTS FOR SUPPOSITORIES
1. VISUAL EXAMINATION
• This includes shape, color, surface condition and odor.
I. SHAPE
• It is advisable to check the shape of the suppository to see if it is
consistent.
II. COLOR
• The intensity, nature and homogeneity of the color should be verified.
The use of color chart is advisable.
III. SURFACE CONDITION
• The surface conditions can be checked for brilliance, dullness, mottling,
cracks, dark regions, axial cavities, bursts, air bubbles, holes, etc.
48
QUALITY CONTROL OF SUPPOSITORIES
SUPPOSITORIES
• Suppositories are medicated solid bodies of various sizes and shapes
suitable for introduction into body cavities for their local and systemic
effect.
• The medicament is incorporated into the base such as coca butter that
melts at body temperature or into one such as glycerol gelatin or PEG
which slowly dissolve into the mucous secretions.
• Suppositories are suited particularly for producing local action but may
also be used to produce a systemic effect or exert a mechanical effect to
facilitate emptying the lower bowel.
CLASSIFICATION OF SUPPOSITORIES
• Rectal suppositories for adults weigh 2 gm and are torpedo shape.
Children's suppositories weigh about 1 gm.
• Vaginal suppositories or Pessaries weigh about 3-5gm and are molded
in globular or oviform shape or compressed on a tablet press into conical
shapes.
• Urethral suppositories called bogies are pencil shape. Those intended
for males weigh 4 gm each and are 100-150 mm long while those for
females are 2 gm each and 60-75 mm in length.
QUALITY CONTROL TESTS FOR SUPPOSITORIES
1. VISUAL EXAMINATION
• This includes shape, color, surface condition and odor.
I. SHAPE
• It is advisable to check the shape of the suppository to see if it is
consistent.
II. COLOR
• The intensity, nature and homogeneity of the color should be verified.
The use of color chart is advisable.
III. SURFACE CONDITION
• The surface conditions can be checked for brilliance, dullness, mottling,
cracks, dark regions, axial cavities, bursts, air bubbles, holes, etc.
48
GM Hamad
IV. ODOR
• A change in the odor may also be indicative of a degradation process.
2. DISSOLUTION TEST
• Dissolution testing is often required for suppositories to test for
hardening and polymorphic transitions of active ingredients and
suppository bases.
• Dissolution testing methods include the paddle method, basket method,
membrane diffusion method/dialysis method, and the continuous
flow/bead method.
• The melting suppositories with the paddle method showed fat floating
rapidly to the surface of the fluid instead of staying below the water
surface. With the basket method, the surfactant produced small droplets
of the fat that were dispersed into the medium almost immediately.
3. WEIGHT UNIFORMITY
• Weigh 20 suppositories individually. w1, w2, w3….w20
• Weigh all the suppositories together = W.
• Calculate the average weight = W/20.
LIMIT
• No suppository should deviate more than 5% from the average weight
except that two may deviate by not more than 7.5% of the average
weight of suppository.
NOTE
• If the weight is found to be too small, it is advisable to check whether
the mold is being well filled and whether there are axial cavities or air
bubbles caused by badly adjusted mechanical stirring or the presence of
an undesirable surfactant.
• If the weight is found to be too high, check that scraping has been
carried out correctly, and also that the mixture is homogeneous.
4. ASSAY OF ACTIVE INGREDIENTS
• Determine the amount of active ingredients in suppositories by the
method described in the Assay.
• It should be according to the specification given in individual
monograph.
49
IV. ODOR
• A change in the odor may also be indicative of a degradation process.
2. DISSOLUTION TEST
• Dissolution testing is often required for suppositories to test for
hardening and polymorphic transitions of active ingredients and
suppository bases.
• Dissolution testing methods include the paddle method, basket method,
membrane diffusion method/dialysis method, and the continuous
flow/bead method.
• The melting suppositories with the paddle method showed fat floating
rapidly to the surface of the fluid instead of staying below the water
surface. With the basket method, the surfactant produced small droplets
of the fat that were dispersed into the medium almost immediately.
3. WEIGHT UNIFORMITY
• Weigh 20 suppositories individually. w1, w2, w3….w20
• Weigh all the suppositories together = W.
• Calculate the average weight = W/20.
LIMIT
• No suppository should deviate more than 5% from the average weight
except that two may deviate by not more than 7.5% of the average
weight of suppository.
NOTE
• If the weight is found to be too small, it is advisable to check whether
the mold is being well filled and whether there are axial cavities or air
bubbles caused by badly adjusted mechanical stirring or the presence of
an undesirable surfactant.
• If the weight is found to be too high, check that scraping has been
carried out correctly, and also that the mixture is homogeneous.
4. ASSAY OF ACTIVE INGREDIENTS
• Determine the amount of active ingredients in suppositories by the
method described in the Assay.
• It should be according to the specification given in individual
monograph.
49
GM Hamad
5. LIQUEFACTION TIME OR SOFTENING TIME TEST
• In this test a U tube is partially immersed in a constant temperature bath
and is maintained at a temperature between 35°C to 37°C. There is a
constriction in the tube in which the suppository is kept and above the
suppository, a glass rod is kept. The time taken for the glass rod to go
through the suppository and reach the constriction is known as the
liquefaction time or softening time.
• Another apparatus is there for finding “softening time” which mimics in
vivo conditions. It uses a cellophane tube, and the temperature is
maintained by water circulation at 37°C. Time taken for the suppository
to melt is noted. Time is 5-25 mins.
6. BREAKING TEST (HARDNESS)
• The breaking test is designed as a method for measuring the fragility or
brittleness of suppository.
• The suppository is placed in the instrument. Add 600 g weight and leave
it for one min. If not broken, add 200 g weight every one minute until
the suppository is broken.
CALCULATIONS
• The hardness of the suppository is calculated by adding the weights
together. But if the suppository is broken before the end of the last min,
the last weight is canceled.
7. MELTING RANGE (MELTING POINT, MELTING ZONE)
• Many suppository bases and medicated suppositories are mixtures, and
so do not have a precise melting point. Melting range or melting zone is
the term often preferred.
• The release rate of the suppository is related to its melting point; it is
therefore critical that this test be evaluated using a non-destructive
method. A number of different techniques are used to study melting
behavior, including the open capillary tube, the U-tube, and the drop
point methods.
• The methods used are similar in principle but include different steps and
techniques. In general, they include the set-up of the equipment,
placement of the suppository dosage unit in the apparatus, followed by
the application of heat and observation for a change in the system, such
as melting or movement. In general, the melting point should be equal
to or less than 37℃. A non-destructive method must be used because if
50
5. LIQUEFACTION TIME OR SOFTENING TIME TEST
• In this test a U tube is partially immersed in a constant temperature bath
and is maintained at a temperature between 35°C to 37°C. There is a
constriction in the tube in which the suppository is kept and above the
suppository, a glass rod is kept. The time taken for the glass rod to go
through the suppository and reach the constriction is known as the
liquefaction time or softening time.
• Another apparatus is there for finding “softening time” which mimics in
vivo conditions. It uses a cellophane tube, and the temperature is
maintained by water circulation at 37°C. Time taken for the suppository
to melt is noted. Time is 5-25 mins.
6. BREAKING TEST (HARDNESS)
• The breaking test is designed as a method for measuring the fragility or
brittleness of suppository.
• The suppository is placed in the instrument. Add 600 g weight and leave
it for one min. If not broken, add 200 g weight every one minute until
the suppository is broken.
CALCULATIONS
• The hardness of the suppository is calculated by adding the weights
together. But if the suppository is broken before the end of the last min,
the last weight is canceled.
7. MELTING RANGE (MELTING POINT, MELTING ZONE)
• Many suppository bases and medicated suppositories are mixtures, and
so do not have a precise melting point. Melting range or melting zone is
the term often preferred.
• The release rate of the suppository is related to its melting point; it is
therefore critical that this test be evaluated using a non-destructive
method. A number of different techniques are used to study melting
behavior, including the open capillary tube, the U-tube, and the drop
point methods.
• The methods used are similar in principle but include different steps and
techniques. In general, they include the set-up of the equipment,
placement of the suppository dosage unit in the apparatus, followed by
the application of heat and observation for a change in the system, such
as melting or movement. In general, the melting point should be equal
to or less than 37℃. A non-destructive method must be used because if
50
GM Hamad
the suppository is melted before a measurement is made, the
suppository constituents may be transformed into a metastable state.
• The melting test consists of placing a suppository on the surface of water
thermostatically controlled at 37℃ and verifying the complete melting
of the suppository in a few minutes. This is not so much a measurement
as an evaluation.
MELTING RANGE TEST
• Determines the time taken by an entire suppository to melt when it is
immersed in a constant temperature bath at 37°C.
• The experiment done by using the USP Tablet Disintegration Apparatus.
PROCEDURE
• The suppository is completely immersed in the constant temperature
water bath, and the time for the entire suppository to melt or disperse
in the surrounding water is measured.
• The suppository is considered disintegrated when:
It is completely dissolved or
Dispersed into its component part.
Become soft “change in shape” with formation of core which is
not resistant to pressure with glass rod.
TYPES
• Melting Range tests are of two types:
Macro-Melting Range Test
▪ Macro-melting range test is applied for entire suppository
and measure the time it takes for complete melting.
Micro-Melting range test
▪ The test is performed for suppository bases only.
8. DISINTEGRATION TEST
APPARATUS
• Cylinder of glass or other transparent material, 60 mm high, 50 mm
internal diameter and thickness of walls is 8 mm.
• The cylinder is fitted with two horizontal parallel perforated stainless-
steel plates. These plates are 30 mm apart.
• A device to maintain the temperature at 37℃ i. e. water bath.
51
the suppository is melted before a measurement is made, the
suppository constituents may be transformed into a metastable state.
• The melting test consists of placing a suppository on the surface of water
thermostatically controlled at 37℃ and verifying the complete melting
of the suppository in a few minutes. This is not so much a measurement
as an evaluation.
MELTING RANGE TEST
• Determines the time taken by an entire suppository to melt when it is
immersed in a constant temperature bath at 37°C.
• The experiment done by using the USP Tablet Disintegration Apparatus.
PROCEDURE
• The suppository is completely immersed in the constant temperature
water bath, and the time for the entire suppository to melt or disperse
in the surrounding water is measured.
• The suppository is considered disintegrated when:
It is completely dissolved or
Dispersed into its component part.
Become soft “change in shape” with formation of core which is
not resistant to pressure with glass rod.
TYPES
• Melting Range tests are of two types:
Macro-Melting Range Test
▪ Macro-melting range test is applied for entire suppository
and measure the time it takes for complete melting.
Micro-Melting range test
▪ The test is performed for suppository bases only.
8. DISINTEGRATION TEST
APPARATUS
• Cylinder of glass or other transparent material, 60 mm high, 50 mm
internal diameter and thickness of walls is 8 mm.
• The cylinder is fitted with two horizontal parallel perforated stainless-
steel plates. These plates are 30 mm apart.
• A device to maintain the temperature at 37℃ i. e. water bath.
51
GM Hamad
• A device which will hold the cylinder 90 mm below the water i. e. surface
of the water and after every 10 mins. the cylinder can be inverted
without emerging from water.
METHOD
• Place a suppository to the lower perforated plate insert the cylinder for
30 mins. Unless otherwise specified in individual monograph.
• The suppository is disintegrated when:
It has completely dissolved except for any insoluble material.
The disintegration product has fallen through the perforated tube
or risen to the surface of water.
Any solid material remaining between the plates melted
completely and no longer has a solid core.
• Repeat the test for two more suppositories. All three should disintegrate
within 30 mins. unless otherwise specified in individual monographs.
9. STABILITY TESTING
• Cocoa butter suppositories on storage, “bloom”; i.e., they form a white
powdery deposit on the surface. This can be avoided by storing the
suppositories at uniform cool temperatures and by wrapping them in
foils.
• Fat based suppositories harden on storage, i.e. there is an upward shift
in melting range due to slow crystallization to the more stable
polymorphic forms of the base.
• The softening time test and differential scanning calorimetry can be used
as stability indicating test methods.
• If we store the suppositories at an elevated temperature, just below its
melting range, immediately after manufacture, the aging process is
speeded up.
TESTS FOR SUPPOSITORY BASES
1. MELTING RANGE
• Since fats do not have sharp melting point, their melting characteristics
are expressed as a range indicating the temperature at which the fat
start to melt and the temperature at which it is completely melted.
2. SOLIDIFICATION POINT
• This value indicates the time required for the base solidification when it
is chilled in the mold. If the interval between the melting range and
52
• A device which will hold the cylinder 90 mm below the water i. e. surface
of the water and after every 10 mins. the cylinder can be inverted
without emerging from water.
METHOD
• Place a suppository to the lower perforated plate insert the cylinder for
30 mins. Unless otherwise specified in individual monograph.
• The suppository is disintegrated when:
It has completely dissolved except for any insoluble material.
The disintegration product has fallen through the perforated tube
or risen to the surface of water.
Any solid material remaining between the plates melted
completely and no longer has a solid core.
• Repeat the test for two more suppositories. All three should disintegrate
within 30 mins. unless otherwise specified in individual monographs.
9. STABILITY TESTING
• Cocoa butter suppositories on storage, “bloom”; i.e., they form a white
powdery deposit on the surface. This can be avoided by storing the
suppositories at uniform cool temperatures and by wrapping them in
foils.
• Fat based suppositories harden on storage, i.e. there is an upward shift
in melting range due to slow crystallization to the more stable
polymorphic forms of the base.
• The softening time test and differential scanning calorimetry can be used
as stability indicating test methods.
• If we store the suppositories at an elevated temperature, just below its
melting range, immediately after manufacture, the aging process is
speeded up.
TESTS FOR SUPPOSITORY BASES
1. MELTING RANGE
• Since fats do not have sharp melting point, their melting characteristics
are expressed as a range indicating the temperature at which the fat
start to melt and the temperature at which it is completely melted.
2. SOLIDIFICATION POINT
• This value indicates the time required for the base solidification when it
is chilled in the mold. If the interval between the melting range and
52
GM Hamad
solidification point is 10℃ or more, the time require for solidification
may have to be shortened for more efficient manufacturing procedure
by augmenting refrigeration.
3. SAPONIFICATION VALUE
• The number of milligrams of potassium hydroxide require to neutralize
the free acids and to saponify the esters contained in 1 g of fat is an
indication of the type of glyceride (mono or tri) as well as the amount of
glyceride present.
4. IODINE VALUE
• The value expresses the number of grams of iodine that react with 100 g
of fat or other unsaturated material.
• The possibility of the decomposition by moisture, acid, or oxygen (leads
to rancidity in fats) increases with high iodine values.
5. WATER NUMBER
• The amount of water in grams which can be incorporated in 100 gram of
fat is expressed by this value.
• The water number can be increased by addition of surface-active agents
(surfactants).
6. ACID VALUE
• The number of milligrams of potassium hydroxide required to neutralize
the free acid in 1 gram of substance is expressed by this value.
• Low acid values or complete absence of acid are important for good
suppository base. Free acids complicate formulation work, because they
react with other ingredients and can also cause irritation when in
contact with mucous membranes.
53
solidification point is 10℃ or more, the time require for solidification
may have to be shortened for more efficient manufacturing procedure
by augmenting refrigeration.
3. SAPONIFICATION VALUE
• The number of milligrams of potassium hydroxide require to neutralize
the free acids and to saponify the esters contained in 1 g of fat is an
indication of the type of glyceride (mono or tri) as well as the amount of
glyceride present.
4. IODINE VALUE
• The value expresses the number of grams of iodine that react with 100 g
of fat or other unsaturated material.
• The possibility of the decomposition by moisture, acid, or oxygen (leads
to rancidity in fats) increases with high iodine values.
5. WATER NUMBER
• The amount of water in grams which can be incorporated in 100 gram of
fat is expressed by this value.
• The water number can be increased by addition of surface-active agents
(surfactants).
6. ACID VALUE
• The number of milligrams of potassium hydroxide required to neutralize
the free acid in 1 gram of substance is expressed by this value.
• Low acid values or complete absence of acid are important for good
suppository base. Free acids complicate formulation work, because they
react with other ingredients and can also cause irritation when in
contact with mucous membranes.
53
GM Hamad
QUALITY CONTROL OF PARENTERAL
DEDINITION
• Parenteral dosage form is used through injection and must be free of
any viable micro-organism, pyrogens and also meeting the other official
criteria of the dosage form including Isotonicity.
QUALITY CONTROL TESTS FOR PARENTERALS
• Three general areas of quality control tests for sterile products are:
Incoming stock
Manufacturing
Finished product
INCOMING STOCK
• For sterile products, incoming stock control encompasses routine
conformation of color, odor, crystalline state, solubility, identification,
melting point, loss on drying, residue on ignition, heavy metal and
specific gravity on all ingredient.
• It also includes special evaluation such as pyrogen test for water for
injection, glass test on containers and identity test on rubber closures.
IN PROCESS QUALITY CONTROL
• In process control, manufacturing of sterile products involves many
tests, readings and observations are made throughout the
manufacturing process of products.
• In-process quality control of sterile product includes:
Conductivity measurement during the distillation of water for
injection.
Confirmation of volume of fill in product containers.
Recording of cycle time and temperature for thermal sterilization
of the product.
Confirming the count and identity of labels for the product.
FINISHED PRODUCT
• The finished product control tests includes the final assay and tests to
which a product is subjected.
54
QUALITY CONTROL OF PARENTERAL
DEDINITION
• Parenteral dosage form is used through injection and must be free of
any viable micro-organism, pyrogens and also meeting the other official
criteria of the dosage form including Isotonicity.
QUALITY CONTROL TESTS FOR PARENTERALS
• Three general areas of quality control tests for sterile products are:
Incoming stock
Manufacturing
Finished product
INCOMING STOCK
• For sterile products, incoming stock control encompasses routine
conformation of color, odor, crystalline state, solubility, identification,
melting point, loss on drying, residue on ignition, heavy metal and
specific gravity on all ingredient.
• It also includes special evaluation such as pyrogen test for water for
injection, glass test on containers and identity test on rubber closures.
IN PROCESS QUALITY CONTROL
• In process control, manufacturing of sterile products involves many
tests, readings and observations are made throughout the
manufacturing process of products.
• In-process quality control of sterile product includes:
Conductivity measurement during the distillation of water for
injection.
Confirmation of volume of fill in product containers.
Recording of cycle time and temperature for thermal sterilization
of the product.
Confirming the count and identity of labels for the product.
FINISHED PRODUCT
• The finished product control tests includes the final assay and tests to
which a product is subjected.
54
GM Hamad
• In addition to usual chemical and biological tests, sterile product should
be subjected to:
Sterility test
Pyrogen test
Leaker test
Clarity test
Final assay of drug
product
1. STERILITY TEST
• It is used for Parenteral, ophthalmic preparations, syringes and implants.
STERILITY TESTING FOR PARENTERAL
BASIC CONCEPTS
NON-STERILE PRODUCTS
• Means that presence of any viable micro-organisms (Bacteria, Fungi,
Yeast etc.) is there.
STERILE PRODUCTS
• Absence of any viable micro-organism in preparation.
ASSUMPTION
• It is assumed that organisms will grow in the given culture medium
although some limitations are there.
LIMITATIONS
• Different organisms have different nutritional need.
• Different temperature for different organisms is required.
• Some micro-organisms especially spores need longer period to grow
than recommended one.
SELECTION OF CULTURE MEDIA
• Various culture media with methods of preparation are given in B.P. and
U.S.P. which should be chosen.
• Any other medium should give equal or more growth of micro-organisms
like Aerobic, Anaerobic or Fungi.
TESTS FOR MEDIA
I. STERILITY
• Prior to testing it is checked the media prepared is sterilized or not.
PROCEDURE
• Incubate the portions of the media for 14 days.
55
• In addition to usual chemical and biological tests, sterile product should
be subjected to:
Sterility test
Pyrogen test
Leaker test
Clarity test
Final assay of drug
product
1. STERILITY TEST
• It is used for Parenteral, ophthalmic preparations, syringes and implants.
STERILITY TESTING FOR PARENTERAL
BASIC CONCEPTS
NON-STERILE PRODUCTS
• Means that presence of any viable micro-organisms (Bacteria, Fungi,
Yeast etc.) is there.
STERILE PRODUCTS
• Absence of any viable micro-organism in preparation.
ASSUMPTION
• It is assumed that organisms will grow in the given culture medium
although some limitations are there.
LIMITATIONS
• Different organisms have different nutritional need.
• Different temperature for different organisms is required.
• Some micro-organisms especially spores need longer period to grow
than recommended one.
SELECTION OF CULTURE MEDIA
• Various culture media with methods of preparation are given in B.P. and
U.S.P. which should be chosen.
• Any other medium should give equal or more growth of micro-organisms
like Aerobic, Anaerobic or Fungi.
TESTS FOR MEDIA
I. STERILITY
• Prior to testing it is checked the media prepared is sterilized or not.
PROCEDURE
• Incubate the portions of the media for 14 days.
55
GM Hamad
• Incubation of media for Bacteria at 30℃– 35℃
• Incubation of media for Fungi at 20℃ – 25℃
• Observe the microbial growth.
RESULT
• If there is no growth of microbes, media is sterilized and ready for
testing.
II. GROWTH PROMOTION TEST OF AEROBES, ANAEROBES AND FUNGI
• The growth promotion test is performed to check whether the media
prepared is good for the microbial growth or not. Test each batch of
ready-prepared medium and each batch of medium prepared either
from dehydrated medium or from the ingredients.
PROCEDURE
• Inoculation of the medium with 100 viable micro-organisms of each of
the following micro-organisms.
An aerobe ------ (Staphylococcus aureus)
Spore forming aerobe --- (Bacillus subtilis)
An anaerobe ----- (Clostridium sporogenes)
A Fungus ----- (Candida albicans)
• Incubation Temperature:
For Bacteria: 30℃ to 35℃
For Fungus: 20℃ to 25℃
• Incubation Period.
Incubation period should not be less than 07 days.
RESULT
• If early and copious growth occurs, the medium contains required
nutritive properties and is suitable.
EFFECTIVENESS OF MEDIA IN THE PRESENCE AND ABSENCE OF THE
PREPARATION BEING EXAMINED
• Two containers of media are prepared for each of aerobe, anaerobe,
fungus or a spore forming aerobe.
• To each container, add preparation. Inoculate each container with 100
viable micro-organisms. Prepare similar sets without preparation.
• Incubate all containers at an appropriate temperature i.e. 30℃ to 35℃
for bacteria and 20℃ to 25℃ for fungus not more than 07 days.
56
• Incubation of media for Bacteria at 30℃– 35℃
• Incubation of media for Fungi at 20℃ – 25℃
• Observe the microbial growth.
RESULT
• If there is no growth of microbes, media is sterilized and ready for
testing.
II. GROWTH PROMOTION TEST OF AEROBES, ANAEROBES AND FUNGI
• The growth promotion test is performed to check whether the media
prepared is good for the microbial growth or not. Test each batch of
ready-prepared medium and each batch of medium prepared either
from dehydrated medium or from the ingredients.
PROCEDURE
• Inoculation of the medium with 100 viable micro-organisms of each of
the following micro-organisms.
An aerobe ------ (Staphylococcus aureus)
Spore forming aerobe --- (Bacillus subtilis)
An anaerobe ----- (Clostridium sporogenes)
A Fungus ----- (Candida albicans)
• Incubation Temperature:
For Bacteria: 30℃ to 35℃
For Fungus: 20℃ to 25℃
• Incubation Period.
Incubation period should not be less than 07 days.
RESULT
• If early and copious growth occurs, the medium contains required
nutritive properties and is suitable.
EFFECTIVENESS OF MEDIA IN THE PRESENCE AND ABSENCE OF THE
PREPARATION BEING EXAMINED
• Two containers of media are prepared for each of aerobe, anaerobe,
fungus or a spore forming aerobe.
• To each container, add preparation. Inoculate each container with 100
viable micro-organisms. Prepare similar sets without preparation.
• Incubate all containers at an appropriate temperature i.e. 30℃ to 35℃
for bacteria and 20℃ to 25℃ for fungus not more than 07 days.
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RESULT
• Equal growth in all the containers indicate that preparation has no
antimicrobial activity.
• Less growth, delayed growth or no growth in containers having
preparation shows antimicrobial activity.
ELIMINATION OF ANTIMICROBIAL ACTIVITY OF PREPARATION
• Antimicrobial activity of preparation is eliminated by:
Dilution
Neutralization of activity
Filtration
• After treatment (removal of activity), it is again checked for
antimicrobial activity as above.
METHODS FOR STERILITY TESTING
• Two methods/techniques of sterility testing are:
Membrane Filtration
Direct Inoculation
I. MEMBRANE FILTRATION
• The method is preferably used for:
Filterable aqueous preparations
Alcoholic or oily preparations
Preparations miscible with or soluble in aqueous or oily solvents
that do not have antimicrobial activity.
• Membrane filters of esters or mixture of cellulose are recommended for
alcoholic or oily preparations.
• Pore size should not be greater than 0.450 micrometer or 450 nm.
Diameter of the filter is 50 mm, if more than 50 mm then adjusted as per
procedure given in the official procedure.
• The apparatus is designed so that the solution to be examined can be
introduced and filtered under aseptic conditions; it permits the aseptic
removal of the membrane for transfer to the medium or it is suitable for
carrying out the incubation after adding the medium to the apparatus
itself.
DIFFERENT DOSAGE FORMS/PREPARATIONS ARE TREATED BEFORE
MEMBRANE FILTRATION
A. AQUEOUS SOLUTION
57
RESULT
• Equal growth in all the containers indicate that preparation has no
antimicrobial activity.
• Less growth, delayed growth or no growth in containers having
preparation shows antimicrobial activity.
ELIMINATION OF ANTIMICROBIAL ACTIVITY OF PREPARATION
• Antimicrobial activity of preparation is eliminated by:
Dilution
Neutralization of activity
Filtration
• After treatment (removal of activity), it is again checked for
antimicrobial activity as above.
METHODS FOR STERILITY TESTING
• Two methods/techniques of sterility testing are:
Membrane Filtration
Direct Inoculation
I. MEMBRANE FILTRATION
• The method is preferably used for:
Filterable aqueous preparations
Alcoholic or oily preparations
Preparations miscible with or soluble in aqueous or oily solvents
that do not have antimicrobial activity.
• Membrane filters of esters or mixture of cellulose are recommended for
alcoholic or oily preparations.
• Pore size should not be greater than 0.450 micrometer or 450 nm.
Diameter of the filter is 50 mm, if more than 50 mm then adjusted as per
procedure given in the official procedure.
• The apparatus is designed so that the solution to be examined can be
introduced and filtered under aseptic conditions; it permits the aseptic
removal of the membrane for transfer to the medium or it is suitable for
carrying out the incubation after adding the medium to the apparatus
itself.
DIFFERENT DOSAGE FORMS/PREPARATIONS ARE TREATED BEFORE
MEMBRANE FILTRATION
A. AQUEOUS SOLUTION
57
GM Hamad
• Membrane is moistened with sterile diluent like 0.1% w/v neutral
solution of meat or casein peptone.
• Volume of the preparation should be according to the specifications.
Dilute the volume of the preparation to about 100 ml with diluent
and filter immediately.
• If preparation has antimicrobial activity, washing is done by three
portions of diluents each of 100 ml.
• Transfer the whole membrane to the culture medium or cut it
aseptically into two equal parts and transfer one half to each of two
suitable media. Incubate the media for not less than 14 days.
• Alternatively, transfer the medium onto the membrane in the
apparatus. Incubate the media for not less than 14 days.
B. SOLUBLE SOLIDS
• Specified quantity dissolved in 0.1% w/v neutral solution of meat or
casein peptone (sterile).
• After filtration, perform the test as for aqueous solution.
C. OILS AND OILY SOLUTION
• Low viscosity oils/oily preparations filtered through dry membrane
directly.
• Viscous preparations are diluted with isopropyl myristate having no
antimicrobial activity.
• After penetration, the oil into membrane, filtrations is facilitated by
pressure or suction. Washing with diluents (sterile neutral meat
solution, 0.1% w/v or casein peptone) containing 0.1 % w/v (4-
tertoctylphenoxy) polyethoxy ethanol or 0.1 % w/v polysorbate 80.
• Complete test as in the case of aqueous solutions.
D. OINTMENTS AND CREAMS
• Ointments in fatty bases or W/O emulsion diluted by heating (40℃ or
up to 45℃) and add diluent (Isopropyl myristate). Filter rapidly.
• Test as for oily preparations.
II. DIRECT INOCULATION
• Dilution of the dosage forms. Liquids are to be diluted 10 folds. Solids
are to be diluted 100 folds. To eliminate antimicrobial activity of the
preparation, larger volume is required for dilution. Either concentrated
medium is to be added to the preparation or preparations are added to
the medium.
OILY LIQUIDS
58
• Membrane is moistened with sterile diluent like 0.1% w/v neutral
solution of meat or casein peptone.
• Volume of the preparation should be according to the specifications.
Dilute the volume of the preparation to about 100 ml with diluent
and filter immediately.
• If preparation has antimicrobial activity, washing is done by three
portions of diluents each of 100 ml.
• Transfer the whole membrane to the culture medium or cut it
aseptically into two equal parts and transfer one half to each of two
suitable media. Incubate the media for not less than 14 days.
• Alternatively, transfer the medium onto the membrane in the
apparatus. Incubate the media for not less than 14 days.
B. SOLUBLE SOLIDS
• Specified quantity dissolved in 0.1% w/v neutral solution of meat or
casein peptone (sterile).
• After filtration, perform the test as for aqueous solution.
C. OILS AND OILY SOLUTION
• Low viscosity oils/oily preparations filtered through dry membrane
directly.
• Viscous preparations are diluted with isopropyl myristate having no
antimicrobial activity.
• After penetration, the oil into membrane, filtrations is facilitated by
pressure or suction. Washing with diluents (sterile neutral meat
solution, 0.1% w/v or casein peptone) containing 0.1 % w/v (4-
tertoctylphenoxy) polyethoxy ethanol or 0.1 % w/v polysorbate 80.
• Complete test as in the case of aqueous solutions.
D. OINTMENTS AND CREAMS
• Ointments in fatty bases or W/O emulsion diluted by heating (40℃ or
up to 45℃) and add diluent (Isopropyl myristate). Filter rapidly.
• Test as for oily preparations.
II. DIRECT INOCULATION
• Dilution of the dosage forms. Liquids are to be diluted 10 folds. Solids
are to be diluted 100 folds. To eliminate antimicrobial activity of the
preparation, larger volume is required for dilution. Either concentrated
medium is to be added to the preparation or preparations are added to
the medium.
OILY LIQUIDS
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GM Hamad
• Use media to which have been added a suitable emulsifying agent at a
concentration shown to be appropriate in the method suitability of the
test, for example, polysorbate 80 at a concentration of 10 g/l.
OINTMENTS AND CREAMS
• Prepare by diluting to about 1 in 10 by emulsifying with the chosen
emulsifying agent in a suitable sterile diluent such as peptone (1 g/l) TS1.
Transfer the diluted product to a medium not containing an emulsifying
agent.
• Incubate the inoculated media for not less than 14 days. Observe the
cultures several times during the incubation period. Shake cultures
containing oily products gently each day.
• For anaerobic micro-organisms, for example Clostridium sporogenes,
mercapto acetate or similar medium is used. Minimum shaking is done
to maintain anaerobic condition.
OBSERVATION AND INTERPRETATION OF RESULTS
• At intervals during the incubation period and at its conclusion examine
the media for macroscopic evidence of microbial growth.
If no evidence of microbial growth is found, the product to be
examined complies with the test for sterility.
If evidence of microbial growth is found, the product, in this case
preserve the culture and repeat the whole procedure, if again a
culture is formed, compare it with the first growth. If it matches
then the product to be examined does not comply with the test
for sterility.
• Conventional microbiological methods are generally satisfactory for
identification of microorganisms recovered from a sterility test. While,
routine microbiological method can demonstrate that 2 isolates are not
identical these methods may not be sufficiently sensitive or reliable
enough to provide unequivocal evidence that two isolates are from the
same source.
• More sensitive tests, for example; Molecular typing with RNA/ DNA
homology, may be necessary to determine that microorganism are
clonally related and have a common origin.
OPHTHALMIC PREPARATIONS
• Ophthalmic preparations include:
59
• Use media to which have been added a suitable emulsifying agent at a
concentration shown to be appropriate in the method suitability of the
test, for example, polysorbate 80 at a concentration of 10 g/l.
OINTMENTS AND CREAMS
• Prepare by diluting to about 1 in 10 by emulsifying with the chosen
emulsifying agent in a suitable sterile diluent such as peptone (1 g/l) TS1.
Transfer the diluted product to a medium not containing an emulsifying
agent.
• Incubate the inoculated media for not less than 14 days. Observe the
cultures several times during the incubation period. Shake cultures
containing oily products gently each day.
• For anaerobic micro-organisms, for example Clostridium sporogenes,
mercapto acetate or similar medium is used. Minimum shaking is done
to maintain anaerobic condition.
OBSERVATION AND INTERPRETATION OF RESULTS
• At intervals during the incubation period and at its conclusion examine
the media for macroscopic evidence of microbial growth.
If no evidence of microbial growth is found, the product to be
examined complies with the test for sterility.
If evidence of microbial growth is found, the product, in this case
preserve the culture and repeat the whole procedure, if again a
culture is formed, compare it with the first growth. If it matches
then the product to be examined does not comply with the test
for sterility.
• Conventional microbiological methods are generally satisfactory for
identification of microorganisms recovered from a sterility test. While,
routine microbiological method can demonstrate that 2 isolates are not
identical these methods may not be sufficiently sensitive or reliable
enough to provide unequivocal evidence that two isolates are from the
same source.
• More sensitive tests, for example; Molecular typing with RNA/ DNA
homology, may be necessary to determine that microorganism are
clonally related and have a common origin.
OPHTHALMIC PREPARATIONS
• Ophthalmic preparations include:
59
GM Hamad
Eye solutions
Eye ointments
Eye creams
• These preparations are sterile and tested for sterility along with other
tests. Tests are:
Sterility (as given in official Books).
Clarity.
Heavy metal particles.
Active contents determination.
pH.
Retention time.
2. PYROGEN TEST
• Pyrogens are products of the growth of micro-organisms especially
molds, bacteria (in particular gram negative), viruses and fungi.
• Debris left after killing micro-organisms. Chemically, pyrogenic materials
are lipid in nature.
SYMPTOMS CAUSED BY PYROGENS
• Febrile reaction in human being.
• Other symptoms include:
Chills, pains in the back and legs and malaise.
ELIMINATION OF PYROGENS
• Heating equipment and material at:
180℃ for 04 hours.
200℃ for 01 hour.
250℃ for 30 to 45 minutes.
650℃ requires only 01 minute to destroy pyrogens.
• Distillation
• Filtration (Reverse osmosis)
• Adsorption (low molecular weight drugs e.g. glucose etc. can only be
used).
METHODS
• Two Methods are used officially
LAL (limulus amebocyte lysate) test. Also, known as the in vitro
testing.
Biological Test. Also, known as the in vivo testing.
I. LAL TEST
60
Eye solutions
Eye ointments
Eye creams
• These preparations are sterile and tested for sterility along with other
tests. Tests are:
Sterility (as given in official Books).
Clarity.
Heavy metal particles.
Active contents determination.
pH.
Retention time.
2. PYROGEN TEST
• Pyrogens are products of the growth of micro-organisms especially
molds, bacteria (in particular gram negative), viruses and fungi.
• Debris left after killing micro-organisms. Chemically, pyrogenic materials
are lipid in nature.
SYMPTOMS CAUSED BY PYROGENS
• Febrile reaction in human being.
• Other symptoms include:
Chills, pains in the back and legs and malaise.
ELIMINATION OF PYROGENS
• Heating equipment and material at:
180℃ for 04 hours.
200℃ for 01 hour.
250℃ for 30 to 45 minutes.
650℃ requires only 01 minute to destroy pyrogens.
• Distillation
• Filtration (Reverse osmosis)
• Adsorption (low molecular weight drugs e.g. glucose etc. can only be
used).
METHODS
• Two Methods are used officially
LAL (limulus amebocyte lysate) test. Also, known as the in vitro
testing.
Biological Test. Also, known as the in vivo testing.
I. LAL TEST
60
GM Hamad
• Innovation in pyrogen testing is the use of an in-vitro limules amebocyte
lysate (LAL) test. The test is capable of detecting the more potent
endotoxin pyrogens.
• Officially it is termed as bacterial endotoxin test (BET). After initial
reservation, this method of test has become established in many
pharmaceuticals companies. The USP has now list the LAL test for
checking pyrogen in distilled water.
SOURCE OF LYSATE
• An extract from the blood cells of horseshoe crab, limules Polyphemus
contains and enzyme system called as limules amebocyte lysate (LAL)
which reacts with pyrogens (endotoxin-lipopolysaccharides from G –ve
bacteria), so that an assay mixture increases the viscosity and opacity
until and opaque gel is formed.
??????�??????���??????�?????? + �??????��????????????� = �����?????? ????????????�
• The tests accomplishes within 15 to 60 minutes, depending upon
concentration of pyrogen after mixing. The concentrated pyrogen makes
the gel more turbid and thicker.
LAL PREPARATION
• LAL is prepared by bleeding healthy mature specimens by heart
puncture. The amebocytes are carefully concentrated, washed, and
lysed by osmotic effects.
• Prior to perform LAL test, lysate assay is carried out with purified
endotoxins and accepted if it detects 0.001 µg/ ml or less concentration
of purified endotoxins.
REQUIREMENTS
• The glassware, such as glass test tubes (10 x 75 mm) used in the test
must be thoroughly cleaned, dried and heat sterilized.
• A buffer solution of potassium phosphate 2mEq/ml is used to adjust the
pH of the test sample at 7.
PROCEDURE
• The pH of the sample if specified is adjusted.
• Mix equal parts of (0.05 – 0.2 ml) of test solution and LAL standardized
61
• Innovation in pyrogen testing is the use of an in-vitro limules amebocyte
lysate (LAL) test. The test is capable of detecting the more potent
endotoxin pyrogens.
• Officially it is termed as bacterial endotoxin test (BET). After initial
reservation, this method of test has become established in many
pharmaceuticals companies. The USP has now list the LAL test for
checking pyrogen in distilled water.
SOURCE OF LYSATE
• An extract from the blood cells of horseshoe crab, limules Polyphemus
contains and enzyme system called as limules amebocyte lysate (LAL)
which reacts with pyrogens (endotoxin-lipopolysaccharides from G –ve
bacteria), so that an assay mixture increases the viscosity and opacity
until and opaque gel is formed.
??????�??????���??????�?????? + �??????��????????????� = �����?????? ????????????�
• The tests accomplishes within 15 to 60 minutes, depending upon
concentration of pyrogen after mixing. The concentrated pyrogen makes
the gel more turbid and thicker.
LAL PREPARATION
• LAL is prepared by bleeding healthy mature specimens by heart
puncture. The amebocytes are carefully concentrated, washed, and
lysed by osmotic effects.
• Prior to perform LAL test, lysate assay is carried out with purified
endotoxins and accepted if it detects 0.001 µg/ ml or less concentration
of purified endotoxins.
REQUIREMENTS
• The glassware, such as glass test tubes (10 x 75 mm) used in the test
must be thoroughly cleaned, dried and heat sterilized.
• A buffer solution of potassium phosphate 2mEq/ml is used to adjust the
pH of the test sample at 7.
PROCEDURE
• The pH of the sample if specified is adjusted.
• Mix equal parts of (0.05 – 0.2 ml) of test solution and LAL standardized
61
GM Hamad
reagent in thoroughly cleans dried and heat sterilized glass test tube (10
x 75 mm).
• The mixture is incubated immediately at 36 – 38℃ for one hour in assay
tube. The assay tube must remain undisturbed completely, as agitation
may irreversibly destroy the gel leading to fast negative results.
• The test tube is observed after the specified time and is examined for
the formation of the opaque gel which represents a positive test end
point.
• The test is performed using a commercial LAL test kit. This kit contains a
lyophilized LAL and Escherichia coli endotoxin and pure water as
standards and these later two and used to check the sensitivity of the
test.
• Table below outlines the criteria for interpreting limulus test result.
LAL
tube
Test sample/Control Result
1 Negative control (pyrogen free saline) Should be negative
2 Positive control (pyrogen) Should be positive
3 Positive internal control (test contain with
endotoxins)
Should be positive
4 Test sample May be positive/negative
II. BIOLOGICAL TEST (THE IN VIVO RABBIT TEST)
• It is an official test. It is known as biological test/the in vivo rabbit test.
• The test consist of measuring the rise in body temperature evoked in
rabbits by the IV injection of a sterile solution of the substance to be
examined. It employs rabbit as test animal because:
Rabbit resembles in body temperature to human being. It is a
docile animal. It has appropriate body weight.
• The in vivo test involves the following steps:
i. Selection and Rejection of animals
ii. Materials used:
▪ Temperature recording device
▪ Syringes, needles and glass wares
▪ Boxes for retaining the animals.
iii. Preliminary Test
▪ It also includes the SHAM test i.e. performing all the test
other than injecting test solution.
iv. Main Test
62
reagent in thoroughly cleans dried and heat sterilized glass test tube (10
x 75 mm).
• The mixture is incubated immediately at 36 – 38℃ for one hour in assay
tube. The assay tube must remain undisturbed completely, as agitation
may irreversibly destroy the gel leading to fast negative results.
• The test tube is observed after the specified time and is examined for
the formation of the opaque gel which represents a positive test end
point.
• The test is performed using a commercial LAL test kit. This kit contains a
lyophilized LAL and Escherichia coli endotoxin and pure water as
standards and these later two and used to check the sensitivity of the
test.
• Table below outlines the criteria for interpreting limulus test result.
LAL
tube
Test sample/Control Result
1 Negative control (pyrogen free saline) Should be negative
2 Positive control (pyrogen) Should be positive
3 Positive internal control (test contain with
endotoxins)
Should be positive
4 Test sample May be positive/negative
II. BIOLOGICAL TEST (THE IN VIVO RABBIT TEST)
• It is an official test. It is known as biological test/the in vivo rabbit test.
• The test consist of measuring the rise in body temperature evoked in
rabbits by the IV injection of a sterile solution of the substance to be
examined. It employs rabbit as test animal because:
Rabbit resembles in body temperature to human being. It is a
docile animal. It has appropriate body weight.
• The in vivo test involves the following steps:
i. Selection and Rejection of animals
ii. Materials used:
▪ Temperature recording device
▪ Syringes, needles and glass wares
▪ Boxes for retaining the animals.
iii. Preliminary Test
▪ It also includes the SHAM test i.e. performing all the test
other than injecting test solution.
iv. Main Test
62
GM Hamad
▪ Preparation and injection of sample
▪ Determination of initial and maximum temperature.
v. Interpretation of results.
I. SELECTION AND REJECTION OF ANIMAL
SELECTION OF ANIMAL
• Rabbit should be healthy and adult of either sex. Weight of each rabbit
should not be less than 1.5 kg. Should be given normal/balanced diet
without any medicines in particular antibiotics.
• They should not show any loss in body weight during the week receding
the test.
REJECTION/EXCLUSION OF THE ANIMAL
• If used for negative Pyrogens test and the period passed was less than
03 days.
• If used for positive Pyrogens test and the period passed was less than 03
weeks.
• If any individual animal having temperature 39.8℃ or 38℃ is excluded
from the test.
II. MATERIAL USED
A. THERMOMETER OR ELECTRICAL DEVICE
• Any device can be used with the precision of ± 0.1°C.
• Should be inserted in the rectum of the rabbit to a depth about 5cm
(B.P.) or 7.5 cm (U.S.P.).
• Depth of insertion should be constant for any one rabbit for any one
test.
• If electrical device is used, it should be inserted at least 90 minutes
before the test start and should remain in position through the test.
B. GLASS WARE, SYRINGES AND NEEDLES
• Should be free of Pyrogens.
• Should be washed with water for injection.
• Should be heated in an oven (Hot air) at 250° for 30 to 45 minutes.
C. RETAINING BOXES
• Where electrical device is used, boxes should be such that the animal
is retained only by loosely fitting neck-stock, the rest of the body
remains relatively free.
• The animal must be put into boxes one hour before test and remain
in them through the test.
63
▪ Preparation and injection of sample
▪ Determination of initial and maximum temperature.
v. Interpretation of results.
I. SELECTION AND REJECTION OF ANIMAL
SELECTION OF ANIMAL
• Rabbit should be healthy and adult of either sex. Weight of each rabbit
should not be less than 1.5 kg. Should be given normal/balanced diet
without any medicines in particular antibiotics.
• They should not show any loss in body weight during the week receding
the test.
REJECTION/EXCLUSION OF THE ANIMAL
• If used for negative Pyrogens test and the period passed was less than
03 days.
• If used for positive Pyrogens test and the period passed was less than 03
weeks.
• If any individual animal having temperature 39.8℃ or 38℃ is excluded
from the test.
II. MATERIAL USED
A. THERMOMETER OR ELECTRICAL DEVICE
• Any device can be used with the precision of ± 0.1°C.
• Should be inserted in the rectum of the rabbit to a depth about 5cm
(B.P.) or 7.5 cm (U.S.P.).
• Depth of insertion should be constant for any one rabbit for any one
test.
• If electrical device is used, it should be inserted at least 90 minutes
before the test start and should remain in position through the test.
B. GLASS WARE, SYRINGES AND NEEDLES
• Should be free of Pyrogens.
• Should be washed with water for injection.
• Should be heated in an oven (Hot air) at 250° for 30 to 45 minutes.
C. RETAINING BOXES
• Where electrical device is used, boxes should be such that the animal
is retained only by loosely fitting neck-stock, the rest of the body
remains relatively free.
• The animal must be put into boxes one hour before test and remain
in them through the test.
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GM Hamad
III. PRELIMINARY TEST
• One to three days before testing the product, the animals are selected
as discussed below:
The animals have not been used for the last two weeks.
They are injected 10 ml per kg of body weight of normal saline
free of Pyrogens. Animals should not be disturbed or excited.
Temperature of test laboratory should not vary more than 3°C
than the building quarter of the rabbit. The animal should be
transferred at least 18 hours before the test start.
Withhold food over-night and through-out the test. Withhold
water during the test.
Temperature of each animal should be recorded at least 90
minutes before the test and continued for 03 hours after injection
of the solution.
Any animal showing temperature variation greater than 0.6°C,
should not be included in pyrogens testing.
IV. MAIN TEST
• Carry out the test using a group of three rabbits. Maximum four groups
of rabbits can be used.
• Following steps are taken to perform main test:
i. Preparation and Injection of the sample.
▪ Warm the liquid to be examined to 38.5° (If required,
product should be dissolved in Pyrogens free sodium
chloride isotonic solution).
▪ Inject I.V.(intravenous) into rabbit by the marginal ear vein
slowly. The injection period should not exceed 04 minutes
(B.P.) or 10 minutes (U.S.P.).
▪ The volume for injection may vary according to the
individual monograph but it should not be less than 0.5
ml/kg of body weight and not more than 10 ml/kg of body
weight.
▪ The temperature of each rabbit is noted at 1, 2 and 3 hours
subsequent to the injection of sample. The difference
between the initial and final temperature is noted. Any
increase in the temperature is taken to be the response of
the sample injected.
64
III. PRELIMINARY TEST
• One to three days before testing the product, the animals are selected
as discussed below:
The animals have not been used for the last two weeks.
They are injected 10 ml per kg of body weight of normal saline
free of Pyrogens. Animals should not be disturbed or excited.
Temperature of test laboratory should not vary more than 3°C
than the building quarter of the rabbit. The animal should be
transferred at least 18 hours before the test start.
Withhold food over-night and through-out the test. Withhold
water during the test.
Temperature of each animal should be recorded at least 90
minutes before the test and continued for 03 hours after injection
of the solution.
Any animal showing temperature variation greater than 0.6°C,
should not be included in pyrogens testing.
IV. MAIN TEST
• Carry out the test using a group of three rabbits. Maximum four groups
of rabbits can be used.
• Following steps are taken to perform main test:
i. Preparation and Injection of the sample.
▪ Warm the liquid to be examined to 38.5° (If required,
product should be dissolved in Pyrogens free sodium
chloride isotonic solution).
▪ Inject I.V.(intravenous) into rabbit by the marginal ear vein
slowly. The injection period should not exceed 04 minutes
(B.P.) or 10 minutes (U.S.P.).
▪ The volume for injection may vary according to the
individual monograph but it should not be less than 0.5
ml/kg of body weight and not more than 10 ml/kg of body
weight.
▪ The temperature of each rabbit is noted at 1, 2 and 3 hours
subsequent to the injection of sample. The difference
between the initial and final temperature is noted. Any
increase in the temperature is taken to be the response of
the sample injected.
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GM Hamad
ii. Determination of initial and maximum temperature
▪ The “initial temperature” for each rabbit is the mean of two
temperature readings recorded for that rabbit at an
interval of 30 minutes in the 40 minutes immediately
preceding the injection of material to be examined.
▪ The “maximum temperature” is the highest temperature
recorded for that rabbit in the 03 hours after the injection.
▪ Note the temperature of each rabbit at intervals of not
more than 30 minutes beginning at least 90 minutes before
the injection of the product to be examined and continuing
3 hours after the injection. The difference between the
maximum temperature and the initial temperature of each
rabbit is taken o be its response.
▪ Rabbits showing temperature variation greater than 0.2℃
between two successive readings in the determination of
the initial temperature are excluded from the test. All
rabbits having an initial temperature higher than 39.8℃ or
less than 38℃ are excluded from the test.
V. INTERPRETATION OF RESULTS
• The material under examination meets the requirements for
apyrogenicity if no rapid shows an individual rise in temperature of 0.6℃
or more above is respective control temperature OR The sum of the
temperature rise of 3 rabbits does not exceed 1.4℃.
• If the results are not within the limits the test is repeated for additional 5
rabbits and the result is considered for the eight rabbits. After repeating
the material under examination meets the requirements if not more
than 3 out of eight rabbits show individual rise in temperature of 0.6℃
OR The sum of rise in the temperature in eight rabbits does not exceed
3.7℃.
• When the difference is negative the result is counted as a zero response
and the sample is considered apyrogenic.
3. CLARITY (PARTICULATE) TESTING
• The test is very important particularly when injections are given I.V.
(intravenously) as the contents of such injections are entering into blood
directly. After administration, the contents are also circulating through
lungs. Bronchioles and other parts of respiratory system may be
obstructed by the presence of high number as well as larger particles.
65
ii. Determination of initial and maximum temperature
▪ The “initial temperature” for each rabbit is the mean of two
temperature readings recorded for that rabbit at an
interval of 30 minutes in the 40 minutes immediately
preceding the injection of material to be examined.
▪ The “maximum temperature” is the highest temperature
recorded for that rabbit in the 03 hours after the injection.
▪ Note the temperature of each rabbit at intervals of not
more than 30 minutes beginning at least 90 minutes before
the injection of the product to be examined and continuing
3 hours after the injection. The difference between the
maximum temperature and the initial temperature of each
rabbit is taken o be its response.
▪ Rabbits showing temperature variation greater than 0.2℃
between two successive readings in the determination of
the initial temperature are excluded from the test. All
rabbits having an initial temperature higher than 39.8℃ or
less than 38℃ are excluded from the test.
V. INTERPRETATION OF RESULTS
• The material under examination meets the requirements for
apyrogenicity if no rapid shows an individual rise in temperature of 0.6℃
or more above is respective control temperature OR The sum of the
temperature rise of 3 rabbits does not exceed 1.4℃.
• If the results are not within the limits the test is repeated for additional 5
rabbits and the result is considered for the eight rabbits. After repeating
the material under examination meets the requirements if not more
than 3 out of eight rabbits show individual rise in temperature of 0.6℃
OR The sum of rise in the temperature in eight rabbits does not exceed
3.7℃.
• When the difference is negative the result is counted as a zero response
and the sample is considered apyrogenic.
3. CLARITY (PARTICULATE) TESTING
• The test is very important particularly when injections are given I.V.
(intravenously) as the contents of such injections are entering into blood
directly. After administration, the contents are also circulating through
lungs. Bronchioles and other parts of respiratory system may be
obstructed by the presence of high number as well as larger particles.
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METHODS OF CLARITY TESTING
• Clarity is checked by either of the two ways:
Visual inspection
Electronic particulate counting
I. VISUAL INSPECTION
• Officially, the test is performed visually by looking at all the unit packed
injectable i.e. each and every filled ampoule and vial are checked visually
for the presence of particle against the required back ground, means
colored material is checked against white and white or colorless packed
material against black back ground. Injectable with particle is rejected.
II. ELECTRONIC PARTICULATE COUNTING
• Now a days, particulate matter is also checked by camera eye. Three
cameras are placed in series and specimen to be checked is spun in front
of each camera. The specimen having particle is identified by any of the
camera and rejected automatically. However, care must be taken that
during spinning process, bubble formation must not occur otherwise,
the cameras may identify it as particle.
• This test is pretty costly and is therefore not observed by many of the
manufacturers. Also, particulate testing by camera eye is not officially
demanded. It is just optional to observe high level of quality of
injectable.
4. LEAKERS TEST
• Leakage test is employed to test the package integrity. Package reflects
ability of a container to keep the product in and to keep potential
contamination out. It is because leakage occurs when discontinuity exist
in the wall of a package that can allow the passage of gas under pressure
or concentration differential existing across the wall.
METHODS OF LEAKER TEST
• Leakage is checked by following ways:
I. VISUAL INSPECTION
• It is the easiest leak test method to perform, but the method is the least
sensitive. To increase the sensitivity of the method the visual inspection
of the sample container may be coupled with the application of vacuum
to make leakage more readily observable.
• This method is easy and inexpensive. However, method is insensitive,
operator dependent and qualitative. Sometimes the method is used in
66
METHODS OF CLARITY TESTING
• Clarity is checked by either of the two ways:
Visual inspection
Electronic particulate counting
I. VISUAL INSPECTION
• Officially, the test is performed visually by looking at all the unit packed
injectable i.e. each and every filled ampoule and vial are checked visually
for the presence of particle against the required back ground, means
colored material is checked against white and white or colorless packed
material against black back ground. Injectable with particle is rejected.
II. ELECTRONIC PARTICULATE COUNTING
• Now a days, particulate matter is also checked by camera eye. Three
cameras are placed in series and specimen to be checked is spun in front
of each camera. The specimen having particle is identified by any of the
camera and rejected automatically. However, care must be taken that
during spinning process, bubble formation must not occur otherwise,
the cameras may identify it as particle.
• This test is pretty costly and is therefore not observed by many of the
manufacturers. Also, particulate testing by camera eye is not officially
demanded. It is just optional to observe high level of quality of
injectable.
4. LEAKERS TEST
• Leakage test is employed to test the package integrity. Package reflects
ability of a container to keep the product in and to keep potential
contamination out. It is because leakage occurs when discontinuity exist
in the wall of a package that can allow the passage of gas under pressure
or concentration differential existing across the wall.
METHODS OF LEAKER TEST
• Leakage is checked by following ways:
I. VISUAL INSPECTION
• It is the easiest leak test method to perform, but the method is the least
sensitive. To increase the sensitivity of the method the visual inspection
of the sample container may be coupled with the application of vacuum
to make leakage more readily observable.
• This method is easy and inexpensive. However, method is insensitive,
operator dependent and qualitative. Sometimes the method is used in
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combination with pressure and/ or temperature cycling to accelerate
leakage to improve sensitivity of the test.
II. BUBBLE TEST
• The test package is submerged in liquid. A differential positive pressure
of Psi is applied inside container for 15 minutes. The container is
observed for bubbles. Sometimes, surfactant added liquid is used for
immersion of test package. Any leakage is evident after the application
of differential pressure as the generation of foaming in immersed liquid.
• The method is simple and economical the location of the leaks can be
observed in this method. However, it is relatively insensitive and the
finds are operator dependent and are qualitative. The optimized
conditions can be achieved by using a surfactant immersion fluid along
with the dark background and high intensity lighting.
III. DYE TEST
• The test container is immersed in dye bath. Vacuum and pressure is
applied for some time. The container is removed from dye bath, washed
and is then inspected for the presence of dye either visually or by means
of UV spectroscopy.
• The dye used may be of blue, green or yellowish green color. The dye
test can be optimized by use of a surfactant of a low viscosity fluid in the
dye solution to increase the capillary migration through the pores.
• It is widely accepted in the industry and is approved. The test is
inexpensive and does not require special equipment required for visual
test detection. However, the test is qualitative, destructive and slow.
Test is used for ampules and vials.
IV. VACUUM IONIZATION TEST
• It is a useful test for testing the leakage in the vials and bottles sealed
under vacuum or for outline testing of the lyophilized products. High
voltage and high frequency field are applied to vials which causes
residual gas if present to glow. Glow intensity is the function of
headspace vacuum level. The blue glow is the indicative of vacuum while
the purple glow is the indicative of no vacuum.
• The sensitivity of this method is not documented. This is online rapid
and is non-destructive test. However, the proteins present in the sample
may be decomposed. The method is used for the lyophilized vials of
biopharmaceuticals.
67
combination with pressure and/ or temperature cycling to accelerate
leakage to improve sensitivity of the test.
II. BUBBLE TEST
• The test package is submerged in liquid. A differential positive pressure
of Psi is applied inside container for 15 minutes. The container is
observed for bubbles. Sometimes, surfactant added liquid is used for
immersion of test package. Any leakage is evident after the application
of differential pressure as the generation of foaming in immersed liquid.
• The method is simple and economical the location of the leaks can be
observed in this method. However, it is relatively insensitive and the
finds are operator dependent and are qualitative. The optimized
conditions can be achieved by using a surfactant immersion fluid along
with the dark background and high intensity lighting.
III. DYE TEST
• The test container is immersed in dye bath. Vacuum and pressure is
applied for some time. The container is removed from dye bath, washed
and is then inspected for the presence of dye either visually or by means
of UV spectroscopy.
• The dye used may be of blue, green or yellowish green color. The dye
test can be optimized by use of a surfactant of a low viscosity fluid in the
dye solution to increase the capillary migration through the pores.
• It is widely accepted in the industry and is approved. The test is
inexpensive and does not require special equipment required for visual
test detection. However, the test is qualitative, destructive and slow.
Test is used for ampules and vials.
IV. VACUUM IONIZATION TEST
• It is a useful test for testing the leakage in the vials and bottles sealed
under vacuum or for outline testing of the lyophilized products. High
voltage and high frequency field are applied to vials which causes
residual gas if present to glow. Glow intensity is the function of
headspace vacuum level. The blue glow is the indicative of vacuum while
the purple glow is the indicative of no vacuum.
• The sensitivity of this method is not documented. This is online rapid
and is non-destructive test. However, the proteins present in the sample
may be decomposed. The method is used for the lyophilized vials of
biopharmaceuticals.
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5. ASSAY OF CONTENT
• Assay of active principle is performed according to the official procedure
given in official books (B.P., U.S.P., E.P., etc.) regarding that particular
active principle.
• Official specifications of active principle (both for API and dosage forms)
is to be that of particular official book from where the procedure is
adopted i.e. if procedure is from B.P. then official specification must also
be of B.P.
6. VOLUME/AMOUNT
• With respect to the volume and amount, tables given in the official
books must be followed.
• Based on volume principle, parenteral are classified as:
Small volume parenteral (SVP) (in each container, volume is up to
100 ml).
Large volume parenteral (LVP) (in each container, volume is more
than 100 ml).
7. LABELLING
• This is also followed as per the specifications given under different types
of parenteral. The specifications and requirements are given in
monographs/tables of different official books.
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5. ASSAY OF CONTENT
• Assay of active principle is performed according to the official procedure
given in official books (B.P., U.S.P., E.P., etc.) regarding that particular
active principle.
• Official specifications of active principle (both for API and dosage forms)
is to be that of particular official book from where the procedure is
adopted i.e. if procedure is from B.P. then official specification must also
be of B.P.
6. VOLUME/AMOUNT
• With respect to the volume and amount, tables given in the official
books must be followed.
• Based on volume principle, parenteral are classified as:
Small volume parenteral (SVP) (in each container, volume is up to
100 ml).
Large volume parenteral (LVP) (in each container, volume is more
than 100 ml).
7. LABELLING
• This is also followed as per the specifications given under different types
of parenteral. The specifications and requirements are given in
monographs/tables of different official books.
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BIOLOGICAL ASSAYS
INTRODUCTION
ASSAY
• An assay is an investigative (analytic) procedure in laboratory
medicine, pharmacology, environmental biology and molecular
biology for qualitatively assessing or quantitatively measuring the
presence or amount or the functional activity of a target entity (the
analyte) which can be a drug or biochemical substance or
organic sample.
BIOLOGICAL ASSAY
• Potency or concentration of an active principle in unit quantity of
preparation by measuring its biological response on living tissues.
CLASSIFICATION OF BIOASSAY
• There are three types of bioassay:
Quantal assay
Graded assay
Effect produced in confined period
QUANTAL
• All or none response in all individuals. E.g. Digitalis induced cardiac
arrest in guinea pigs, hypoglycemic convulsions in mice by insulin.
GRADED BIOASSAY
• Graded response - response is proportional to the dose and response
may lie between no response and the maximum response.
PREPARATION OF STANDARD
• A selective representative sample of a substance for which it is to serve
as a basis of the measurement is called standard preparation.
Uniform quality
Stable
TYPES OF STANDARD PREPARATION
• Two types of standard preparation:
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BIOLOGICAL ASSAYS
INTRODUCTION
ASSAY
• An assay is an investigative (analytic) procedure in laboratory
medicine, pharmacology, environmental biology and molecular
biology for qualitatively assessing or quantitatively measuring the
presence or amount or the functional activity of a target entity (the
analyte) which can be a drug or biochemical substance or
organic sample.
BIOLOGICAL ASSAY
• Potency or concentration of an active principle in unit quantity of
preparation by measuring its biological response on living tissues.
CLASSIFICATION OF BIOASSAY
• There are three types of bioassay:
Quantal assay
Graded assay
Effect produced in confined period
QUANTAL
• All or none response in all individuals. E.g. Digitalis induced cardiac
arrest in guinea pigs, hypoglycemic convulsions in mice by insulin.
GRADED BIOASSAY
• Graded response - response is proportional to the dose and response
may lie between no response and the maximum response.
PREPARATION OF STANDARD
• A selective representative sample of a substance for which it is to serve
as a basis of the measurement is called standard preparation.
Uniform quality
Stable
TYPES OF STANDARD PREPARATION
• Two types of standard preparation:
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1. International standard and reference standard USP units (highly
recognized-able and authorized standard)
2. British standard and reference standard
• Country want to have its own standard preparation, then used according
to its own law.
• For specific biological activity small quantity of standard preparation are
used.
POTENCY
• Measurement of drug activity expressed in terms of amount required to
produce an effect of given intensity.
EXAMPLES
• Highly potent drugs like morphine, alprazolam, chlorpromazine etc.
produce high response at low concentration.
• Low potent drugs like ibuprofen and acetylsalicylic acid produce low
response at low concentration.
BIOASSAY OF ANTIBIOTICS
• The potency of a sample of an antibiotic is determined by comparing the
dose which inhibits the growth of a suitable susceptible micro-organism
with the dose of standard preparation of that antibiotic that produces
the same degree of inhibition.
STANDARD PREPARATIONS AND UNITS
• The standard preparations are supplied as dry powders in sealed
ampoules. The potency of an antibiotic is usually described as the units
contained in 1mg of the powder of the standard preparation or as the
milligrams of the standard antibiotic powder that contains 1 unit.
• The standard preparations and units for some antibiotics are given in the
following table:
Standard
Preparation
Units contained
in 1 mg
Mg containing 1
unit
Quantity
Supplied
Amphotericin b 940 0.001064 100mg
Streptomycin 780 0.001282 175mg
Tetracycline 982 0.0010183 75mg
Erythromycin 950 0.001053 200mg
Vancomycin 1007 0.000993 50mg
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1. International standard and reference standard USP units (highly
recognized-able and authorized standard)
2. British standard and reference standard
• Country want to have its own standard preparation, then used according
to its own law.
• For specific biological activity small quantity of standard preparation are
used.
POTENCY
• Measurement of drug activity expressed in terms of amount required to
produce an effect of given intensity.
EXAMPLES
• Highly potent drugs like morphine, alprazolam, chlorpromazine etc.
produce high response at low concentration.
• Low potent drugs like ibuprofen and acetylsalicylic acid produce low
response at low concentration.
BIOASSAY OF ANTIBIOTICS
• The potency of a sample of an antibiotic is determined by comparing the
dose which inhibits the growth of a suitable susceptible micro-organism
with the dose of standard preparation of that antibiotic that produces
the same degree of inhibition.
STANDARD PREPARATIONS AND UNITS
• The standard preparations are supplied as dry powders in sealed
ampoules. The potency of an antibiotic is usually described as the units
contained in 1mg of the powder of the standard preparation or as the
milligrams of the standard antibiotic powder that contains 1 unit.
• The standard preparations and units for some antibiotics are given in the
following table:
Standard
Preparation
Units contained
in 1 mg
Mg containing 1
unit
Quantity
Supplied
Amphotericin b 940 0.001064 100mg
Streptomycin 780 0.001282 175mg
Tetracycline 982 0.0010183 75mg
Erythromycin 950 0.001053 200mg
Vancomycin 1007 0.000993 50mg
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METHOD
• Petri dishes or rectangular trays are filled to a depth of 3 – 4mm with a
nutrient agar medium which has previously been inoculated with 1
percent v/v of a suitable inoculums of a susceptible test organism (list of
organisms used for particular antibiotics are given in the table under).
The inoculated plates are allowed to dry for 30 minutes at room
temperature or solidified using a refrigerator. Small holes with diameter
between 5 – 8mm are bored in the solidified agar.
• Solutions of standard preparations of known concentrations
(concentrations used are given in the following table) are made and
solutions of the antibiotic being tested are made of the same
concentrations. The solutions are made with a sterile solvent and
dilutions made unless otherwise stated in the monographs. The
solutions are placed in the holes bored in the agar using a pipette which
delivers a uniform and equal amount of test solution and standard
solution in their respective holes.
• The plates are then maintained at room temperature for 4 hours to
allow the antibiotic solutions to diffuse into the agar, the plates are then
incubated at a suitable temperature for approximately 16 hours and the
diameters of the zones of inhibition produced by various concentrations
of the test and standard solutions are measured carefully.
• From the results the potency of the test solution is estimated using
standard statistical methods.
Antibiotics Micro-organisms
Potency of
Solutions (u/ml)
Incubation
Temperature ℃
Amphotericin b Saccharomyces
cerevisiae
1 – 4 35 – 37
Streptomycin Bacillus subtilis 5 – 20 37 – 39
Tetracycline Bacillus pumilus 2 – 20 37 – 39
Erythromycin Bacillus pumilus 5 – 25 37 – 39
Vancomycin Bacillus subtilis 20 – 200 37 – 39
BIOASSAY OF INSULIN INJECTION
• Insulin is a hormone that is synthesized and secreted by the β-cells of
the pancreatic islets and is stored in intracellular granules. Human
insulin has the molecular weight of 5807 and is made up of two
polypeptide chains linked together by disulfide linkages.
• Insulin preparations are used to control blood – glucose levels in people
71
METHOD
• Petri dishes or rectangular trays are filled to a depth of 3 – 4mm with a
nutrient agar medium which has previously been inoculated with 1
percent v/v of a suitable inoculums of a susceptible test organism (list of
organisms used for particular antibiotics are given in the table under).
The inoculated plates are allowed to dry for 30 minutes at room
temperature or solidified using a refrigerator. Small holes with diameter
between 5 – 8mm are bored in the solidified agar.
• Solutions of standard preparations of known concentrations
(concentrations used are given in the following table) are made and
solutions of the antibiotic being tested are made of the same
concentrations. The solutions are made with a sterile solvent and
dilutions made unless otherwise stated in the monographs. The
solutions are placed in the holes bored in the agar using a pipette which
delivers a uniform and equal amount of test solution and standard
solution in their respective holes.
• The plates are then maintained at room temperature for 4 hours to
allow the antibiotic solutions to diffuse into the agar, the plates are then
incubated at a suitable temperature for approximately 16 hours and the
diameters of the zones of inhibition produced by various concentrations
of the test and standard solutions are measured carefully.
• From the results the potency of the test solution is estimated using
standard statistical methods.
Antibiotics Micro-organisms
Potency of
Solutions (u/ml)
Incubation
Temperature ℃
Amphotericin b Saccharomyces
cerevisiae
1 – 4 35 – 37
Streptomycin Bacillus subtilis 5 – 20 37 – 39
Tetracycline Bacillus pumilus 2 – 20 37 – 39
Erythromycin Bacillus pumilus 5 – 25 37 – 39
Vancomycin Bacillus subtilis 20 – 200 37 – 39
BIOASSAY OF INSULIN INJECTION
• Insulin is a hormone that is synthesized and secreted by the β-cells of
the pancreatic islets and is stored in intracellular granules. Human
insulin has the molecular weight of 5807 and is made up of two
polypeptide chains linked together by disulfide linkages.
• Insulin preparations are used to control blood – glucose levels in people
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with diabetes mellitus, which results from an inadequate secretion of
insulin from the pancreas. The insulin preparations used are mostly
human insulin prepared by recombinant DNA or enzymatically modified
insulin isolated from porcine pancreas.
• The potency of a sample insulin injection is estimated by comparing the
hypoglycemic effect it produces with that produced by a standard
preparation of insulin under conditions of a suitable method of assay.
STANDARD PREPARATION AND UNIT
• The standard preparation consists of recrystallized purified insulin. It is
supplied in ampoules containing approximately 110 to 125 mg. One unit
is contained in 0.04167 mg of standard preparation.
PREPARATION OF THE SOLUTION OF THE STANDARD PREPARATION
• A quantity of the standard preparation is dissolved in normal saline such
that 1ml of the solution contains 20 Units.
METHOD
• Not less than 96 mice which have been well fed are selected and are
divided into 4 groups equally at random. The test animals are deprived
of food for 2 hours prior to the test. Two groups receive SC injections of
2 dilutions of the standard preparation and the other 2 groups receive
one dilution each of the sample preparation having the same dilutions.
Suitable doses for mice weighing about 20g are 0.015 Units and 0.03
Units.
• The mice after injection are kept at a uniform temperature between 29 –
35℃ in an incubator with a transparent front. The mice are observed for
1½ hour after injection. The number of mice which are dead, convulsed
or were lying on their back for more than 2 – 3 seconds are noted for
each group. The results for the sample preparation are compared with
those of the standard preparation and the assay results are calculated
using standard statistical methods.
BIOASSAY OF PREPARED DIGITALIS
• The pharmacodynamic properties of the crude drug extract or galenical
preparation of digitalis cannot be measured adequately by chemical
assays because of the presence of complex mixture of substances
containing varying structures and activity. Hence, biological assay
techniques are applied.
72
with diabetes mellitus, which results from an inadequate secretion of
insulin from the pancreas. The insulin preparations used are mostly
human insulin prepared by recombinant DNA or enzymatically modified
insulin isolated from porcine pancreas.
• The potency of a sample insulin injection is estimated by comparing the
hypoglycemic effect it produces with that produced by a standard
preparation of insulin under conditions of a suitable method of assay.
STANDARD PREPARATION AND UNIT
• The standard preparation consists of recrystallized purified insulin. It is
supplied in ampoules containing approximately 110 to 125 mg. One unit
is contained in 0.04167 mg of standard preparation.
PREPARATION OF THE SOLUTION OF THE STANDARD PREPARATION
• A quantity of the standard preparation is dissolved in normal saline such
that 1ml of the solution contains 20 Units.
METHOD
• Not less than 96 mice which have been well fed are selected and are
divided into 4 groups equally at random. The test animals are deprived
of food for 2 hours prior to the test. Two groups receive SC injections of
2 dilutions of the standard preparation and the other 2 groups receive
one dilution each of the sample preparation having the same dilutions.
Suitable doses for mice weighing about 20g are 0.015 Units and 0.03
Units.
• The mice after injection are kept at a uniform temperature between 29 –
35℃ in an incubator with a transparent front. The mice are observed for
1½ hour after injection. The number of mice which are dead, convulsed
or were lying on their back for more than 2 – 3 seconds are noted for
each group. The results for the sample preparation are compared with
those of the standard preparation and the assay results are calculated
using standard statistical methods.
BIOASSAY OF PREPARED DIGITALIS
• The pharmacodynamic properties of the crude drug extract or galenical
preparation of digitalis cannot be measured adequately by chemical
assays because of the presence of complex mixture of substances
containing varying structures and activity. Hence, biological assay
techniques are applied.
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• The activity of a sample prepared is determined by comparing its activity
on the cardiac muscles of guinea – pig or pigeons with that of a standard
preparation of prepared digitalis.
STANDARD PREPARATION AND UNIT
• The standard preparation of digitalis consists of dry powdered leaves of
Digitalis purpurea. It is supplied in ampoules containing approximately
2.5g of the standard digitalis. The Unit is contained in 76mg of that
standard preparation and hence, 1 mg contains 0.01316 Units.
PREPARATION OF THE EXTRACT FOR STANDARD PREPARATION AND SAMPLE
PREPARATION
• The extracts of the standard and test samples are prepared in the same
way. First, the amount of the dry powder is added to a previously
weighed stoppered glass bottle. The amount of the powdered digitalis
added is noted. 10 ml of 80% alcohol is added to the stoppered bottle
for each gram of the powder.
• The container is continuously shaken for 24 hours at 20 – 30℃ or for 48
hours at 10 – 20℃. The mixture is then centrifuged or filtered through
sintered glass filter taking care to avoid evaporation of the solvent. The
extract is stored at a temperature of -5 to 5℃ and should be used within
a month after preparation.
METHODS
I. BY INJECTION INTO GUINEA-PIGS
• Not fewer than 12 guinea pigs each weighing 200 – 600 grams are
distributed at random into two equal groups. The weight of the heaviest
and the lightest animals should not differ more than 100 grams and the
mean body weight of the two groups should not vary more than 10%.
One group is used for the standard preparation and the other is used for
the test preparation. Both the test and standard preparations are diluted
with normal saline until the concentration of previously anesthetized
vein of the test animal.
• The duration of the injection may be 20 – 40 minutes. The injection is
continued until the heart of the animal is arrested, which can be
measured by recording the electrical activity of the heart. The volume of
the extract administered is taken as the lethal dose. The mean log lethal
dose in mL/Kg of body weight is determined for each group.
• The result of the assay is calculated by standard statistical methods.
73
• The activity of a sample prepared is determined by comparing its activity
on the cardiac muscles of guinea – pig or pigeons with that of a standard
preparation of prepared digitalis.
STANDARD PREPARATION AND UNIT
• The standard preparation of digitalis consists of dry powdered leaves of
Digitalis purpurea. It is supplied in ampoules containing approximately
2.5g of the standard digitalis. The Unit is contained in 76mg of that
standard preparation and hence, 1 mg contains 0.01316 Units.
PREPARATION OF THE EXTRACT FOR STANDARD PREPARATION AND SAMPLE
PREPARATION
• The extracts of the standard and test samples are prepared in the same
way. First, the amount of the dry powder is added to a previously
weighed stoppered glass bottle. The amount of the powdered digitalis
added is noted. 10 ml of 80% alcohol is added to the stoppered bottle
for each gram of the powder.
• The container is continuously shaken for 24 hours at 20 – 30℃ or for 48
hours at 10 – 20℃. The mixture is then centrifuged or filtered through
sintered glass filter taking care to avoid evaporation of the solvent. The
extract is stored at a temperature of -5 to 5℃ and should be used within
a month after preparation.
METHODS
I. BY INJECTION INTO GUINEA-PIGS
• Not fewer than 12 guinea pigs each weighing 200 – 600 grams are
distributed at random into two equal groups. The weight of the heaviest
and the lightest animals should not differ more than 100 grams and the
mean body weight of the two groups should not vary more than 10%.
One group is used for the standard preparation and the other is used for
the test preparation. Both the test and standard preparations are diluted
with normal saline until the concentration of previously anesthetized
vein of the test animal.
• The duration of the injection may be 20 – 40 minutes. The injection is
continued until the heart of the animal is arrested, which can be
measured by recording the electrical activity of the heart. The volume of
the extract administered is taken as the lethal dose. The mean log lethal
dose in mL/Kg of body weight is determined for each group.
• The result of the assay is calculated by standard statistical methods.
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II. BY INJECTION INTO PIGEONS
• Not fewer than 12 healthy pigeons are taken and are divided at random
into 2 groups. One group is for the standard preparation and the other
for the sample preparation. The weight of the heaviest pigeon should
not me more than twice as much as the lightest pigeon. Also, the mean
weight of both the groups of the pigeons should not vary more than
30%.
• Food, but not water is withheld from the test animals for 16 – 28 hours
before the test. The extracts of both the sample and standard
preparation are diluted with normal saline such that the estimated lethal
dose per kilogram body weight of the pigeon must not exceed more
than 15ml. The diluted extract is administered through a cannula into a
previously anesthetized vein of an immobilized pigeon. The dose of
1ml/kg of the body weight is administered within a few seconds and
repeated after every 5 minutes till the heart of the test animal is
arrested.
• If the average number of doses received before the heart arrests is less
than 13 or more than 19, or the average number of doses of the 2
groups vary more than four, the test is repeated using freshly prepared
and accurate dilutions.
• The lethal dose per kilogram body weight of the pigeon is equal to the
dose administered. The result of the assay is calculated using standard
statistical methods.
BIOASSAY OF VITAMIN D
• The activity of a preparation of vitamin D is determined by comparing its
antirachitic (ability to prevent rickets) activity with that of the
antirachitic activity of a standard preparation.
• A fundamental requirement in a microbial assay for the activity of a
vitamin is the inability of the test organism to produce that vitamin.
Furthermore, the test organism must require the factor being assayed
for normal growth and should be sensitive to very small amounts of that
factor.
STANDARD PREPARATION AND UNIT
• It consists of activated crystalline 7 – dehydrocholesterol. It is supplied in
bottles as a solution in vegetable oil. One unit is contained in 0.000025
mg of the pure substance.
74
II. BY INJECTION INTO PIGEONS
• Not fewer than 12 healthy pigeons are taken and are divided at random
into 2 groups. One group is for the standard preparation and the other
for the sample preparation. The weight of the heaviest pigeon should
not me more than twice as much as the lightest pigeon. Also, the mean
weight of both the groups of the pigeons should not vary more than
30%.
• Food, but not water is withheld from the test animals for 16 – 28 hours
before the test. The extracts of both the sample and standard
preparation are diluted with normal saline such that the estimated lethal
dose per kilogram body weight of the pigeon must not exceed more
than 15ml. The diluted extract is administered through a cannula into a
previously anesthetized vein of an immobilized pigeon. The dose of
1ml/kg of the body weight is administered within a few seconds and
repeated after every 5 minutes till the heart of the test animal is
arrested.
• If the average number of doses received before the heart arrests is less
than 13 or more than 19, or the average number of doses of the 2
groups vary more than four, the test is repeated using freshly prepared
and accurate dilutions.
• The lethal dose per kilogram body weight of the pigeon is equal to the
dose administered. The result of the assay is calculated using standard
statistical methods.
BIOASSAY OF VITAMIN D
• The activity of a preparation of vitamin D is determined by comparing its
antirachitic (ability to prevent rickets) activity with that of the
antirachitic activity of a standard preparation.
• A fundamental requirement in a microbial assay for the activity of a
vitamin is the inability of the test organism to produce that vitamin.
Furthermore, the test organism must require the factor being assayed
for normal growth and should be sensitive to very small amounts of that
factor.
STANDARD PREPARATION AND UNIT
• It consists of activated crystalline 7 – dehydrocholesterol. It is supplied in
bottles as a solution in vegetable oil. One unit is contained in 0.000025
mg of the pure substance.
74
GM Hamad
METHOD
• Not fewer than 40 young rats of either sex, recently weaned are chosen
and divided into 4 groups of 10. The weight of the heaviest rat must not
vary more than 10 grams of the weight of the lightest rat. They are fed
for 3 weeks on a rachitogenic diet, which consists mostly of
carbohydrates, proteins and electrolytes and no fats. The development
of the necessary degree of rickets is determined in each rat under light
anesthesia by the X–ray of the proximal ends of the tibia or the distal
ends of the radius and ulna.
• Then the rats in the 2 groups receive dose x and nx of the standard
preparation respectively and the other two groups receive doses in the
same ratio of the sample preparation. Suitable doses may vary from 2 –
8 units, i.e. where x = 2 and n = 1, 2, 3, 4. Each rat may receive the whole
of its dose at once or the dose maybe divided into 8 daily doses. 10 – 14
days after that, the rats are killed and the extent to which rickets has
been cured is estimated by means of X–ray photographs.
• The result of the assay of the sample preparation is calculated by
comparing it with the results of the standard preparation by means of
standard statistical tests.
75
METHOD
• Not fewer than 40 young rats of either sex, recently weaned are chosen
and divided into 4 groups of 10. The weight of the heaviest rat must not
vary more than 10 grams of the weight of the lightest rat. They are fed
for 3 weeks on a rachitogenic diet, which consists mostly of
carbohydrates, proteins and electrolytes and no fats. The development
of the necessary degree of rickets is determined in each rat under light
anesthesia by the X–ray of the proximal ends of the tibia or the distal
ends of the radius and ulna.
• Then the rats in the 2 groups receive dose x and nx of the standard
preparation respectively and the other two groups receive doses in the
same ratio of the sample preparation. Suitable doses may vary from 2 –
8 units, i.e. where x = 2 and n = 1, 2, 3, 4. Each rat may receive the whole
of its dose at once or the dose maybe divided into 8 daily doses. 10 – 14
days after that, the rats are killed and the extent to which rickets has
been cured is estimated by means of X–ray photographs.
• The result of the assay of the sample preparation is calculated by
comparing it with the results of the standard preparation by means of
standard statistical tests.
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GM Hamad
ALCOHOL DETERMINATION
DEFINITION
• Alcohol content determination is performed to check the purity and
quantity of product in various industries.
SPECIAL TREATMENTS
1. VOLATILE ACIDS AND BASES
• Render the preparations containing volatile bases slightly acidic with
dilute H2SO4 before distillation.
• If volatile acids are present, render the preparation slightly alkaline
with NaOH test solution.
• Acidifying the bases or alkalinizing the acids causes them to be
converted to ionized form and form salts. These salts then do not
interfere with ethanol evaporation in distillation process.
2. GLYCERINE
• To preparation containing glycerin, add sufficient water so that after
the distillation, the residue contains not less than 50% of water.
3. IODINE
• Solution containing iodine must be treated with:
Powdered zinc (zinc iodide will be formed)
Decolorize with sufficient sodium thiosulfate solution followed
by few drops of NaOH.
4. OTHER VOLATILE SUBSTANCES
• Spirits, Tinctures, Elixirs and similar preparations containing
appreciable proportion of volatile substances like chloroform, ether,
camphor, etc. require special treatment as below:
A. FOR LIQUIDS PRESUMED TO BE CONTAINING 50% ALCOHOL OR LESS
• Mix 25ml sample with equal volume of water in a separator.
• Saturate with NaCl and add 25ml hexane and shake well to remove
any interfering volatile substances.
• Draw off the separated lower layer into the second separator and
repeat the extraction twice with hexane (2, 50ml portions)
76
ALCOHOL DETERMINATION
DEFINITION
• Alcohol content determination is performed to check the purity and
quantity of product in various industries.
SPECIAL TREATMENTS
1. VOLATILE ACIDS AND BASES
• Render the preparations containing volatile bases slightly acidic with
dilute H2SO4 before distillation.
• If volatile acids are present, render the preparation slightly alkaline
with NaOH test solution.
• Acidifying the bases or alkalinizing the acids causes them to be
converted to ionized form and form salts. These salts then do not
interfere with ethanol evaporation in distillation process.
2. GLYCERINE
• To preparation containing glycerin, add sufficient water so that after
the distillation, the residue contains not less than 50% of water.
3. IODINE
• Solution containing iodine must be treated with:
Powdered zinc (zinc iodide will be formed)
Decolorize with sufficient sodium thiosulfate solution followed
by few drops of NaOH.
4. OTHER VOLATILE SUBSTANCES
• Spirits, Tinctures, Elixirs and similar preparations containing
appreciable proportion of volatile substances like chloroform, ether,
camphor, etc. require special treatment as below:
A. FOR LIQUIDS PRESUMED TO BE CONTAINING 50% ALCOHOL OR LESS
• Mix 25ml sample with equal volume of water in a separator.
• Saturate with NaCl and add 25ml hexane and shake well to remove
any interfering volatile substances.
• Draw off the separated lower layer into the second separator and
repeat the extraction twice with hexane (2, 50ml portions)
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GM Hamad
• Extract this solution (combined solvent hexane) thrice with 10ml
portions of saturated saline.
• Combine these portions and distill as usual.
• Take volume having simple ratio to original specimen.
B. FOR LIQUIDS PRESUMED TO BE CONTAINING MORE THAN 50%
ALCOHOL
• Adjust the concentration of alcohol to approx. 25% v/v by diluting
with water.
• Perform the same procedure as above beginning with “saturate this
mixture with NaCl.”
METHODS OF ALCOHOL CONTENT DETERMINATION
1. Distillation method
2. Gas chromatography method
3. High performance liquid chromatography
1. DISTILLATION METHOD
INTRODUCTION
• It must be used for the determination of alcohol, unless otherwise
specified in individual monograph.
• This method is useful for examining most of the fluid elixirs and
tinctures, provided:
The capacity of distilling flask is sufficient (commonly 2-4x the
volume of liquid to be heated)
Rate of distillation is such that clear distillates are produced.
PROBLEM DURING DISTILLATION OF ALCOHOL
• Distillate should be clear, if cloudy add the talc or CaCO3 and filter,
adjust temperature and determine alcohol content from the specific
gravity.
• Tread the liquids that froth to a troublesome extent during distillation
by:
Rendering them strongly acidic with H2SO4, H2PO4 and tannic acid.
Treating with a slight excess of calcium chloride solution or with
small amount of paraffin or silicon oil before starting distillation.
• Loss of alcohol by evaporation should be minimized.
77
• Extract this solution (combined solvent hexane) thrice with 10ml
portions of saturated saline.
• Combine these portions and distill as usual.
• Take volume having simple ratio to original specimen.
B. FOR LIQUIDS PRESUMED TO BE CONTAINING MORE THAN 50%
ALCOHOL
• Adjust the concentration of alcohol to approx. 25% v/v by diluting
with water.
• Perform the same procedure as above beginning with “saturate this
mixture with NaCl.”
METHODS OF ALCOHOL CONTENT DETERMINATION
1. Distillation method
2. Gas chromatography method
3. High performance liquid chromatography
1. DISTILLATION METHOD
INTRODUCTION
• It must be used for the determination of alcohol, unless otherwise
specified in individual monograph.
• This method is useful for examining most of the fluid elixirs and
tinctures, provided:
The capacity of distilling flask is sufficient (commonly 2-4x the
volume of liquid to be heated)
Rate of distillation is such that clear distillates are produced.
PROBLEM DURING DISTILLATION OF ALCOHOL
• Distillate should be clear, if cloudy add the talc or CaCO3 and filter,
adjust temperature and determine alcohol content from the specific
gravity.
• Tread the liquids that froth to a troublesome extent during distillation
by:
Rendering them strongly acidic with H2SO4, H2PO4 and tannic acid.
Treating with a slight excess of calcium chloride solution or with
small amount of paraffin or silicon oil before starting distillation.
• Loss of alcohol by evaporation should be minimized.
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GM Hamad
• Prevent bumping during distillation by adding porous chips of insoluble
material such as small pieces of broken glass, glass beads, porous chips
or silicon carbide.
DISTILLATION METHOD
I. FOR LIQUID PRESUMED TO CONTAIN LESS THAN 30% V/V ALCOHOL
• Take 25ml sample in a suitable distilling apparatus and note the
temperature at which the volume was measured.
• Add equal volume of water and distill.
• Collect distillate 2ml less than original volume of the test liquid (23ml),
adjust the temperature at which the original test liquid measured, add
water to make 25ml and mix.
• The distillate must be clear, not cloudy and should not contain more
than traces of volatile substance other than alcohol and water.
• Find specific gravity at 25℃ (as specified in pharmacopoeia) or refractive
index.
• From the alcoholometric table find the %age of alcohol.
II. FOR LIQUID PRESUMED TO CONTAIN MORE THAN 30% V/V ALCOHOL
• Take 25ml sample in a suitable distilling apparatus and note the
temperature at which the volume was measured.
• Add 50ml of water and distill.
• Collect distillate 2ml less than original volume of the test liquid (48ml),
adjust the temperature at which the original test liquid was measured,
add water to make 50ml and a mix.
• The distillate must be clear, not cloudy and should not contain more
than traces of volatile substances other than alcohol and water.
• Find specific gravity at 25℃ (as stated in pharmacopoeia) or refractive
index.
• From alcoholometric table find %age of alcohol.
2. GAS LIQUID CHROMATOGRAPHY METHOD
CHROMATOGRAPHIC CONDITIONS
• Carrier gas: Helium He.
• Column packing: Silica particles coated with polyethylene glycol.
• Detector: Flame Ionization Detector FID.
• Column temperature: Temperature of the column is maintained at
120℃.
• Temperature of the injection port: 210℃.
78
• Prevent bumping during distillation by adding porous chips of insoluble
material such as small pieces of broken glass, glass beads, porous chips
or silicon carbide.
DISTILLATION METHOD
I. FOR LIQUID PRESUMED TO CONTAIN LESS THAN 30% V/V ALCOHOL
• Take 25ml sample in a suitable distilling apparatus and note the
temperature at which the volume was measured.
• Add equal volume of water and distill.
• Collect distillate 2ml less than original volume of the test liquid (23ml),
adjust the temperature at which the original test liquid measured, add
water to make 25ml and mix.
• The distillate must be clear, not cloudy and should not contain more
than traces of volatile substance other than alcohol and water.
• Find specific gravity at 25℃ (as specified in pharmacopoeia) or refractive
index.
• From the alcoholometric table find the %age of alcohol.
II. FOR LIQUID PRESUMED TO CONTAIN MORE THAN 30% V/V ALCOHOL
• Take 25ml sample in a suitable distilling apparatus and note the
temperature at which the volume was measured.
• Add 50ml of water and distill.
• Collect distillate 2ml less than original volume of the test liquid (48ml),
adjust the temperature at which the original test liquid was measured,
add water to make 50ml and a mix.
• The distillate must be clear, not cloudy and should not contain more
than traces of volatile substances other than alcohol and water.
• Find specific gravity at 25℃ (as stated in pharmacopoeia) or refractive
index.
• From alcoholometric table find %age of alcohol.
2. GAS LIQUID CHROMATOGRAPHY METHOD
CHROMATOGRAPHIC CONDITIONS
• Carrier gas: Helium He.
• Column packing: Silica particles coated with polyethylene glycol.
• Detector: Flame Ionization Detector FID.
• Column temperature: Temperature of the column is maintained at
120℃.
• Temperature of the injection port: 210℃.
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GM Hamad
STANDARD SOLUTION
A. SAMPLES PRESUMED TO CONTAIN 10% ALCOHOL
• Two standard solutions of alcohol are prepared:
Standard solution I containing 5% more alcohol than presumed
alcohol.
Standard solution II containing 5% less alcohol than presumed
alcohol.
B. SAMPLES PRESUMED TO CONTAIN LESS THAN 10% OF ALCOHOL
• Two standard solutions of alcohol are prepared:
Standard solution I containing 1% more alcohol than presumed
alcohol.
Standard solution II containing 1% less alcohol than presumed
alcohol.
PROCEDURE
• Transfer 25ml of sample ,SS-I and II separately to glass containers.
• Add known concentration of internal standard (acetone or acetonitrile
otherwise specified) in each.
• Shake and inject 5µ each of the test preparation and standard
preparation, in duplicate, into gas chromatography.
• Compare the peak response ratio.
Ratio=
Alcohol peaks (for sample,SI and SII)
Internal standard peak area
• Determine the percentage of alcohol by following equation:
%age of alcohol=
SI (Y−Z) + SII (Z−X)
(Y−X)
• Where,
SI = % alcohol in standard solution I (standard with lower alcohol
level)
SII = % alcohol in standard solution II (standard with higher alcohol
level )
X = Value for standard I
Y = Value for standard II
Z = Value of sample
79
STANDARD SOLUTION
A. SAMPLES PRESUMED TO CONTAIN 10% ALCOHOL
• Two standard solutions of alcohol are prepared:
Standard solution I containing 5% more alcohol than presumed
alcohol.
Standard solution II containing 5% less alcohol than presumed
alcohol.
B. SAMPLES PRESUMED TO CONTAIN LESS THAN 10% OF ALCOHOL
• Two standard solutions of alcohol are prepared:
Standard solution I containing 1% more alcohol than presumed
alcohol.
Standard solution II containing 1% less alcohol than presumed
alcohol.
PROCEDURE
• Transfer 25ml of sample ,SS-I and II separately to glass containers.
• Add known concentration of internal standard (acetone or acetonitrile
otherwise specified) in each.
• Shake and inject 5µ each of the test preparation and standard
preparation, in duplicate, into gas chromatography.
• Compare the peak response ratio.
Ratio=
Alcohol peaks (for sample,SI and SII)
Internal standard peak area
• Determine the percentage of alcohol by following equation:
%age of alcohol=
SI (Y−Z) + SII (Z−X)
(Y−X)
• Where,
SI = % alcohol in standard solution I (standard with lower alcohol
level)
SII = % alcohol in standard solution II (standard with higher alcohol
level )
X = Value for standard I
Y = Value for standard II
Z = Value of sample
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GM Hamad
3. HPLC METHOD
PREPARATION OF SOLUTIONS
• Standard solution: Dilute 5ml dehydrated alcohol to 250ml (2% v/v)
• Test solution: Dilute specimen stepwise to conc. of alcohol (2% v/v)
• Internal standard: Dilute 5ml acetonitrile with water to 250ml (2% v/v)
• Test preparation: Pipette 10ml test solution and internal standard in
100ml flask, make volume with water.
• Standard preparation: Pipette 10ml standard solution and internal
standard in 100ml flask, make volume with water.
PROCEDURE
• Inject 5µl each of test preparation and standard preparation in duplicate
to column.
• Record the chromatogram.
• Determine peak response ratios.
• Calculate %age of alcohol on specimen by the formula:
%age of alcohol = 2(
R
u
R
s
)D
• Where,
D=Dilution factor
Ru/Rs=Peaks ratio
80
3. HPLC METHOD
PREPARATION OF SOLUTIONS
• Standard solution: Dilute 5ml dehydrated alcohol to 250ml (2% v/v)
• Test solution: Dilute specimen stepwise to conc. of alcohol (2% v/v)
• Internal standard: Dilute 5ml acetonitrile with water to 250ml (2% v/v)
• Test preparation: Pipette 10ml test solution and internal standard in
100ml flask, make volume with water.
• Standard preparation: Pipette 10ml standard solution and internal
standard in 100ml flask, make volume with water.
PROCEDURE
• Inject 5µl each of test preparation and standard preparation in duplicate
to column.
• Record the chromatogram.
• Determine peak response ratios.
• Calculate %age of alcohol on specimen by the formula:
%age of alcohol = 2(
R
u
R
s
)D
• Where,
D=Dilution factor
Ru/Rs=Peaks ratio
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GM Hamad
ALKALOIDAL DRUG ASSAY
INTRODUCTION
• “Basic Nitrogenous compounds of natural origin having some definite
pharmacological/physiological activity.”
• They are alkali like compounds therefore they are called Alkaloids.
PROPERTIES OF ALKALOIDS
PHYSICAL PROPERTIES
SOLUBILITY
• Insoluble or sparingly soluble in water.
• Soluble in organic solvents.
• Alkaloidal salts are soluble in water and insoluble in organic solvents.
CONDITION
• Most are crystalline solids.
• A few are amorphous solids.
• Some are liquids that are either:
Volatile: E.g. Nicotine and Coniine.
Non-volatile: E.g. Pilocarpine and hyoscine
• Alkaloidal salts are crystalline.
COLOR
• Most of alkaloids are colorless but some are colored.
CHEMICAL PROPERTIES
NITROGEN
• Primary amines R-NH2 E.g. Norephedrine
• Secondary amines R2-NH E.g. Ephedrine
• Tertiary amines R3-N E.g. Atropine
• Quaternary NH4 salts R4-N E.g. d-Tubocurarine
BASICITY
• R2-NH > R-NH2 > R3-N
• Saturated hexacyclic amines are more basic than aromatic amines.
81
ALKALOIDAL DRUG ASSAY
INTRODUCTION
• “Basic Nitrogenous compounds of natural origin having some definite
pharmacological/physiological activity.”
• They are alkali like compounds therefore they are called Alkaloids.
PROPERTIES OF ALKALOIDS
PHYSICAL PROPERTIES
SOLUBILITY
• Insoluble or sparingly soluble in water.
• Soluble in organic solvents.
• Alkaloidal salts are soluble in water and insoluble in organic solvents.
CONDITION
• Most are crystalline solids.
• A few are amorphous solids.
• Some are liquids that are either:
Volatile: E.g. Nicotine and Coniine.
Non-volatile: E.g. Pilocarpine and hyoscine
• Alkaloidal salts are crystalline.
COLOR
• Most of alkaloids are colorless but some are colored.
CHEMICAL PROPERTIES
NITROGEN
• Primary amines R-NH2 E.g. Norephedrine
• Secondary amines R2-NH E.g. Ephedrine
• Tertiary amines R3-N E.g. Atropine
• Quaternary NH4 salts R4-N E.g. d-Tubocurarine
BASICITY
• R2-NH > R-NH2 > R3-N
• Saturated hexacyclic amines are more basic than aromatic amines.
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GM Hamad
ALKALOIDAL SALT PREPARATION
• The drug is converted into “alkaloidal base (free) by the addition of alkali
in immiscible solvent (chloroform).
• The alkaloidal base is then treated with dilute acid and is converted into
“acid alkaloidal salt” in aqueous solution.
• The alkaloidal salt is treated with "alkali” to form “alkaloidal base” (free)
in immiscible solvent like chloroform.
• Chloroform is evaporated giving “Free Alkaloid”.
• Finally, Free Alkaloid treated with excess dilute acid to form “Alkaloidal
salt”.
• The excess salt is back titrated with alkali.
• The amount of alkaloid is either weighed or determined volumetrically.
PREPARATION OF THE DRUG FOR THE ASSAY
• The preparation of the drug for the assay includes:
Pulverization of drug substance
Weighing for assay
Extraction of alkaloids
Purification of alkaloids
Determination of alkaloids
PULVERIZATION OF DRUG SUBSTANCE
• Pulverization of drug means, the drugs is crushed, minced or powdered.
The drug should be pulverized to the required finesse-required mesh.
• The care should be taken to avoid the loss of water from the drug during
the pulverization.
WEIGHING FOR ASSAY
• The accuracy should be within 10 mg for quantities equal to 5g or over.
EXTRACTION OF ALKALOIDS
• Extraction of the alkaloids is carried out by either of the following ways:
Maceration
Percolation
Continuous extraction
MACERATION
82
ALKALOIDAL SALT PREPARATION
• The drug is converted into “alkaloidal base (free) by the addition of alkali
in immiscible solvent (chloroform).
• The alkaloidal base is then treated with dilute acid and is converted into
“acid alkaloidal salt” in aqueous solution.
• The alkaloidal salt is treated with "alkali” to form “alkaloidal base” (free)
in immiscible solvent like chloroform.
• Chloroform is evaporated giving “Free Alkaloid”.
• Finally, Free Alkaloid treated with excess dilute acid to form “Alkaloidal
salt”.
• The excess salt is back titrated with alkali.
• The amount of alkaloid is either weighed or determined volumetrically.
PREPARATION OF THE DRUG FOR THE ASSAY
• The preparation of the drug for the assay includes:
Pulverization of drug substance
Weighing for assay
Extraction of alkaloids
Purification of alkaloids
Determination of alkaloids
PULVERIZATION OF DRUG SUBSTANCE
• Pulverization of drug means, the drugs is crushed, minced or powdered.
The drug should be pulverized to the required finesse-required mesh.
• The care should be taken to avoid the loss of water from the drug during
the pulverization.
WEIGHING FOR ASSAY
• The accuracy should be within 10 mg for quantities equal to 5g or over.
EXTRACTION OF ALKALOIDS
• Extraction of the alkaloids is carried out by either of the following ways:
Maceration
Percolation
Continuous extraction
MACERATION
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GM Hamad
• It is the most commonly adopted method for the organized drugs.
Organized drugs are those drugs which have specific cell structure like
root, stem, leaves and flowers.
PROCEDURE
• Accurately weigh the powdered drug.
• Take whole of the solvent and add crude drug to the specified amount of
solvent or mixture of solvents.
• Then made it alkaline with ammonia TS solution and mix well.
• Macerate for 12 – 24 hours with occasional shaking in a closed vessel.
• Afterwards allows the solvent to settle.
• Press the marc and combined the filtrate and expressed liquid.
• Combined liquid is allowed to stand so that colloidal particles are settled
down. Decant or filter this combined liquid.
• The liquid is used for the analysis.
PERCOLATION
• It is process in which crude drug is packed into a column and solvent is
allowed to pass through it till the complete extraction is accomplished
tested by certain tests.
PERCOLATOR
• The apparatus used for percolation process is a percolator. A percolator
is a conical vessel having a lid at the top and is provided with a fake
bottom on which cotton wool or filter paper is placed to support the
column of the drug and help in the escape of the percolate. The base of
the percolator is fitted with a tap from which the percolate is collected.
PROCEDURE
• Place accurately weighed powdered drug is in a suitable container,
imbibe or moistened the drug with suitable solvent for 5 minutes, so
that the drugs swells up.
• Make it alkaline with Ammonia solution and mix thoroughly.
• Now pack the imbibe drug evenly in the percolator.
• After packing of the crude drug sufficient amount of the solvent is added
to saturate the material and close the percolator with a lid. Maintain the
large of solvent over it for uniform and continuous percolation.
• Allow the drug to macerate for 10 – 12 hours.
83
• It is the most commonly adopted method for the organized drugs.
Organized drugs are those drugs which have specific cell structure like
root, stem, leaves and flowers.
PROCEDURE
• Accurately weigh the powdered drug.
• Take whole of the solvent and add crude drug to the specified amount of
solvent or mixture of solvents.
• Then made it alkaline with ammonia TS solution and mix well.
• Macerate for 12 – 24 hours with occasional shaking in a closed vessel.
• Afterwards allows the solvent to settle.
• Press the marc and combined the filtrate and expressed liquid.
• Combined liquid is allowed to stand so that colloidal particles are settled
down. Decant or filter this combined liquid.
• The liquid is used for the analysis.
PERCOLATION
• It is process in which crude drug is packed into a column and solvent is
allowed to pass through it till the complete extraction is accomplished
tested by certain tests.
PERCOLATOR
• The apparatus used for percolation process is a percolator. A percolator
is a conical vessel having a lid at the top and is provided with a fake
bottom on which cotton wool or filter paper is placed to support the
column of the drug and help in the escape of the percolate. The base of
the percolator is fitted with a tap from which the percolate is collected.
PROCEDURE
• Place accurately weighed powdered drug is in a suitable container,
imbibe or moistened the drug with suitable solvent for 5 minutes, so
that the drugs swells up.
• Make it alkaline with Ammonia solution and mix thoroughly.
• Now pack the imbibe drug evenly in the percolator.
• After packing of the crude drug sufficient amount of the solvent is added
to saturate the material and close the percolator with a lid. Maintain the
large of solvent over it for uniform and continuous percolation.
• Allow the drug to macerate for 10 – 12 hours.
83
GM Hamad
• Percolate it slowly until percolate is alkaloid free.
ALKALOIDAL TEST
• Take 4 ml percolate, evaporate and dissolve the residue in 0.5N HCl and
add a drop of mercuric chloride.
• Inference: If turbidity is not produced, percolate is free from alkaloid.
CONTINUOUS EXTRACTION
• Continuous extraction combines two operations i.e. evaporation and
condensation. Condensed liquid is returned to the drug to continue
extraction. So, with the help of temperature smaller volumes of solvent
may be used and process is called “continuous extraction.”
• Counter current extraction using Soxhlet extractor is done.
PURIFICATION OF ALKALOID
• Purification of the alkaloidal extract is carried out by:
Crystallization.
By use of immiscible solvents.
Removal of associated alkaloids by chemical methods.
DETERMINATION OF ALKALOIDS
• Determination of alkaloids is done by following methods:
Gravimetric method
Titrimetric method
GRAVIMETRIC METHOD
• In this method, the solution containing the extract is evaporated to
dryness in a tared container.
• The increase in weight of the container represents the weight of the
alkaloid in the sample.
VOLUMETRIC METHOD
• In this method, the solvent is evaporated carefully to a small volume and
an excess of standard acid plus a small amount of alcohol is added and
the evaporation continued.
• The residual titration method is used.
• Methyl Red TS is used as indicator in volumetric determinations.
84
• Percolate it slowly until percolate is alkaloid free.
ALKALOIDAL TEST
• Take 4 ml percolate, evaporate and dissolve the residue in 0.5N HCl and
add a drop of mercuric chloride.
• Inference: If turbidity is not produced, percolate is free from alkaloid.
CONTINUOUS EXTRACTION
• Continuous extraction combines two operations i.e. evaporation and
condensation. Condensed liquid is returned to the drug to continue
extraction. So, with the help of temperature smaller volumes of solvent
may be used and process is called “continuous extraction.”
• Counter current extraction using Soxhlet extractor is done.
PURIFICATION OF ALKALOID
• Purification of the alkaloidal extract is carried out by:
Crystallization.
By use of immiscible solvents.
Removal of associated alkaloids by chemical methods.
DETERMINATION OF ALKALOIDS
• Determination of alkaloids is done by following methods:
Gravimetric method
Titrimetric method
GRAVIMETRIC METHOD
• In this method, the solution containing the extract is evaporated to
dryness in a tared container.
• The increase in weight of the container represents the weight of the
alkaloid in the sample.
VOLUMETRIC METHOD
• In this method, the solvent is evaporated carefully to a small volume and
an excess of standard acid plus a small amount of alcohol is added and
the evaporation continued.
• The residual titration method is used.
• Methyl Red TS is used as indicator in volumetric determinations.
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GM Hamad
SPECIAL TREATMENT
USE OF ADSORBENT
• For fluid extracts and tinctures having alkaloidal drugs, it is necessary to
evaporate them to dryness. To facilitate evaporation and reduce loss,
extracts are added on an adsorbent – previously acid or alkali washed –
then made neutral by washing with water and dried before use.
EMULSIONS
• Avoid shaking of immiscible solvent with water. If emulsion forms, add
excess of either of the solvents. It will result in the breakdown of
emulsion.
• Emulsion can also be broken down by adding anhydrous sodium
sulphate.
85
SPECIAL TREATMENT
USE OF ADSORBENT
• For fluid extracts and tinctures having alkaloidal drugs, it is necessary to
evaporate them to dryness. To facilitate evaporation and reduce loss,
extracts are added on an adsorbent – previously acid or alkali washed –
then made neutral by washing with water and dried before use.
EMULSIONS
• Avoid shaking of immiscible solvent with water. If emulsion forms, add
excess of either of the solvents. It will result in the breakdown of
emulsion.
• Emulsion can also be broken down by adding anhydrous sodium
sulphate.
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QUALITY CONTROL OF VACCINES
VACCINE
• A vaccine is a biological preparation that provides active acquired
immunity to a particular infectious disease. A vaccine typically contains
an agent that resembles a disease-causing microorganism and is often
made from weakened or killed forms of the microbe, its toxins, or one of
its surface proteins.
AVAILABLE VACCINES
• Hepatitis A vaccine
• Inactivated Polio vaccine
• Diphtheria vaccine
• Human Tetanus
Immunoglobulin
• Acellular Pertussis vaccine
• Tetanus/Diphtheria Vaccine
• Tetanus Toxoid
• Tetanus vaccine
• Rabies vaccine
DIFFERENCE BETWEEN VACCINES AND CHEMICAL DRUGS
• Different starting materials and manufacturing process
• Highly complex products – large proteins molecules
• Test methods needed to characterize products are biological in nature
Bioassays
Potency (activity)
Immunogenicity
Safety
• Not meeting the definition of generics – a specific terminology,
biosimilar is used.
QUALITY CONTROL OF VACCINES
• Testing requires microbiology, virology, molecular biology, cell biology,
and toxicology ensures your quality policy.
• Cell substrates (viral contamination of cell banks and biological products)
• Cultivation and harvesting
• Downstream processing
• Detailed product characterization
• Viral validation studies
• Testing and release
86
QUALITY CONTROL OF VACCINES
VACCINE
• A vaccine is a biological preparation that provides active acquired
immunity to a particular infectious disease. A vaccine typically contains
an agent that resembles a disease-causing microorganism and is often
made from weakened or killed forms of the microbe, its toxins, or one of
its surface proteins.
AVAILABLE VACCINES
• Hepatitis A vaccine
• Inactivated Polio vaccine
• Diphtheria vaccine
• Human Tetanus
Immunoglobulin
• Acellular Pertussis vaccine
• Tetanus/Diphtheria Vaccine
• Tetanus Toxoid
• Tetanus vaccine
• Rabies vaccine
DIFFERENCE BETWEEN VACCINES AND CHEMICAL DRUGS
• Different starting materials and manufacturing process
• Highly complex products – large proteins molecules
• Test methods needed to characterize products are biological in nature
Bioassays
Potency (activity)
Immunogenicity
Safety
• Not meeting the definition of generics – a specific terminology,
biosimilar is used.
QUALITY CONTROL OF VACCINES
• Testing requires microbiology, virology, molecular biology, cell biology,
and toxicology ensures your quality policy.
• Cell substrates (viral contamination of cell banks and biological products)
• Cultivation and harvesting
• Downstream processing
• Detailed product characterization
• Viral validation studies
• Testing and release
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GM Hamad
• Cell bank/bacterial host/other expression systems
• Cell culture/fermentation
• Separation and purification of product
• Bulk product testing
• Characterization of resulting protein and testing for other modifications
• Final product testing
QUALITY CONTROL OF VACCINES - ISSUES
• Different bacterial/virus strains are used by different manufacturers for
same vaccines, e.g., measles vaccine can be produced from Schwartz or
Edmonston Zagreb
• Vaccines can be produced in one fermentation tank or in several bottles
• Same host can be grown in one type of cell in one company and
different in others
• Different stabilizers or preservatives for different companies.
QUALITY CONTROL OF VACCINES
• Starting materials
Raw materials
Labelling
Packaging
Qualifications of suppliers
• Intermediate products
• Finished products
QUALITY CONTROL OF INTERMEDIATE PRODUCTS
1. Gross bacterial contamination test
2. Testing aerobic contaminants
3. Testing for anaerobic contaminants
4. Testing for Salmonella species
5. Testing for contamination with fungi
6. Stability study
I. GROSS BACTERIAL CONTAMINATION
• Measured immediately after harvesting to test contamination in fluid
with extraneous microorganisms.
• Involves inoculation and incubation of a general-purpose sterile broth
(9ml) with wet vaccine (1ml).
• Allowed to stand for overnight.
87
• Cell bank/bacterial host/other expression systems
• Cell culture/fermentation
• Separation and purification of product
• Bulk product testing
• Characterization of resulting protein and testing for other modifications
• Final product testing
QUALITY CONTROL OF VACCINES - ISSUES
• Different bacterial/virus strains are used by different manufacturers for
same vaccines, e.g., measles vaccine can be produced from Schwartz or
Edmonston Zagreb
• Vaccines can be produced in one fermentation tank or in several bottles
• Same host can be grown in one type of cell in one company and
different in others
• Different stabilizers or preservatives for different companies.
QUALITY CONTROL OF VACCINES
• Starting materials
Raw materials
Labelling
Packaging
Qualifications of suppliers
• Intermediate products
• Finished products
QUALITY CONTROL OF INTERMEDIATE PRODUCTS
1. Gross bacterial contamination test
2. Testing aerobic contaminants
3. Testing for anaerobic contaminants
4. Testing for Salmonella species
5. Testing for contamination with fungi
6. Stability study
I. GROSS BACTERIAL CONTAMINATION
• Measured immediately after harvesting to test contamination in fluid
with extraneous microorganisms.
• Involves inoculation and incubation of a general-purpose sterile broth
(9ml) with wet vaccine (1ml).
• Allowed to stand for overnight.
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• If free of growth, vaccines can be divided into fractions and stored as a
wet vaccine or as freeze dried.
II. TESTING AEROBIC CONTAMINANTS
• Use a general-purpose broth culture medium that supports growth of
most likely contaminants, e.g., Tryptic soy broth or Nutrient broth.
• Inoculate 1 mL of wet or reconstituted freeze-dried vaccine into 9 mL of
broth.
• Incubation at 30°C to 37°C for 24 hours.
• If gross contamination is found after 24 hours, retest in twice number of
cultures.
• If contamination is evident after second culture, discard vaccine.
III. TESTING ANAEROBIC CONTAMINANTS
• Anaerobic bacteria have fastidious growth requirements.
• Using aseptic technique minimizes environmental contamination of
harvesting fluid by anaerobes during harvesting.
IV. TESTING SALMONELLA SPECIES
• Inoculate 9 mL of one of liquid media with 1 mL of vaccine
Liquid media (Selenite broth, Tetrathionate broth)
• Incubate at 37°C for 18 to 24 hours
• Inoculate a loopful of incubated broth onto one of solid media
Solid media: MacConkey agar, Salmonella Shigella agar, Brilliant
Green agar, Deoxycholate citrate agar, Xylose Lysine
Deoxycholate, XLD agar
• Incubate 18 to 24 hours.
• If no Salmonella growth is detected, incubate again for 18 to 24 hours
• Discard vaccine if Salmonella are detected.
V. TESTING CONTAMINATION WITH FUNGI
• Inoculate dextrose agar with 100 mL of vaccine and spread over plate.
• Add 0.05 g/L chloramphenicol to inhibit bacterial growth
• Inoculate a control plate with diluent only.
• Incubate at 25°C to 30°C for one week.
• Observe daily and record results.
• Growth on control plate invalidates test, which should then be repeated.
VI. TESTING SAFETY OR SUSCEPTIBILITY
• Administration of certain doses of vaccine to appropriate living organism
(animals, rodents, chickens or birds)
88
• If free of growth, vaccines can be divided into fractions and stored as a
wet vaccine or as freeze dried.
II. TESTING AEROBIC CONTAMINANTS
• Use a general-purpose broth culture medium that supports growth of
most likely contaminants, e.g., Tryptic soy broth or Nutrient broth.
• Inoculate 1 mL of wet or reconstituted freeze-dried vaccine into 9 mL of
broth.
• Incubation at 30°C to 37°C for 24 hours.
• If gross contamination is found after 24 hours, retest in twice number of
cultures.
• If contamination is evident after second culture, discard vaccine.
III. TESTING ANAEROBIC CONTAMINANTS
• Anaerobic bacteria have fastidious growth requirements.
• Using aseptic technique minimizes environmental contamination of
harvesting fluid by anaerobes during harvesting.
IV. TESTING SALMONELLA SPECIES
• Inoculate 9 mL of one of liquid media with 1 mL of vaccine
Liquid media (Selenite broth, Tetrathionate broth)
• Incubate at 37°C for 18 to 24 hours
• Inoculate a loopful of incubated broth onto one of solid media
Solid media: MacConkey agar, Salmonella Shigella agar, Brilliant
Green agar, Deoxycholate citrate agar, Xylose Lysine
Deoxycholate, XLD agar
• Incubate 18 to 24 hours.
• If no Salmonella growth is detected, incubate again for 18 to 24 hours
• Discard vaccine if Salmonella are detected.
V. TESTING CONTAMINATION WITH FUNGI
• Inoculate dextrose agar with 100 mL of vaccine and spread over plate.
• Add 0.05 g/L chloramphenicol to inhibit bacterial growth
• Inoculate a control plate with diluent only.
• Incubate at 25°C to 30°C for one week.
• Observe daily and record results.
• Growth on control plate invalidates test, which should then be repeated.
VI. TESTING SAFETY OR SUSCEPTIBILITY
• Administration of certain doses of vaccine to appropriate living organism
(animals, rodents, chickens or birds)
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• Collection of serum and testing with appropriate method for presence of
maternal antibodies to test virus of disease
• To pass test, animals should not have maternal antibodies to virus of
disease or clinical signs of disease after 21 days.
QUALITY CONTROL OF FINISHED PRODUCTS
1. Testing extraneous microorganisms and safety
2. Test of inactivation
3. Testing in-vivo potency (efficacy)
4. Testing pyrogens
5. Stability test of diluents and reconstituted vaccine
6. Safety testing
a. Residual toxicity or reversion to toxicity
b. Testing neurovirulence
c. Chromogenic assay
I. TESTING EXTRANEOUS MICROORGANISMS AND SAFETY
• Extraneous microorganisms and safety tests are employed on finished
products as stated in previous slides.
II. TEST OF INACTIVATION OF VIRULENT VIRUSES
• Test for ability of inactivated pathogen to cause disease
• Real time PCR, which quantify amount of nucleic acids, cannot provide a
direct measure of virulence (because the RNA/DNA of a dead pathogen
would still show up, even though it cannot cause disease).
• Traditionally virulence is tested by injecting a given organism (rabbits,
pigs, chickens, etc.) and checked colonization/spread (and consequently)
production of disease.
• Alternatively, same pathogen can be inoculated into a cell culture
system.
• Cultured cells are observed to determine how many have been infected
or
obtain a bleed from animal and titer how much pathogen is present (and
then compared it to how much was inoculated).
III. TESTING IN-VIVO POTENCY
• Testing in-vivo potency/efficacy/assay or virus content of vaccine to
ensure that vaccinated vaccine recipient receives enough viruses to
induce a protective immunity.
• Potency is quantitative measure of biological activity.
89
• Collection of serum and testing with appropriate method for presence of
maternal antibodies to test virus of disease
• To pass test, animals should not have maternal antibodies to virus of
disease or clinical signs of disease after 21 days.
QUALITY CONTROL OF FINISHED PRODUCTS
1. Testing extraneous microorganisms and safety
2. Test of inactivation
3. Testing in-vivo potency (efficacy)
4. Testing pyrogens
5. Stability test of diluents and reconstituted vaccine
6. Safety testing
a. Residual toxicity or reversion to toxicity
b. Testing neurovirulence
c. Chromogenic assay
I. TESTING EXTRANEOUS MICROORGANISMS AND SAFETY
• Extraneous microorganisms and safety tests are employed on finished
products as stated in previous slides.
II. TEST OF INACTIVATION OF VIRULENT VIRUSES
• Test for ability of inactivated pathogen to cause disease
• Real time PCR, which quantify amount of nucleic acids, cannot provide a
direct measure of virulence (because the RNA/DNA of a dead pathogen
would still show up, even though it cannot cause disease).
• Traditionally virulence is tested by injecting a given organism (rabbits,
pigs, chickens, etc.) and checked colonization/spread (and consequently)
production of disease.
• Alternatively, same pathogen can be inoculated into a cell culture
system.
• Cultured cells are observed to determine how many have been infected
or
obtain a bleed from animal and titer how much pathogen is present (and
then compared it to how much was inoculated).
III. TESTING IN-VIVO POTENCY
• Testing in-vivo potency/efficacy/assay or virus content of vaccine to
ensure that vaccinated vaccine recipient receives enough viruses to
induce a protective immunity.
• Potency is quantitative measure of biological activity.
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• Virus content/load is a measure of amount of infective disease virus in
vaccine.
• In this test, infectivity titer of vaccine is measured. For killed
bacteria/virus vaccine, potency assay is performed using animals.
Potency of test vaccine may be expressed as a percentage of potency of
standard vaccine.
• Amount of vaccine required to protect animals from a defined challenge
dose of pathogen is compared with the amount of a standard vaccines
that is required to provide same protection. Number of survivors in each
group is used to calculate potency of test vaccine relative to the potency
of standard vaccine by statistical method.
• Test animals are divided into two groups of 8-10 animals per group and
housed separately.
One group of animals (adult mice, suckling mice and guinea-pigs,
control eggs) is vaccinated.
Other group remained unvaccinated to act as control group.
• Serum of animals from both groups is collected two weeks after
vaccination. Antibody response of animals to vaccine is measured.
Serum is tested for presence of antibodies to disease virus.
• An adequate antibody response to vaccine can be measured in two
ways:
i. Testing serum collected from vaccinated animal (serological test)
for level of antibodies titers.
ii. Challenging vaccinated animal with a virulent virus strain of
disease under test.
• Method 1 is carried mostly.
• Titer level indicates that animal would be protected against disease if
challenged in method 2.
• Further testing of efficacy of vaccine may be carried out by challenging
both groups of animals with virulent disease virus.
• Select animals having antibody levels which is indicative of unexposed
animals to test disease virus. Divide the animals into two groups, a
control group and a treatment group. House the two groups separately
and arrange one attendant for each group of chickens.
• Implement reasonable biosecurity measures to minimize the risk of
uncontrolled introduction of disease virus to either of the groups.
90
• Virus content/load is a measure of amount of infective disease virus in
vaccine.
• In this test, infectivity titer of vaccine is measured. For killed
bacteria/virus vaccine, potency assay is performed using animals.
Potency of test vaccine may be expressed as a percentage of potency of
standard vaccine.
• Amount of vaccine required to protect animals from a defined challenge
dose of pathogen is compared with the amount of a standard vaccines
that is required to provide same protection. Number of survivors in each
group is used to calculate potency of test vaccine relative to the potency
of standard vaccine by statistical method.
• Test animals are divided into two groups of 8-10 animals per group and
housed separately.
One group of animals (adult mice, suckling mice and guinea-pigs,
control eggs) is vaccinated.
Other group remained unvaccinated to act as control group.
• Serum of animals from both groups is collected two weeks after
vaccination. Antibody response of animals to vaccine is measured.
Serum is tested for presence of antibodies to disease virus.
• An adequate antibody response to vaccine can be measured in two
ways:
i. Testing serum collected from vaccinated animal (serological test)
for level of antibodies titers.
ii. Challenging vaccinated animal with a virulent virus strain of
disease under test.
• Method 1 is carried mostly.
• Titer level indicates that animal would be protected against disease if
challenged in method 2.
• Further testing of efficacy of vaccine may be carried out by challenging
both groups of animals with virulent disease virus.
• Select animals having antibody levels which is indicative of unexposed
animals to test disease virus. Divide the animals into two groups, a
control group and a treatment group. House the two groups separately
and arrange one attendant for each group of chickens.
• Implement reasonable biosecurity measures to minimize the risk of
uncontrolled introduction of disease virus to either of the groups.
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• The control group is not vaccinated. The treatment group receives one
vaccine dose per animal. Bleed both groups at weekly intervals.
• Test serum to measure the antibody response to vaccination and to
indicate levels of protection after 2 to 3 weeks of vaccination. Make
daily observations of the animal/chickens and record the results.
• At least 90 percent of the vaccinated chickens should survive the
challenge and show no clinical signs of the Newcastle disease. All
unvaccinated control chickens should die of disease.
• Checking serum for antibodies is called serological test. Results of
biological assays is usually expressed in units of activity calibrated
against a reference standard. Match the titers with that which is
considered protective.
• ELISA for in vitro potency testing of hepatitis A.
• Invitro-assay of Hepatitis A vaccine (method A) carried out by an
immunochemical determination of antigen content.
• In vivo assay of Hepatitis A vaccine (method B), by comparing its capacity
to induce specific antibodies in mice with same capacity of a reference
preparation.
• Vaccine containing live microorganisms are generally tested for potency
by determining their content of viable particles.
• Dilutions of vaccine are prepared in media. Fixed volumes of diluted
vaccines are dropped on to solid media capable of supporting
viral/bacterial growth. After fortnight, the colonies generated by drops
are counted and the live count of undiluted vaccine is calculated.
IV. TESTING PYROGEN
• There are two methods of pyrogen testing
Endotoxin LAL test
Rabbit test
• Both methods are limited in products and range of pyrogens which can
be detected, e.g., LAL test can only detect endotoxins.
• Both also have a high level of animal consumption.
• Problematic in testing of products that affect body temperature
regulation (e.g., antipyretic drugs and steroids), drugs that cause
immunological reactions (e.g., immunoglobulins)
MAT TEST
• Human whole blood Monocyte- Activation Test (MAT), introduced into
European Pharmacopoeia (EP) in 2010 overcome limitations of above
91
• The control group is not vaccinated. The treatment group receives one
vaccine dose per animal. Bleed both groups at weekly intervals.
• Test serum to measure the antibody response to vaccination and to
indicate levels of protection after 2 to 3 weeks of vaccination. Make
daily observations of the animal/chickens and record the results.
• At least 90 percent of the vaccinated chickens should survive the
challenge and show no clinical signs of the Newcastle disease. All
unvaccinated control chickens should die of disease.
• Checking serum for antibodies is called serological test. Results of
biological assays is usually expressed in units of activity calibrated
against a reference standard. Match the titers with that which is
considered protective.
• ELISA for in vitro potency testing of hepatitis A.
• Invitro-assay of Hepatitis A vaccine (method A) carried out by an
immunochemical determination of antigen content.
• In vivo assay of Hepatitis A vaccine (method B), by comparing its capacity
to induce specific antibodies in mice with same capacity of a reference
preparation.
• Vaccine containing live microorganisms are generally tested for potency
by determining their content of viable particles.
• Dilutions of vaccine are prepared in media. Fixed volumes of diluted
vaccines are dropped on to solid media capable of supporting
viral/bacterial growth. After fortnight, the colonies generated by drops
are counted and the live count of undiluted vaccine is calculated.
IV. TESTING PYROGEN
• There are two methods of pyrogen testing
Endotoxin LAL test
Rabbit test
• Both methods are limited in products and range of pyrogens which can
be detected, e.g., LAL test can only detect endotoxins.
• Both also have a high level of animal consumption.
• Problematic in testing of products that affect body temperature
regulation (e.g., antipyretic drugs and steroids), drugs that cause
immunological reactions (e.g., immunoglobulins)
MAT TEST
• Human whole blood Monocyte- Activation Test (MAT), introduced into
European Pharmacopoeia (EP) in 2010 overcome limitations of above
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tests.
• MAT is an alternative to Rabbit Pyrogen/ Endotoxin test and is used to
detects or quantifies endotoxin and non-endotoxin pyrogens.
• MAT is based on principle of the human immune system response
• In human when pyrogens come into contact with blood stream, host’s
innate immune defense mechanisms cause monocytes/macrophages to
produce prostaglandins and pro-inflammatory cytokines such as
interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-
α)
• An in vitro pyrogen detection test mimics above biological response by
utilizing cryo-preserved human whole blood as a source of monocytes
and determining pyrogenicity of sample by measuring Interleukin-1β
production in an immunological assay (ELISA).
• It simulates human fever reaction better than any animal-based pyrogen
test. MAT generates an interleukin-1ß response when pyrogens are
present.
• Using whole human blood (cryo blood or fresh blood), MAT initiates
innate immune response to a fever reaction caused by pyrogens.
• MAT is highly robust and provides a high level of reliability due to
including both a positive and a negative control.
• MAT assay detects Gram-positive and Gram-negative organisms and
parasitic, viral, fungi and other biological pyrogens (e.g., yeast) and non-
biological pyrogens.
V. SAFETY TESTING
• Bacterial vaccines containing killed bacteria/bacterial products must
show that they are completely free from the living microorganisms used
in the production process
• Vaccines prepared from toxins require a test system capable of revealing
inadequately detoxified toxins
• To prove that, test toxin sensitive ginea pigs are inoculated
• Observation for the symptoms of disease
• In killed vaccines, tested the incomplete virus inactivation, a consequent
presence of residual live virus
• With attenuated viral vaccine, tested the reversion of virus during
production to a degree of virulence capable of causing disease in
recipients.
92
tests.
• MAT is an alternative to Rabbit Pyrogen/ Endotoxin test and is used to
detects or quantifies endotoxin and non-endotoxin pyrogens.
• MAT is based on principle of the human immune system response
• In human when pyrogens come into contact with blood stream, host’s
innate immune defense mechanisms cause monocytes/macrophages to
produce prostaglandins and pro-inflammatory cytokines such as
interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-
α)
• An in vitro pyrogen detection test mimics above biological response by
utilizing cryo-preserved human whole blood as a source of monocytes
and determining pyrogenicity of sample by measuring Interleukin-1β
production in an immunological assay (ELISA).
• It simulates human fever reaction better than any animal-based pyrogen
test. MAT generates an interleukin-1ß response when pyrogens are
present.
• Using whole human blood (cryo blood or fresh blood), MAT initiates
innate immune response to a fever reaction caused by pyrogens.
• MAT is highly robust and provides a high level of reliability due to
including both a positive and a negative control.
• MAT assay detects Gram-positive and Gram-negative organisms and
parasitic, viral, fungi and other biological pyrogens (e.g., yeast) and non-
biological pyrogens.
V. SAFETY TESTING
• Bacterial vaccines containing killed bacteria/bacterial products must
show that they are completely free from the living microorganisms used
in the production process
• Vaccines prepared from toxins require a test system capable of revealing
inadequately detoxified toxins
• To prove that, test toxin sensitive ginea pigs are inoculated
• Observation for the symptoms of disease
• In killed vaccines, tested the incomplete virus inactivation, a consequent
presence of residual live virus
• With attenuated viral vaccine, tested the reversion of virus during
production to a degree of virulence capable of causing disease in
recipients.
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MISCELLANEOUS DETERMINATIONS AND
TESTS
Determination of:
• Weight/ml
• Water/Moisture content
• Loss on Drying
• Evaluation of Ointments
• Ash contents
• Alkalinity of glass
• Toxicity testing
• Total solids
WEIGHT PER ML
DEFINITION
• The weight per milliliter of a liquid is the weight, in gram, of 1 mL of a
liquid when weighed in air at 20°C, unless otherwise specified in the
monograph. -British pharmacopoeia, 2012 online (Appendix V G.)
• Simply, it may be defined as the weight, expressed in grams, of 1 mL of a
liquid when weighed in air at the specific temperature.
REASON OF DETERMINATION
• Batch to batch thickness maintenance (variables are like water, actives,
excipients, impurities etc.) of liquid preparations such as suspensions,
syrups and semi-liquid preparations e.g. ointments.
• Preparations/ formulations for which assays are not possible i.e.
formulation or preparation is a complex mixture. E.g. tea
FORMULATIONS EXPOSED TO WEIGHT/ML TEST
• Extracts
• Tinctures
• Oral solutions
• Oral suspension
• Syrups
• Galenicals
• Complex mixtures (containing
more than 1 actives)
• The quantity of the oral solution or suspension taken for assay purposes
is weighed accurately and the weight per ml determined in order to
calculate the content as weight in volume (g per ml).
93
MISCELLANEOUS DETERMINATIONS AND
TESTS
Determination of:
• Weight/ml
• Water/Moisture content
• Loss on Drying
• Evaluation of Ointments
• Ash contents
• Alkalinity of glass
• Toxicity testing
• Total solids
WEIGHT PER ML
DEFINITION
• The weight per milliliter of a liquid is the weight, in gram, of 1 mL of a
liquid when weighed in air at 20°C, unless otherwise specified in the
monograph. -British pharmacopoeia, 2012 online (Appendix V G.)
• Simply, it may be defined as the weight, expressed in grams, of 1 mL of a
liquid when weighed in air at the specific temperature.
REASON OF DETERMINATION
• Batch to batch thickness maintenance (variables are like water, actives,
excipients, impurities etc.) of liquid preparations such as suspensions,
syrups and semi-liquid preparations e.g. ointments.
• Preparations/ formulations for which assays are not possible i.e.
formulation or preparation is a complex mixture. E.g. tea
FORMULATIONS EXPOSED TO WEIGHT/ML TEST
• Extracts
• Tinctures
• Oral solutions
• Oral suspension
• Syrups
• Galenicals
• Complex mixtures (containing
more than 1 actives)
• The quantity of the oral solution or suspension taken for assay purposes
is weighed accurately and the weight per ml determined in order to
calculate the content as weight in volume (g per ml).
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PROCEDURE
• Take a clean, dry pycnometer.
• Calibrate the pycnometer by filling it with a freshly boiled and cooled
water at 20°C and weighing the contents.
• Calculate the capacity of pycnometer by assuming that the weight of 1ml
of water at 20°C when weighed in air of density 0.0012g/ml is 0.997 g.
• Adjust the temperature of the substance under investigation to 20°C and
fill the pycnometer with it in such a way that no bubble formation takes
place.
• Adjust the temperature of filled pycnometer to 20°C, remove the excess
of liquid and weigh.
• Subtract the tare weight of pycnometer from the filled weight of
pycnometer.
• Calculate the weight per milliliter by dividing the weight (in g) of the
quantity of liquid that fills a pycnometer at the specified temperature by
the capacity, expressed in ml, of the pycnometer at the same
temperature.
CALCULATIONS
• Weight of pycnometer −−−−−−−−−−−−− = W1 A grams
• Weight of pycnometer + Liquid sample −−−− = W2 B grams
• Weight of liquid sample −−−−−−−−−−−− = W3 C = B – A
• Capacity of pycnometer −−−−−−−−−−−− = V
• Weight/ml −−−−−−−−−−−−−−−−−−−− E = C/V
??????�??????�ℎ� ��� ��=
??????����??????�?????? �� �??????��??????� ??????� �??????���� �??????�������� (�)
�����??????�?????? �� �??????�������� (��)
BRITISH PHARMACOPOEIA TABLE
• The weight of a liter of water at specified temperatures when weighed
against brass weights in air of density 0.0012g per mL is given in the
following table.
Temperature℃ Weight of a liter of water (g)
20 997.18
25 996.02
30 994.62
94
PROCEDURE
• Take a clean, dry pycnometer.
• Calibrate the pycnometer by filling it with a freshly boiled and cooled
water at 20°C and weighing the contents.
• Calculate the capacity of pycnometer by assuming that the weight of 1ml
of water at 20°C when weighed in air of density 0.0012g/ml is 0.997 g.
• Adjust the temperature of the substance under investigation to 20°C and
fill the pycnometer with it in such a way that no bubble formation takes
place.
• Adjust the temperature of filled pycnometer to 20°C, remove the excess
of liquid and weigh.
• Subtract the tare weight of pycnometer from the filled weight of
pycnometer.
• Calculate the weight per milliliter by dividing the weight (in g) of the
quantity of liquid that fills a pycnometer at the specified temperature by
the capacity, expressed in ml, of the pycnometer at the same
temperature.
CALCULATIONS
• Weight of pycnometer −−−−−−−−−−−−− = W1 A grams
• Weight of pycnometer + Liquid sample −−−− = W2 B grams
• Weight of liquid sample −−−−−−−−−−−− = W3 C = B – A
• Capacity of pycnometer −−−−−−−−−−−− = V
• Weight/ml −−−−−−−−−−−−−−−−−−−− E = C/V
??????�??????�ℎ� ��� ��=
??????����??????�?????? �� �??????��??????� ??????� �??????���� �??????�������� (�)
�����??????�?????? �� �??????�������� (��)
BRITISH PHARMACOPOEIA TABLE
• The weight of a liter of water at specified temperatures when weighed
against brass weights in air of density 0.0012g per mL is given in the
following table.
Temperature℃ Weight of a liter of water (g)
20 997.18
25 996.02
30 994.62
94
GM Hamad
WATER / MOISTURE CONTENT
DEFINITION
• This test determines the amount of water (as the volatile matter) that is
driven of under specified conditions. -British pharmacopoeia, 2012
online (Appendix V G.).
IMPORTANCE OF DETERMINATION
• Many Pharmacopeial articles either are hydrates or contain water in
adsorbed form. As a result, the determination of the water content is
important in demonstrating compliance with the Pharmacopeial
standards.
• Presence of water beyond the acceptance limits interferes with quality
of the drug since its presence may affect:
Drug stability
Microbial degradation of drug.
FORMULATIONS EXPOSED TO THE TEST
• When water is the only volatile component, this test is usually employed
for:
Water extracts
Tinctures
Powders
Granules
Tablets
Capsules
PROCEDURE
Moisture
content
Titrimetric
Volumetric
titration
Direct
titration
Residual
titration
Coulometric
titration
AzeotropicGravimetric
95
WATER / MOISTURE CONTENT
DEFINITION
• This test determines the amount of water (as the volatile matter) that is
driven of under specified conditions. -British pharmacopoeia, 2012
online (Appendix V G.).
IMPORTANCE OF DETERMINATION
• Many Pharmacopeial articles either are hydrates or contain water in
adsorbed form. As a result, the determination of the water content is
important in demonstrating compliance with the Pharmacopeial
standards.
• Presence of water beyond the acceptance limits interferes with quality
of the drug since its presence may affect:
Drug stability
Microbial degradation of drug.
FORMULATIONS EXPOSED TO THE TEST
• When water is the only volatile component, this test is usually employed
for:
Water extracts
Tinctures
Powders
Granules
Tablets
Capsules
PROCEDURE
Moisture
content
Titrimetric
Volumetric
titration
Direct
titration
Residual
titration
Coulometric
titration
AzeotropicGravimetric
95
GM Hamad
1. TITRIMETRIC METHOD
• It is also known as Karl Fischer method.
• It is a method of water determination in the technical products (oil,
plastics and gases), in cosmetic products, in pharmaceutical products
and in food industry. It is a standard technique for low moisture
containing products.
KARL FISCHER TITRATION METHODS
• The test specimen may be titrated with the reagent directly or
analysis may be carried out by a residual titration procedure.
I. VOLUMETRIC TITRATION
BASIC PRINCIPLE
• The titrimetric determination of water is based on quantitative
reaction of water with anhydrous solution of sulfur dioxide and
iodine in the presence of methanol and pyridine.
• Range of application 0.1% − 100% depends on sample size.
• Step 1: H2O + I2 + SO2 + 3C5H5N → 2[C5H5N+H]I + C5H5NSO3
• Step 2: C5H5NSO3 + CH3OH → [C5H5N+H]O.SO2.OCH3
• Dissolve Iodine in a mixture of pyridine, Methanol and SO2.
A. DIRECT TITRATION
• Fill titration vessel with solvent
• Pre-titration with KF reagent
• Add the sample
• Titrate with KF reagent
• Concentration of KF Reagent = mg H2O /ml of Reagent
• % H2O = [Concentration x ml Reagent/mg of Sample] x 100
B. RESIDUAL TITRATION
• In the residual titration, excess reagent is added to the test specimen,
sufficient time is allowed for the reaction to reach completion, and
the unconsumed reagent is titrated with a standard solution of water
in a solvent such as methanol.
II. COULOMETRIC TITRATION
• The main compartment of the titration cell contains:
The anode solution (A solution containing alcohol, a base, SO2 and
I2)
The analyte
Cathode immersed in the anode solution.
96
1. TITRIMETRIC METHOD
• It is also known as Karl Fischer method.
• It is a method of water determination in the technical products (oil,
plastics and gases), in cosmetic products, in pharmaceutical products
and in food industry. It is a standard technique for low moisture
containing products.
KARL FISCHER TITRATION METHODS
• The test specimen may be titrated with the reagent directly or
analysis may be carried out by a residual titration procedure.
I. VOLUMETRIC TITRATION
BASIC PRINCIPLE
• The titrimetric determination of water is based on quantitative
reaction of water with anhydrous solution of sulfur dioxide and
iodine in the presence of methanol and pyridine.
• Range of application 0.1% − 100% depends on sample size.
• Step 1: H2O + I2 + SO2 + 3C5H5N → 2[C5H5N+H]I + C5H5NSO3
• Step 2: C5H5NSO3 + CH3OH → [C5H5N+H]O.SO2.OCH3
• Dissolve Iodine in a mixture of pyridine, Methanol and SO2.
A. DIRECT TITRATION
• Fill titration vessel with solvent
• Pre-titration with KF reagent
• Add the sample
• Titrate with KF reagent
• Concentration of KF Reagent = mg H2O /ml of Reagent
• % H2O = [Concentration x ml Reagent/mg of Sample] x 100
B. RESIDUAL TITRATION
• In the residual titration, excess reagent is added to the test specimen,
sufficient time is allowed for the reaction to reach completion, and
the unconsumed reagent is titrated with a standard solution of water
in a solvent such as methanol.
II. COULOMETRIC TITRATION
• The main compartment of the titration cell contains:
The anode solution (A solution containing alcohol, a base, SO2 and
I2)
The analyte
Cathode immersed in the anode solution.
96
GM Hamad
• The two compartments (anode and cathode) are separated by an ion-
permeable membrane.
• The Pt anode generates I2 when current is provided through the electric
circuit. One mole of I2 is consumed for each mole of H2O. In other words,
2 moles of electrons are consumed per mole of water.
• The end point is detected most commonly by immersing a second pair of
Pt electrodes in the anode solution. The detector circuit maintains a
constant current between the two detector electrodes during titration.
Prior to the equivalence point, the solution contains I but little I2.
• At the equivalence point, excess I2 appears and an abrupt voltage drop
marks the end point. The amount of current needed to generate I2 and
reach the end point can then be used to calculate the amount of water
in the original sample.
• Coulometry is best suited for determination of water content in the
range of 1ppm – 5ppm.
2. AZEOTROPIC METHOD
• It is useful for the detection of the water in oily and semi-solid
preparations.
• As the test substance is heated, any water present in the sample
vaporizes.
• The water vapors are then condensed and collected in a graduated
collection tube, such that the volume of water produced by
distillation can be measured as a function of the total volume of oil
used.
3. GRAVIMETRIC METHOD
• Unless otherwise prescribed in the original monograph, weigh
accurately 2g of the powder material in a tarred silica crucible.
• Dry the powder in an oven at 105°C for 30 min and cool it at room
temperature in desiccators until constant weight is obtained.
• Calculate the moisture contents difference in the weight of powdered
drug before and after drying.
• The results are expressed as a percentage of dry powdered drug.
CALCULATIONS
• Weight of bottle and contents before drying ----- = W1 A grams
• Weight of bottle and contents after drying ------- = W2 B grams
• Loss on drying (%) ---------------------------------------- = W3 C = (A – B)* 100
97
• The two compartments (anode and cathode) are separated by an ion-
permeable membrane.
• The Pt anode generates I2 when current is provided through the electric
circuit. One mole of I2 is consumed for each mole of H2O. In other words,
2 moles of electrons are consumed per mole of water.
• The end point is detected most commonly by immersing a second pair of
Pt electrodes in the anode solution. The detector circuit maintains a
constant current between the two detector electrodes during titration.
Prior to the equivalence point, the solution contains I but little I2.
• At the equivalence point, excess I2 appears and an abrupt voltage drop
marks the end point. The amount of current needed to generate I2 and
reach the end point can then be used to calculate the amount of water
in the original sample.
• Coulometry is best suited for determination of water content in the
range of 1ppm – 5ppm.
2. AZEOTROPIC METHOD
• It is useful for the detection of the water in oily and semi-solid
preparations.
• As the test substance is heated, any water present in the sample
vaporizes.
• The water vapors are then condensed and collected in a graduated
collection tube, such that the volume of water produced by
distillation can be measured as a function of the total volume of oil
used.
3. GRAVIMETRIC METHOD
• Unless otherwise prescribed in the original monograph, weigh
accurately 2g of the powder material in a tarred silica crucible.
• Dry the powder in an oven at 105°C for 30 min and cool it at room
temperature in desiccators until constant weight is obtained.
• Calculate the moisture contents difference in the weight of powdered
drug before and after drying.
• The results are expressed as a percentage of dry powdered drug.
CALCULATIONS
• Weight of bottle and contents before drying ----- = W1 A grams
• Weight of bottle and contents after drying ------- = W2 B grams
• Loss on drying (%) ---------------------------------------- = W3 C = (A – B)* 100
97
GM Hamad
LOSS ON DRYING
INTRODUCTION
• The test is usually carried out to determine the amount of volatile
matter of any kind that is driven of under specified conditions.
PROCEDURE
• Take 1 – 2g of substance, reduce particle size if necessary, in a dried
glass stoppered, weighed bottle.
• Weigh and shake to evenly distribute the substance.
• Place the loaded bottle in drying chamber, remove stopper and place it
also in the chamber.
• Dry the sample at temperature specified in monograph, close the bottle.
Cool in desiccator and weigh.
• If the substance melts at a temperature at which loss on drying is to be
performed, it is kept at 5 − 10℃ for 1 – 2 hours for drying.
• For substance requiring thermogravimetric analysis a sensitive electro-
balance is used.
• For vacuum drying vacuum desiccator or vacuum drying pistol are used.
CALCULATION
%���� �� ��????????????�� (�??????�)=
���� �� ??????���� ??????� ������ (��)
??????���� ���� �� ??????�� ������ (��)
×100
EVALUATION OF OINTMENTS
OINTMENTS
• Ointments are semisolid dosage forms in which one or more drug
substances are dissolved or dispersed or emulsified in a suitable
ointment base and are meant for application on skin or mucous
membrane where it exhibit local or systemic effects.
EVALUATION TESTS FOR OINTMENTS
PHYSICAL METHODS
• Rate of absorption • Non irritancy
98
LOSS ON DRYING
INTRODUCTION
• The test is usually carried out to determine the amount of volatile
matter of any kind that is driven of under specified conditions.
PROCEDURE
• Take 1 – 2g of substance, reduce particle size if necessary, in a dried
glass stoppered, weighed bottle.
• Weigh and shake to evenly distribute the substance.
• Place the loaded bottle in drying chamber, remove stopper and place it
also in the chamber.
• Dry the sample at temperature specified in monograph, close the bottle.
Cool in desiccator and weigh.
• If the substance melts at a temperature at which loss on drying is to be
performed, it is kept at 5 − 10℃ for 1 – 2 hours for drying.
• For substance requiring thermogravimetric analysis a sensitive electro-
balance is used.
• For vacuum drying vacuum desiccator or vacuum drying pistol are used.
CALCULATION
%���� �� ��????????????�� (�??????�)=
���� �� ??????���� ??????� ������ (��)
??????���� ���� �� ??????�� ������ (��)
×100
EVALUATION OF OINTMENTS
OINTMENTS
• Ointments are semisolid dosage forms in which one or more drug
substances are dissolved or dispersed or emulsified in a suitable
ointment base and are meant for application on skin or mucous
membrane where it exhibit local or systemic effects.
EVALUATION TESTS FOR OINTMENTS
PHYSICAL METHODS
• Rate of absorption • Non irritancy
98
GM Hamad
• Rate of penetration
• Rate of drug release
• Rheological properties
• Content uniformity
MICROBIAL METHODS
• Preservative efficacy • Test of microbial content
1. TEST OF RATE OF ABSORPTION
• The diadermic ointment should be evaluated for the rate of
absorption of drug into the blood stream. This test can be done in-
vivo only.
• The ointment should be applied over a definite area of the skin by
rubbing.
• At regular intervals of time, serum and urine samples should be
analyzed for the quantity of drug absorbed.
• The rate of absorption i.e. the amount of drug absorbed per unit time
should be more.
2. TEST OF NON-IRRITANCY
• The bases used in the formulation of ointments may cause irritation
or allergic reactions.
• Non-irritancy of the preparation is evaluated by patch test.
• In this test 24 human volunteers are selected.
• Definite quantity of ointment is applied under occlusion daily on the
back or volar forearm for 21 days.
• Daily the type of pharmacological action observed is noted.
• No visible reaction or erythema or intense erythema with edema and
vesicular erosion should occur.
• A good ointment base shows no visible reaction.
3. TEST OF RATE OF PENETRATION
• The rate of penetration of a semisolid dosage form is crucial in the
onset and duration of action of the drug.
• Weighed quantity of the preparation should be applied over selected
area of the skin for a definite period of time.
• Then the preparation left over is collected and weighed.
• The difference between the initial and the final weights of the
preparation gives the amount of preparation penetrated through the
skin and this when divided by the area and time period of application
gives the rate of penetration of the preparation.
• The test should be repeated twice or thrice.
99
• Rate of penetration
• Rate of drug release
• Rheological properties
• Content uniformity
MICROBIAL METHODS
• Preservative efficacy • Test of microbial content
1. TEST OF RATE OF ABSORPTION
• The diadermic ointment should be evaluated for the rate of
absorption of drug into the blood stream. This test can be done in-
vivo only.
• The ointment should be applied over a definite area of the skin by
rubbing.
• At regular intervals of time, serum and urine samples should be
analyzed for the quantity of drug absorbed.
• The rate of absorption i.e. the amount of drug absorbed per unit time
should be more.
2. TEST OF NON-IRRITANCY
• The bases used in the formulation of ointments may cause irritation
or allergic reactions.
• Non-irritancy of the preparation is evaluated by patch test.
• In this test 24 human volunteers are selected.
• Definite quantity of ointment is applied under occlusion daily on the
back or volar forearm for 21 days.
• Daily the type of pharmacological action observed is noted.
• No visible reaction or erythema or intense erythema with edema and
vesicular erosion should occur.
• A good ointment base shows no visible reaction.
3. TEST OF RATE OF PENETRATION
• The rate of penetration of a semisolid dosage form is crucial in the
onset and duration of action of the drug.
• Weighed quantity of the preparation should be applied over selected
area of the skin for a definite period of time.
• Then the preparation left over is collected and weighed.
• The difference between the initial and the final weights of the
preparation gives the amount of preparation penetrated through the
skin and this when divided by the area and time period of application
gives the rate of penetration of the preparation.
• The test should be repeated twice or thrice.
99
GM Hamad
• This procedure is tedious and not followed anymore.
• Using flow-through diffusion cell or micro-dialysis method, the rate of
penetration of the preparation can be estimated. Animal or human
skin of definite area should be collected and tied to the holder
present in a diffusion cell.
• The diffusion cell is placed in a fluid bath. Measured quantity of the
preparation is applied over the skin and the amount of drug passed
into the fluid is measured at regular intervals by analyzing the
aliquots of fluid using a spectrophotometer.
4. TEST OF RATE OF DRUG RELEASE
• A clean test tube is taken and the internal surface is coated with the
preparation as a thin layer. Saline or serum is poured into the test
tube. After a certain period of time, the saline is analyzed for the
quantity of the drug. The amount of drug when divided by the time
period gives the rate of drug release.
• To assess the rate of release of medicament, small amount of the
ointment can be placed on the surface of nutrient agar contained in a
petri dish or alternately in a small cup cut in the agar surface.
• If the medicament is bactericidal the agar plate is previously seeded
with a suitable organism like Staphylococcus aureus.
• After a suitable period of incubation, the zone of inhibition is
measured and correlated with the rate of release.
5. TEST OF RHEOLOGICAL PROPERTIES
• The viscosity of the preparation should be such that the product can
be easily removed from the container and easily applied to the skin.
• Using cone and plate viscometer the viscosity of the preparation is
determined.
6. TEST OF CONTENT UNIFORMITY
• The net weight of contents of ten filled ointment containers is
determined.
• The results should match each other and with the labeled quantity.
• This test is also called minimum fill test.
7. TEST OF PRESERVATIVE EFFICACY
• Using pour plate technique, the number of micro-organisms initially
present in the preparation are determined.
100
• This procedure is tedious and not followed anymore.
• Using flow-through diffusion cell or micro-dialysis method, the rate of
penetration of the preparation can be estimated. Animal or human
skin of definite area should be collected and tied to the holder
present in a diffusion cell.
• The diffusion cell is placed in a fluid bath. Measured quantity of the
preparation is applied over the skin and the amount of drug passed
into the fluid is measured at regular intervals by analyzing the
aliquots of fluid using a spectrophotometer.
4. TEST OF RATE OF DRUG RELEASE
• A clean test tube is taken and the internal surface is coated with the
preparation as a thin layer. Saline or serum is poured into the test
tube. After a certain period of time, the saline is analyzed for the
quantity of the drug. The amount of drug when divided by the time
period gives the rate of drug release.
• To assess the rate of release of medicament, small amount of the
ointment can be placed on the surface of nutrient agar contained in a
petri dish or alternately in a small cup cut in the agar surface.
• If the medicament is bactericidal the agar plate is previously seeded
with a suitable organism like Staphylococcus aureus.
• After a suitable period of incubation, the zone of inhibition is
measured and correlated with the rate of release.
5. TEST OF RHEOLOGICAL PROPERTIES
• The viscosity of the preparation should be such that the product can
be easily removed from the container and easily applied to the skin.
• Using cone and plate viscometer the viscosity of the preparation is
determined.
6. TEST OF CONTENT UNIFORMITY
• The net weight of contents of ten filled ointment containers is
determined.
• The results should match each other and with the labeled quantity.
• This test is also called minimum fill test.
7. TEST OF PRESERVATIVE EFFICACY
• Using pour plate technique, the number of micro-organisms initially
present in the preparation are determined.
100
GM Hamad
• Solutions of different samples of the preparation are made and mixed
with Tryptone Azolectin (TAT) broth separately.
• All cultures of the micro-organisms are added into each mixture,
under aseptic conditions. All mixtures are incubated.
• The number of micro-organisms in each sample are counted on 7
th
,
14
th
,21
st
, and 28
th
days of inoculation.
MICROBIAL LIMITS
• On 14
th
day, the number of vegetative cells should not be more than
0.1% of initial concentration.
• On 28
th
day, the number of organisms should be below or equal to
initial concentration.
8. TEST OF MICROBIAL CONTENT
• Micro-organisms like pseudomonas aeruginosa and staphylococcus
aureus may contaminate the preparation and finally infect the skin. So,
ointments should be tested for the absence of such micro-organisms.
• Solutions of different samples of the preparation are made. Each sample
is inoculated into separate volumes of 0.5 ml of rabbit's plasma under
aseptic conditions and incubated at 37℃ for 1-4 hours. No formation of
the clot in the incubated mass indicates the absence of the micro-
organisms.
DETERMINATION OF ASH CONTENT
INTRODUCTION
• Ash refers to the inorganic residue remaining after either ignition or
complete oxidation of organic matter in a food sample.
• The analysis of ash content in foods is simply the burning away of
organic content, leaving inorganic minerals.
• This helps to determine the amount and type of minerals in food.
IMPORTANCE
• It is important because the amount of minerals can determine:
Physiochemical properties of foods
As well as retard the growth of microorganisms.
101
• Solutions of different samples of the preparation are made and mixed
with Tryptone Azolectin (TAT) broth separately.
• All cultures of the micro-organisms are added into each mixture,
under aseptic conditions. All mixtures are incubated.
• The number of micro-organisms in each sample are counted on 7
th
,
14
th
,21
st
, and 28
th
days of inoculation.
MICROBIAL LIMITS
• On 14
th
day, the number of vegetative cells should not be more than
0.1% of initial concentration.
• On 28
th
day, the number of organisms should be below or equal to
initial concentration.
8. TEST OF MICROBIAL CONTENT
• Micro-organisms like pseudomonas aeruginosa and staphylococcus
aureus may contaminate the preparation and finally infect the skin. So,
ointments should be tested for the absence of such micro-organisms.
• Solutions of different samples of the preparation are made. Each sample
is inoculated into separate volumes of 0.5 ml of rabbit's plasma under
aseptic conditions and incubated at 37℃ for 1-4 hours. No formation of
the clot in the incubated mass indicates the absence of the micro-
organisms.
DETERMINATION OF ASH CONTENT
INTRODUCTION
• Ash refers to the inorganic residue remaining after either ignition or
complete oxidation of organic matter in a food sample.
• The analysis of ash content in foods is simply the burning away of
organic content, leaving inorganic minerals.
• This helps to determine the amount and type of minerals in food.
IMPORTANCE
• It is important because the amount of minerals can determine:
Physiochemical properties of foods
As well as retard the growth of microorganisms.
101
GM Hamad
ASH CONTENT DETERMINATION
• Ash content determination can be done in four following ways:
General ash determination
Acid insoluble ash
Water soluble ash
Sulphate ash determination
1. GENERAL ASH DETERMINATION TEST FOR CRUDE DRUGS
• Incinerate (reduce to ash) 2 – 3 grams of the ground drug in a tared
platinum or silica dish at a temperature not exceeding 450℃ until
free from carbon.
• Cool and weigh. If carbon free ash cannot be obtained in this way,
exhaust the charred mass with hot water.
• Collect the residue on the ash-less filter paper. Incinerate the residue
and filter paper. Add the filtrate, evaporate to dryness and ignite at a
temperature not exceeding 450℃.
• Calculate the %age of ash with reference to air dried drug.
%age of ash=
weight of ash
weight of air dried drug
×100
2. ACID INSOLUBLE ASH
• Use above method, unless otherwise indicated in the monograph.
• Boil the ash (obtained in above method) for 5 min with 25ml of 2M
HCl.
• Collect the insoluble matter in a sintered glass crucible or on an ash-
less filter paper. Wash with hot water and ignite.
• Calculate the percentage of acid insoluble ash with reference to the
air-dried drug.
%��� �� ��ℎ=
??????�??????�ℎ� �� ��ℎ
??????�??????�ℎ� �� �??????� ��??????�� ����
×100
3. WATER SOLUBLE ASH
• Boil the ash (obtained as treated above) for 5 minutes with 25ml of
water.
• Collect the insoluble matter in a crucible or on ash-less filter paper.
• Wash with hot water and ignite for 15 minutes at a temperature not
exceeding 450℃.
• Subtract the weight of insoluble matter from the weight of ash.
102
ASH CONTENT DETERMINATION
• Ash content determination can be done in four following ways:
General ash determination
Acid insoluble ash
Water soluble ash
Sulphate ash determination
1. GENERAL ASH DETERMINATION TEST FOR CRUDE DRUGS
• Incinerate (reduce to ash) 2 – 3 grams of the ground drug in a tared
platinum or silica dish at a temperature not exceeding 450℃ until
free from carbon.
• Cool and weigh. If carbon free ash cannot be obtained in this way,
exhaust the charred mass with hot water.
• Collect the residue on the ash-less filter paper. Incinerate the residue
and filter paper. Add the filtrate, evaporate to dryness and ignite at a
temperature not exceeding 450℃.
• Calculate the %age of ash with reference to air dried drug.
%age of ash=
weight of ash
weight of air dried drug
×100
2. ACID INSOLUBLE ASH
• Use above method, unless otherwise indicated in the monograph.
• Boil the ash (obtained in above method) for 5 min with 25ml of 2M
HCl.
• Collect the insoluble matter in a sintered glass crucible or on an ash-
less filter paper. Wash with hot water and ignite.
• Calculate the percentage of acid insoluble ash with reference to the
air-dried drug.
%��� �� ��ℎ=
??????�??????�ℎ� �� ��ℎ
??????�??????�ℎ� �� �??????� ��??????�� ����
×100
3. WATER SOLUBLE ASH
• Boil the ash (obtained as treated above) for 5 minutes with 25ml of
water.
• Collect the insoluble matter in a crucible or on ash-less filter paper.
• Wash with hot water and ignite for 15 minutes at a temperature not
exceeding 450℃.
• Subtract the weight of insoluble matter from the weight of ash.
102
GM Hamad
• The difference in weight represents the water-soluble ash.
• Calculate the percentage of water-soluble ash with reference to air
dried drug.
??????���� ������� ��ℎ =??????�??????�ℎ� �� ??????�������� ������ − ??????�??????�ℎ� �� ��ℎ
4. SULPHATED ASH DETERMINATION
• Use the method unless otherwise given in monograph.
• Heat the platinum dish to redness for 10 min.
• Allow to cool in a desiccator and weigh.
• Place one gram of the substance being examined, in the dish,
moisten with H2SO4 and ignite generally and gently.
• Again. moisten with H2SO4 and ignite at about 800℃.
• Cool and weigh again.
• Ignite for 15 min and repeat the procedure until two successive
weighing do not differ by more than 0.5mg.
• Calculate the %age of sulphated ash with reference to air dried drug.
DETERMINATION OF ALKALINITY OF GLASS
INTRODUCTION
• This test determines the resistance of new glass to water attack. The
degree of attack is determined by amount of alkali released (which is
usually very small) under the influence of attacking medium under
specified conditions.
ALKALINITY
• Alkalinity is a measure of the ability of a solution to neutralize acids to
the equivalence point of carbonate or bicarbonate.
REASON OF ALKALINITY OF GLASS
• In glass, the sodium and potassium oxides are hygroscopic; therefore,
the surface of the glass absorbs moisture from the air. The absorbed
moisture and exposure to carbon dioxide causes the NaO2 or NaOH and
KO2 or KOH to convert to sodium or potassium carbonate.
• In water, especially salt water, the Na and K carbonates in unstable glass
may leach out, leaving only fragile, porous hydrated silica (SiO2)
103
• The difference in weight represents the water-soluble ash.
• Calculate the percentage of water-soluble ash with reference to air
dried drug.
??????���� ������� ��ℎ =??????�??????�ℎ� �� ??????�������� ������ − ??????�??????�ℎ� �� ��ℎ
4. SULPHATED ASH DETERMINATION
• Use the method unless otherwise given in monograph.
• Heat the platinum dish to redness for 10 min.
• Allow to cool in a desiccator and weigh.
• Place one gram of the substance being examined, in the dish,
moisten with H2SO4 and ignite generally and gently.
• Again. moisten with H2SO4 and ignite at about 800℃.
• Cool and weigh again.
• Ignite for 15 min and repeat the procedure until two successive
weighing do not differ by more than 0.5mg.
• Calculate the %age of sulphated ash with reference to air dried drug.
DETERMINATION OF ALKALINITY OF GLASS
INTRODUCTION
• This test determines the resistance of new glass to water attack. The
degree of attack is determined by amount of alkali released (which is
usually very small) under the influence of attacking medium under
specified conditions.
ALKALINITY
• Alkalinity is a measure of the ability of a solution to neutralize acids to
the equivalence point of carbonate or bicarbonate.
REASON OF ALKALINITY OF GLASS
• In glass, the sodium and potassium oxides are hygroscopic; therefore,
the surface of the glass absorbs moisture from the air. The absorbed
moisture and exposure to carbon dioxide causes the NaO2 or NaOH and
KO2 or KOH to convert to sodium or potassium carbonate.
• In water, especially salt water, the Na and K carbonates in unstable glass
may leach out, leaving only fragile, porous hydrated silica (SiO2)
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network. This causes the glass to crack, flake and frosty appearance.
TYPES OF GLASS
• USP classifies glass in to 4 types according to the amount of alkali
released from the glass when attacked by water under specified
conditions, which are:
Type1 glass: Neutral or borosilicate glass.
Type 2 glass: Soda glass with a surface treatment.
Type 3 glass: Soda glass of limited alkalinity.
Type 4 glass: European Type 4 or NP glass.
TESTS FOR DETERMINATION OF GLASS ALKALINITY
• Following are tests which are used for the determination of glass
alkalinity:
Powdered glass test
Water attack test
APPARATUS FOR TESTS
• Autoclave: having thermometer, pressure gauge, vent cock and rack for
at least 12 containers.
• Mortar & pestle: Use a hardened – steel mortar and pestle.
• Other equipment: including sieves of various mesh with pan and cover,
conical flasks, hammer, magnet and volumetric apparatus.
REAGENTS FOR TESTS
I. SPECIAL DISILLED WATER
• This type of water is prepared by redistilling once distilled water.
• Prior to distillation add to each 1000ml of water, 1 drop of phosphoric
acid, previously diluted with equal volume of water then boiled to point
of appearance of dense fumes.
• Reject first 10-15% of distilled water and retain the next 70%.
II. METHYL RED SOLUTION
• Dissolve 24 mg of methyl red sodium in purified water to make 100ml.
• Add 1 drop 0.02 N NaOH and check sensitivity of acid, methyl red
solution.
• Color changes from red to yellow.
• Now add 1 drop of 0.02 N H2SO4 so to rechange color from yellow to
red.
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network. This causes the glass to crack, flake and frosty appearance.
TYPES OF GLASS
• USP classifies glass in to 4 types according to the amount of alkali
released from the glass when attacked by water under specified
conditions, which are:
Type1 glass: Neutral or borosilicate glass.
Type 2 glass: Soda glass with a surface treatment.
Type 3 glass: Soda glass of limited alkalinity.
Type 4 glass: European Type 4 or NP glass.
TESTS FOR DETERMINATION OF GLASS ALKALINITY
• Following are tests which are used for the determination of glass
alkalinity:
Powdered glass test
Water attack test
APPARATUS FOR TESTS
• Autoclave: having thermometer, pressure gauge, vent cock and rack for
at least 12 containers.
• Mortar & pestle: Use a hardened – steel mortar and pestle.
• Other equipment: including sieves of various mesh with pan and cover,
conical flasks, hammer, magnet and volumetric apparatus.
REAGENTS FOR TESTS
I. SPECIAL DISILLED WATER
• This type of water is prepared by redistilling once distilled water.
• Prior to distillation add to each 1000ml of water, 1 drop of phosphoric
acid, previously diluted with equal volume of water then boiled to point
of appearance of dense fumes.
• Reject first 10-15% of distilled water and retain the next 70%.
II. METHYL RED SOLUTION
• Dissolve 24 mg of methyl red sodium in purified water to make 100ml.
• Add 1 drop 0.02 N NaOH and check sensitivity of acid, methyl red
solution.
• Color changes from red to yellow.
• Now add 1 drop of 0.02 N H2SO4 so to rechange color from yellow to
red.
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POWDERED GLASS TEST
• The various steps involved are mentioned below;
STEP I – PREPARATION OF SAMPLE
• Take 6 or more containers, rinse thoroughly with purified water, and dry
them with dry air.
• Crush them into pieces of about 25mm.
• Divide 100g of this into 3 portions, place one portion in mortar and crush
with pestle.
• Nest the sieves and empty the mortar into No. 20 sieve.
• Repeat the operation with 2 remaining portions.
• Shake the sieve and remove the glass from No. 20 and 40 sieve and
crush it again.
• Shake for 5 minutes, take the portion retained on No. 50 sieve.
STEP II – REMOVING OF IMPURITIES
• Take 10g of sample, spread on glazed paper and pass magnet through it.
• Transfer it to 250ml conical flask and wash with six 30ml portions of
acetone swirling each time and decanting.
• Dry the flask and contents for 20 minutes at 140℃.
• Transfer to desiccator, use within 48 hours.
STEP III – PROCEDURE (WATER TREATMENT)
• Transfer 10g prepared sample to a 250ml conical flask.
• Add 50ml of high purity water to this flask and likewise prepare a blank.
• Cap all flasks with BS beakers to fit snugly.
• Autoclave it at 121℃ for 30 min.
• Cool the flasks at once in running water.
• Decant the water in clean vessels, wash the residual glass with four 1ml
portions of HPW and adding the decanted washing to main portion.
STEP IV – TITRATION
• Add 5 drops of indicator and titrate immediately with 0.02N sulfuric
acid.
• Note the volume of acid used.
• Compare the results as in table.
WATER ATTACK TEST
• Take 3 or more containers, rinse them thoroughly with HPW.
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POWDERED GLASS TEST
• The various steps involved are mentioned below;
STEP I – PREPARATION OF SAMPLE
• Take 6 or more containers, rinse thoroughly with purified water, and dry
them with dry air.
• Crush them into pieces of about 25mm.
• Divide 100g of this into 3 portions, place one portion in mortar and crush
with pestle.
• Nest the sieves and empty the mortar into No. 20 sieve.
• Repeat the operation with 2 remaining portions.
• Shake the sieve and remove the glass from No. 20 and 40 sieve and
crush it again.
• Shake for 5 minutes, take the portion retained on No. 50 sieve.
STEP II – REMOVING OF IMPURITIES
• Take 10g of sample, spread on glazed paper and pass magnet through it.
• Transfer it to 250ml conical flask and wash with six 30ml portions of
acetone swirling each time and decanting.
• Dry the flask and contents for 20 minutes at 140℃.
• Transfer to desiccator, use within 48 hours.
STEP III – PROCEDURE (WATER TREATMENT)
• Transfer 10g prepared sample to a 250ml conical flask.
• Add 50ml of high purity water to this flask and likewise prepare a blank.
• Cap all flasks with BS beakers to fit snugly.
• Autoclave it at 121℃ for 30 min.
• Cool the flasks at once in running water.
• Decant the water in clean vessels, wash the residual glass with four 1ml
portions of HPW and adding the decanted washing to main portion.
STEP IV – TITRATION
• Add 5 drops of indicator and titrate immediately with 0.02N sulfuric
acid.
• Note the volume of acid used.
• Compare the results as in table.
WATER ATTACK TEST
• Take 3 or more containers, rinse them thoroughly with HPW.
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• Fill each container to 90% of its capacity, cap as previously and autoclave
similarly.
• Empty the contents in 100ml cylinder, combine the contents if required
to get 100ml volume.
• Transfer it to 250ml conical flask, add indicator and titrate against 0.02N
sulphuric acid.
• Note the volume used corrected by titrating with 100ml HPW as blank.
TEST LIMITS
Type of glass Type of test Size Vol. of sulphuric acid
Type I – highly
resistant, BS glass
Powdered glass test All 1.0 ml
Type II – Treated
soda lime glass
water attack
≤100ml
≥100ml
0.7 ml
0.2 ml
Type III – soda-
lime glass
powdered glass test All 8.5 ml
Type IV – NP glass powdered glass test All 15.0 ml
TOXICITY TESTING
INTRODUCTION
TOXICITY
• It means the presence of toxic substances or toxins in the preparation.
• Toxicity can also be defined as “The symptoms of disease produced by
the injection of any specific bacterial toxoid.”
TOXICITY TESTING
• Toxicity testing refers to “the quality of being poisonous or presence of
toxins.”
• To determine the presence of toxins in the preparation, the toxicity tests
are carried out.
• Toxicity test is performed for all vaccines, their containers and stoppers.
TOXINS
• A poison especially protein or conjugated protein produced by higher
plants, some animals and pathogenic bacteria i.e. highly poisonous for
other living microorganisms.
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• Fill each container to 90% of its capacity, cap as previously and autoclave
similarly.
• Empty the contents in 100ml cylinder, combine the contents if required
to get 100ml volume.
• Transfer it to 250ml conical flask, add indicator and titrate against 0.02N
sulphuric acid.
• Note the volume used corrected by titrating with 100ml HPW as blank.
TEST LIMITS
Type of glass Type of test Size Vol. of sulphuric acid
Type I – highly
resistant, BS glass
Powdered glass test All 1.0 ml
Type II – Treated
soda lime glass
water attack
≤100ml
≥100ml
0.7 ml
0.2 ml
Type III – soda-
lime glass
powdered glass test All 8.5 ml
Type IV – NP glass powdered glass test All 15.0 ml
TOXICITY TESTING
INTRODUCTION
TOXICITY
• It means the presence of toxic substances or toxins in the preparation.
• Toxicity can also be defined as “The symptoms of disease produced by
the injection of any specific bacterial toxoid.”
TOXICITY TESTING
• Toxicity testing refers to “the quality of being poisonous or presence of
toxins.”
• To determine the presence of toxins in the preparation, the toxicity tests
are carried out.
• Toxicity test is performed for all vaccines, their containers and stoppers.
TOXINS
• A poison especially protein or conjugated protein produced by higher
plants, some animals and pathogenic bacteria i.e. highly poisonous for
other living microorganisms.
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TOXOID
• A toxin treated by heat or chemical agent to destroy its deleterious
properties without destroying its ability to stimulate antibody
production.
TOXICITY TEST FOR DIPHTHERIA TOXOID
INTRODUCTION
• It is a sterile product of formaldehyde-treated product of the growth of
“Corynebacterium diphtheria" used as an active immunizing agent.
USP TEST
• Four guinea pigs each weighing 300g to 400g.
• Injecting S/C a dose with a volume 5 times to that of intended human
immunizing dose to each of these guinea pigs.
• Dose must not be less than 2 ml.
FINDINGS/RESULT
• No local or general symptoms of diphtheria toxin should appear within
30 days.
TOXICITY TEST FOR TETANUS TOXOID
• Inject SC 4 healthy guinea pigs (300-400g) with a TT that is at least 5
times the human immunizing dose (not less than 2ml).
• Check the symptoms of tetanus toxin poisoning after 21 days.
TOXICITY TEST FOR RUBBER CLOSURES
• Two Tests for extracts of Rubber Closures.
I. AN ACUTE SYSTEMIC TOXICITY TEST
• Mice as test animal.
• I/P and IV injections are given.
• Mortality or grass toxicity following the injection if appears, then “the
closures fail the toxicity test”.
II. AN INTRACUTANEOUS REACTIVITY TEST
• Albino rabbits are used as test animals.
• Intracutaneous injection is given.
• At the site of injection, no sign of tissue reaction i. e. no redness or no
necrosis.
• If redness or necrosis appears, then the test fails.
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TOXOID
• A toxin treated by heat or chemical agent to destroy its deleterious
properties without destroying its ability to stimulate antibody
production.
TOXICITY TEST FOR DIPHTHERIA TOXOID
INTRODUCTION
• It is a sterile product of formaldehyde-treated product of the growth of
“Corynebacterium diphtheria" used as an active immunizing agent.
USP TEST
• Four guinea pigs each weighing 300g to 400g.
• Injecting S/C a dose with a volume 5 times to that of intended human
immunizing dose to each of these guinea pigs.
• Dose must not be less than 2 ml.
FINDINGS/RESULT
• No local or general symptoms of diphtheria toxin should appear within
30 days.
TOXICITY TEST FOR TETANUS TOXOID
• Inject SC 4 healthy guinea pigs (300-400g) with a TT that is at least 5
times the human immunizing dose (not less than 2ml).
• Check the symptoms of tetanus toxin poisoning after 21 days.
TOXICITY TEST FOR RUBBER CLOSURES
• Two Tests for extracts of Rubber Closures.
I. AN ACUTE SYSTEMIC TOXICITY TEST
• Mice as test animal.
• I/P and IV injections are given.
• Mortality or grass toxicity following the injection if appears, then “the
closures fail the toxicity test”.
II. AN INTRACUTANEOUS REACTIVITY TEST
• Albino rabbits are used as test animals.
• Intracutaneous injection is given.
• At the site of injection, no sign of tissue reaction i. e. no redness or no
necrosis.
• If redness or necrosis appears, then the test fails.
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TOXICITY TEST FOR PLASTIC CONTAINERS
INTRODUCTION
• Plastics are majorly involved in the packing and packaging of the
pharmaceuticals, but these plastics sometimes leads to toxic
manifestations. Therefore, their toxicity is evaluated to avoid hazards.
PROCEDURE
• The toxicity of the plastic containers can be checked by the following
procedure:
Extract of plastic containers used for parenteral preparations is
prepared.
The extract is prepared in such a way that, the plastic is cut into
pieces and boiled in the water.
The water is collected and used for texting.
The extract is injected into the mice.
Its IV injection to mice determine its gross toxicity and mortality.
Alternatively, we give IC into dorsal shaved skin of rabbit or the
standardized plastic strips are implanted.
The local reaction is observed for toxicity.
TOTAL SOLIDS
INTRODUCTION
• The term total solids is applied to the residue obtained when the
prescribed amount of the preparation is dried to constant weight under
the conditions specified.
• Total solids usually include extractives, both soluble in alcoholic or
hydroalcoholic preparations as tinctures and extracts.
APPARATUS
• The apparatus used is a hollow flat bottom, flanged dish 75mm in
diameter and 25mm deep. It is made up of nickel or other metal.
PROCEDURE
• Place an accurately measured weight or quantity of the preparation
after thorough shaking, say 10 ml in a tarred dish.
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TOXICITY TEST FOR PLASTIC CONTAINERS
INTRODUCTION
• Plastics are majorly involved in the packing and packaging of the
pharmaceuticals, but these plastics sometimes leads to toxic
manifestations. Therefore, their toxicity is evaluated to avoid hazards.
PROCEDURE
• The toxicity of the plastic containers can be checked by the following
procedure:
Extract of plastic containers used for parenteral preparations is
prepared.
The extract is prepared in such a way that, the plastic is cut into
pieces and boiled in the water.
The water is collected and used for texting.
The extract is injected into the mice.
Its IV injection to mice determine its gross toxicity and mortality.
Alternatively, we give IC into dorsal shaved skin of rabbit or the
standardized plastic strips are implanted.
The local reaction is observed for toxicity.
TOTAL SOLIDS
INTRODUCTION
• The term total solids is applied to the residue obtained when the
prescribed amount of the preparation is dried to constant weight under
the conditions specified.
• Total solids usually include extractives, both soluble in alcoholic or
hydroalcoholic preparations as tinctures and extracts.
APPARATUS
• The apparatus used is a hollow flat bottom, flanged dish 75mm in
diameter and 25mm deep. It is made up of nickel or other metal.
PROCEDURE
• Place an accurately measured weight or quantity of the preparation
after thorough shaking, say 10 ml in a tarred dish.
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• Evaporate at as low a temperature as possible until the alcohol is
removed, and heat on a water bath until the residue is apparently dry.
• Transfer to oven and dry to a constant weight at 105℃ or dry in
desiccator.
• Weigh the dish containing residue.
• Difference in two weights is the amount of total solid per 10ml.
• Determine total solid as g/100ml.
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• Evaporate at as low a temperature as possible until the alcohol is
removed, and heat on a water bath until the residue is apparently dry.
• Transfer to oven and dry to a constant weight at 105℃ or dry in
desiccator.
• Weigh the dish containing residue.
• Difference in two weights is the amount of total solid per 10ml.
• Determine total solid as g/100ml.
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STANDARDIZATION OF PHARMACEUTICALS
GOOD MANUFACTURING PRACTICE
• GMP is that part of Quality Assurance which ensures that the products
are consistently manufactured and controlled to the quality standards
appropriate for their intended use.
• A set of principles and procedures which, when followed by
manufacturers for therapeutic goods, helps to ensure that the products
manufactured will have the required quality.
• A basic tenet of GMP is that quality cannot be tested into a batch of
product but must be built into each batch of product during all stages of
the manufacturing process.
• It is designed to minimize the risks involved in any pharmaceutical
production that cannot be eliminated through testing the final product.
• Some of the main risks are:
Unexpected contamination of products, causing damage to health
or even death.
Incorrect labels on containers, which could mean that patients
receive the wrong medicine.
Insufficient or too much active ingredient, resulting in ineffective
treatment or adverse effects.
ASPECTS COVERED BY GMP
• All aspects of production; from the starting materials, premises and
equipment to the training and personal hygiene of staff.
• Detailed, written procedures are essential for each process that could
affect the quality of the finished product.
• There must be systems to provide documented proof that correct
procedures are consistently followed at each step in the manufacturing
process - every time a product is made.
GMP GUIDELINES
• GMP guidelines are provided by following authorities:
GMP as per World Health Organization (WHO)
GMP as per Drug Regulatory Authority Pakistan (DRAP)
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STANDARDIZATION OF PHARMACEUTICALS
GOOD MANUFACTURING PRACTICE
• GMP is that part of Quality Assurance which ensures that the products
are consistently manufactured and controlled to the quality standards
appropriate for their intended use.
• A set of principles and procedures which, when followed by
manufacturers for therapeutic goods, helps to ensure that the products
manufactured will have the required quality.
• A basic tenet of GMP is that quality cannot be tested into a batch of
product but must be built into each batch of product during all stages of
the manufacturing process.
• It is designed to minimize the risks involved in any pharmaceutical
production that cannot be eliminated through testing the final product.
• Some of the main risks are:
Unexpected contamination of products, causing damage to health
or even death.
Incorrect labels on containers, which could mean that patients
receive the wrong medicine.
Insufficient or too much active ingredient, resulting in ineffective
treatment or adverse effects.
ASPECTS COVERED BY GMP
• All aspects of production; from the starting materials, premises and
equipment to the training and personal hygiene of staff.
• Detailed, written procedures are essential for each process that could
affect the quality of the finished product.
• There must be systems to provide documented proof that correct
procedures are consistently followed at each step in the manufacturing
process - every time a product is made.
GMP GUIDELINES
• GMP guidelines are provided by following authorities:
GMP as per World Health Organization (WHO)
GMP as per Drug Regulatory Authority Pakistan (DRAP)
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GMP as per Therapeutic Good Affairs (TGA)
GMP as per US Food and Drug Administration (FDA)
GMP as per Ministry of Health and Regulatory Affairs (MHRA)
GMP as per International Conference on Harmonization (ICH)
GMP IN PHARMACEUTICAL INDUSTRIES
• GMP in solid dosage forms
• GMP in semisolid dosage forms
• GMP in liquid orals
• GMP in parenteral production
• GMP in ayurvedic medicines
• GMP in bio technological products
• GMP in nutraceuticals and cosmeceuticals
PRINCIPLES OF GMP
1. Design and construct the facilities and equipment properly.
2. Follow written procedures and Instructions.
3. Document work.
4. Validate work.
5. Monitor facilities and equipment.
6. Write step by step operating procedures and work on instructions.
7. Design, develop and demonstrate job competence.
8. Protect against contamination.
9. Control components and product related processes.
10. Conduct planned and periodic audits.
IMPORTANT DOCUMENTS IN GMP
• Policies
• SOP (Standard Operating Procedure)
• Specifications
• MFR (Master Formula Record)
• BMR (Batch Manufacturing Record)
• Manuals
• Master plans / files
• Validation protocols
• Forms and Formats
• Records
111
GMP as per Therapeutic Good Affairs (TGA)
GMP as per US Food and Drug Administration (FDA)
GMP as per Ministry of Health and Regulatory Affairs (MHRA)
GMP as per International Conference on Harmonization (ICH)
GMP IN PHARMACEUTICAL INDUSTRIES
• GMP in solid dosage forms
• GMP in semisolid dosage forms
• GMP in liquid orals
• GMP in parenteral production
• GMP in ayurvedic medicines
• GMP in bio technological products
• GMP in nutraceuticals and cosmeceuticals
PRINCIPLES OF GMP
1. Design and construct the facilities and equipment properly.
2. Follow written procedures and Instructions.
3. Document work.
4. Validate work.
5. Monitor facilities and equipment.
6. Write step by step operating procedures and work on instructions.
7. Design, develop and demonstrate job competence.
8. Protect against contamination.
9. Control components and product related processes.
10. Conduct planned and periodic audits.
IMPORTANT DOCUMENTS IN GMP
• Policies
• SOP (Standard Operating Procedure)
• Specifications
• MFR (Master Formula Record)
• BMR (Batch Manufacturing Record)
• Manuals
• Master plans / files
• Validation protocols
• Forms and Formats
• Records
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API MANUFACTURING PROCESS
SECONDARY MANUFACTURING PROCESS – TABLETS
SECONDARY MANUFACTURING PROCESS – STERILE PARENTERAL FOR
INJECTION
Introduction of the
API Starting material
Production of
Intermediates
Isolation and
Purification
Physical Processing
and Packaging
Raw Material A
Raw Material B
Reactor Filtration
Crystallization
Centrifuge
Drying Milling
Packaging API
Introduction of the
API and Excipients
Compounding Creation of Dosage
Form
Primary Packaging
API
Excipients
Mixing
Granulation
Drying
Pressing
Coating Blister Pack
112
API MANUFACTURING PROCESS
SECONDARY MANUFACTURING PROCESS – TABLETS
SECONDARY MANUFACTURING PROCESS – STERILE PARENTERAL FOR
INJECTION
Introduction of the
API Starting material
Production of
Intermediates
Isolation and
Purification
Physical Processing
and Packaging
Raw Material A
Raw Material B
Reactor Filtration
Crystallization
Centrifuge
Drying Milling
Packaging API
Introduction of the
API and Excipients
Compounding Creation of Dosage
Form
Primary Packaging
API
Excipients
Mixing
Granulation
Drying
Pressing
Coating Blister Pack
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BIOTECHNOLOGY MANUFACTURING PROCESS
CURRENT GOOD MANUFACTURING (cGMP)
• cGMP refers to the Current Good Manufacturing Practice regulations
enforced by the US Food and Drug Administration (FDA).
• cGMP provide for systems that assure proper design, monitoring and
control of manufacturing processes and facilities.
• Adherence to the cGMP regulations assures the identity, strength,
Introduction of the
API and Excipients
Compounding Creation of Dosage
Form / Primary
Packaging
API
Excipient
s
Dissolution
0.22 Micron
Filter
Vial Filling
Lyophilization
(freeze drying)
Sterile Stopped
Vial
Shipped and stored in this
form then reconstituted
prior to injection
Introduction of the
Starting material
Production of
Intermediates
Isolation and
Purification
Physical Processing
and Packing
Seed Cells from
Mater cell
Culture
Fermenter Filtration
Centrifuge
Purification
Evaporation
Solvent wash
Freeze Drying
Packaging
API
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BIOTECHNOLOGY MANUFACTURING PROCESS
CURRENT GOOD MANUFACTURING (cGMP)
• cGMP refers to the Current Good Manufacturing Practice regulations
enforced by the US Food and Drug Administration (FDA).
• cGMP provide for systems that assure proper design, monitoring and
control of manufacturing processes and facilities.
• Adherence to the cGMP regulations assures the identity, strength,
Introduction of the
API and Excipients
Compounding Creation of Dosage
Form / Primary
Packaging
API
Excipient
s
Dissolution
0.22 Micron
Filter
Vial Filling
Lyophilization
(freeze drying)
Sterile Stopped
Vial
Shipped and stored in this
form then reconstituted
prior to injection
Introduction of the
Starting material
Production of
Intermediates
Isolation and
Purification
Physical Processing
and Packing
Seed Cells from
Mater cell
Culture
Fermenter Filtration
Centrifuge
Purification
Evaporation
Solvent wash
Freeze Drying
Packaging
API
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quality and purity of drug products by requiring that manufacturers of
medications adequately control manufacturing operations.
IMPORTANCE OF cGMP
• A consumer usually cannot detect (through smell, touch, or sight) that a
drug product is safe or if it will work.
• While cGMPs require testing, testing alone is not adequate to ensure
quality.
• In most instances testing is done on a small sample of a batch (for
example, a drug manufacturer may test 1000 tablets from a batch that
contains 2 million tablets), so that most of the batch can be used for
patients rather than destroyed by testing.
PACKAGING
PRIMARY PACKAGING
• Primary packaging is used to form a container for the product and which
is in direct contact with the product (sterile vial, medicine bottle, tablet
blister pack)
EXAMPLE PRIMARY PACKAGING – TABLETS
SECONDARY PACKAGING
• Secondary packaging is any subsequent packaging which helps to inform
about display and protect the product. It includes all required labelling
and information leaflets.
EXAMPLE SECONDARY PACKAGING – TABLETS
TERTIARY PACKAGING
• Tertiary packaging is any final packaging grouping the products
Fill blister
pack with
tablets
Check
Seal
blister
packs
Print
batch no.
/ Expiry
no.
Separate
packs
Tablet in
blister pack
Cut carton
net
Fill carton
Information
leaflet
Weight to
check carton
is correctly
filled
Print batch
no. / Expiry
Close
carton
Fold net in
carton
114
quality and purity of drug products by requiring that manufacturers of
medications adequately control manufacturing operations.
IMPORTANCE OF cGMP
• A consumer usually cannot detect (through smell, touch, or sight) that a
drug product is safe or if it will work.
• While cGMPs require testing, testing alone is not adequate to ensure
quality.
• In most instances testing is done on a small sample of a batch (for
example, a drug manufacturer may test 1000 tablets from a batch that
contains 2 million tablets), so that most of the batch can be used for
patients rather than destroyed by testing.
PACKAGING
PRIMARY PACKAGING
• Primary packaging is used to form a container for the product and which
is in direct contact with the product (sterile vial, medicine bottle, tablet
blister pack)
EXAMPLE PRIMARY PACKAGING – TABLETS
SECONDARY PACKAGING
• Secondary packaging is any subsequent packaging which helps to inform
about display and protect the product. It includes all required labelling
and information leaflets.
EXAMPLE SECONDARY PACKAGING – TABLETS
TERTIARY PACKAGING
• Tertiary packaging is any final packaging grouping the products
Fill blister
pack with
tablets
Check
Seal
blister
packs
batch no.
/ Expiry
no.
Separate
packs
Tablet in
blister pack
Cut carton
net
Fill carton
Information
leaflet
Weight to
check carton
is correctly
filled
Print batch
no. / Expiry
Close
carton
Fold net in
carton
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for storage and transportation.
EXAMPLE TERTIARY PACKAGING – TABLETS
PACKAGING AND HOLDING OF DRUGS
• Care shall be taken when using automatic tablet and capsule counting,
strip and blister packaging equipment to ensure that all ‘rogue’ tablets,
capsules or foils from packaging operation are removed before a new
packaging operation is commenced.
• There shall be an independent recorded check of the equipment before
a new batch of tablets or capsules is handled.
FINISHED PHARMACEUTICALS
• Appropriate specifications for finished products shall include:
The designated name of the product and the code reference.
The formula or a reference to the formula and the pharmacopeial
reference.
Directions for sampling and testing or a reference to procedures.
GENERAL PROVISIONS
• The processing of dry materials and products creates problems of dust
control and cross contamination. Special attention is therefore, needed
in the design, maintenance and use of premises and equipment in order
to overcome these problems. Wherever required, enclosed dust control
manufacturing systems shall be employed.
CURRENT GOOD MANUFACTURING PRACTICE REGULATIONS
1) ORGANIZATION AND PERSONNEL REQUIREMENTS
RESPONSIBILITIES OF QUALITY CONTROL DEPARTMENT
• The organization and personnel section of the regulation deals with the
responsibilities of the quality control unit, employees, and consultants.
• The regulations require that a quality control unit have the authority and
responsibility for all the functions that may affect product quality.
• This includes accepting or rejecting product components, product
specifications, finished products, packaging and labeling.
Cartons
Shrink wrap
in batches
of 20
Pack into
boxes
Stack onto
pallets
115
for storage and transportation.
EXAMPLE TERTIARY PACKAGING – TABLETS
PACKAGING AND HOLDING OF DRUGS
• Care shall be taken when using automatic tablet and capsule counting,
strip and blister packaging equipment to ensure that all ‘rogue’ tablets,
capsules or foils from packaging operation are removed before a new
packaging operation is commenced.
• There shall be an independent recorded check of the equipment before
a new batch of tablets or capsules is handled.
FINISHED PHARMACEUTICALS
• Appropriate specifications for finished products shall include:
The designated name of the product and the code reference.
The formula or a reference to the formula and the pharmacopeial
reference.
Directions for sampling and testing or a reference to procedures.
GENERAL PROVISIONS
• The processing of dry materials and products creates problems of dust
control and cross contamination. Special attention is therefore, needed
in the design, maintenance and use of premises and equipment in order
to overcome these problems. Wherever required, enclosed dust control
manufacturing systems shall be employed.
CURRENT GOOD MANUFACTURING PRACTICE REGULATIONS
1) ORGANIZATION AND PERSONNEL REQUIREMENTS
RESPONSIBILITIES OF QUALITY CONTROL DEPARTMENT
• The organization and personnel section of the regulation deals with the
responsibilities of the quality control unit, employees, and consultants.
• The regulations require that a quality control unit have the authority and
responsibility for all the functions that may affect product quality.
• This includes accepting or rejecting product components, product
specifications, finished products, packaging and labeling.
Cartons
Shrink wrap
in batches
of 20
Pack into
boxes
Stack onto
pallets
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• Adequate laboratory facilities shall be provided, written procedures
followed, and all records are maintained.
PERSONNEL QUALIFICATION AND RESPONSIBILITIES
• All personnel engaged in the manufacturing processing, packing or
holding of a drug product, including those in supervisory positions are
required to have the education, training or experience needed to fulfill
the assigned responsibility.
• Appropriate programs of skill development, continuing educations are
essential for maintaining quality assurance.
CONSULTANTS
• Any consultants advising on scientific and technical matters should
possess requisite qualifications for the tasks.
2) BUILDINGS AND FACILITIES
DESIGN AND CONSTRUCTION FEATURES
• The regulations of this section include the design, structural features,
and functional aspects of buildings and facilities.
• Each building’s structure, space, design, and placement of equipment
must be such to enable thorough cleaning, inspection, and safe and
effective use for the designated operations.
• Proper consideration must be given to such factors as water quality
standards, security, materials used for floors, walls and ceilings, lighting,
segregated quarantine areas for raw materials and product components
subject to quality control approval; holding areas for rejected
components, storage area for released components, weighing and
measuring rooms, flammable materials storage areas, finished product
storage, control of heat, humidity, temperature and ventilation, waste
handling, employee facilities and safety procedures in compliance with
occupational safety and health administration regulations and
procedures and practice of personal sanitation.
MAINTENANCE
• A log of building maintenances must be kept documenting this
component of the regulations.
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• Adequate laboratory facilities shall be provided, written procedures
followed, and all records are maintained.
PERSONNEL QUALIFICATION AND RESPONSIBILITIES
• All personnel engaged in the manufacturing processing, packing or
holding of a drug product, including those in supervisory positions are
required to have the education, training or experience needed to fulfill
the assigned responsibility.
• Appropriate programs of skill development, continuing educations are
essential for maintaining quality assurance.
CONSULTANTS
• Any consultants advising on scientific and technical matters should
possess requisite qualifications for the tasks.
2) BUILDINGS AND FACILITIES
DESIGN AND CONSTRUCTION FEATURES
• The regulations of this section include the design, structural features,
and functional aspects of buildings and facilities.
• Each building’s structure, space, design, and placement of equipment
must be such to enable thorough cleaning, inspection, and safe and
effective use for the designated operations.
• Proper consideration must be given to such factors as water quality
standards, security, materials used for floors, walls and ceilings, lighting,
segregated quarantine areas for raw materials and product components
subject to quality control approval; holding areas for rejected
components, storage area for released components, weighing and
measuring rooms, flammable materials storage areas, finished product
storage, control of heat, humidity, temperature and ventilation, waste
handling, employee facilities and safety procedures in compliance with
occupational safety and health administration regulations and
procedures and practice of personal sanitation.
MAINTENANCE
• A log of building maintenances must be kept documenting this
component of the regulations.
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3) EQUIPMENT
EQUIPMENT DESIGN, SIZE AND LOCATION
• Each piece of equipment must be of appropriate design and size and
suitably located to facilitate operations for its indented use, cleaning and
maintenance.
• The equipment’s surfaces and parts must not interact with the processes
or product’s components to alter the purity, strength or quality.
EQUIPMENT CLEANING AND MAINTENANCE
• Standard operating procedures must be written and followed for the
proper use, maintenance, and cleaning of each piece of equipment, and
appropriate logs and records must be kept.
AUTOMATIC AND ELECTRICAL EQUIPMENT
• Automated equipment and computers used in the processes must be
routinely calibrated, maintained and validated for accuracy.
4) CONTROL OF COMPONENTS, CONTAINERS, AND CLOSURES
GENERAL REQUIREMENTS
• Written procedures describing the receipt, identification, storage,
handling, sampling, testing and approval or rejection of all drug product
components, product containers, and closures must be maintained and
followed.
• Bulk pharmaceutical chemicals, containers, and closures must meet the
exact physical and chemical specifications established with the supplier
at the time of ordering.
RAW MATERIAL INSPECTION
• An incoming raw materials inspection program is a GMP requirement.
There should be written procedures describing all actions of the raw
material inspection program covering a minimum the parameters listed
in the following table.
RAW MATERIAL INSPECTION PROGRAM
1. Describe how materials are received.
2. Describe how they are identified.
a. Each lot of each shipment must be uniquely identified with
traceability to the supplier manufacturing lot number.
b. Each lot is to be identified with its status: quarantine, approved,
rejected.
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3) EQUIPMENT
EQUIPMENT DESIGN, SIZE AND LOCATION
• Each piece of equipment must be of appropriate design and size and
suitably located to facilitate operations for its indented use, cleaning and
maintenance.
• The equipment’s surfaces and parts must not interact with the processes
or product’s components to alter the purity, strength or quality.
EQUIPMENT CLEANING AND MAINTENANCE
• Standard operating procedures must be written and followed for the
proper use, maintenance, and cleaning of each piece of equipment, and
appropriate logs and records must be kept.
AUTOMATIC AND ELECTRICAL EQUIPMENT
• Automated equipment and computers used in the processes must be
routinely calibrated, maintained and validated for accuracy.
4) CONTROL OF COMPONENTS, CONTAINERS, AND CLOSURES
GENERAL REQUIREMENTS
• Written procedures describing the receipt, identification, storage,
handling, sampling, testing and approval or rejection of all drug product
components, product containers, and closures must be maintained and
followed.
• Bulk pharmaceutical chemicals, containers, and closures must meet the
exact physical and chemical specifications established with the supplier
at the time of ordering.
RAW MATERIAL INSPECTION
• An incoming raw materials inspection program is a GMP requirement.
There should be written procedures describing all actions of the raw
material inspection program covering a minimum the parameters listed
in the following table.
RAW MATERIAL INSPECTION PROGRAM
1. Describe how materials are received.
2. Describe how they are identified.
a. Each lot of each shipment must be uniquely identified with
traceability to the supplier manufacturing lot number.
b. Each lot is to be identified with its status: quarantine, approved,
rejected.
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3. Describe how they are stored and what are the various storage
conditions.
a. Quarantine until tested or examined and dispositioned as
approved for manufacturing use by the quality unit.
b. Storage should prevent contamination.
4. Describe how raw materials are handled, sampled, and tested.
a. Representative samples are to be taken: the number of
containers sampled and the sample amount is to be statistically
appropriate.
b. Sampling technique should prevent contamination.
c. Samples are to be appropriately identified.
d. A minimum of at least one identity test is to be conducted.
5. Describe what the approval and rejection process is for raw materials.
• Each raw material should have a corresponding written specification
that was developed to ensure the appropriate quality of material is used
in the manufacturing process. In order to be released the materials must
meet those specifications.
• Once raw materials are approved for use, the materials management
department is responsible for using the oldest material first. This is
known as FIFO = first in, first out. There should be a procedure in place
that describes how the quality assurance unit will handle taw material
rejection.
• When raw materials are received, they should go directly in quarantine
until they have been tested and approved for manufacturing use. Raw
materials are brought in by the receiving department.
• This group should check the obvious damage to the shipping containers
and match up the type and quantity of the material to the purchase
order. If this information is correct, the material is moved to designated
quarantine area. When it has been determined that the material is
suitable for identification testing and any other testing requirements,
the lot is appropriately identified with a quarantine sticker.
• At this time, quality assurance or the incoming raw material inspectors
are notified of receipt and its quarantine status. The material will remain
in quarantine until it has been approved for use in manufacturing.
ITEMS NEEDED TO IDENTIFY MATERIALS
• The inspector uses raw material inspection sheet document which
consists of following information:
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3. Describe how they are stored and what are the various storage
conditions.
a. Quarantine until tested or examined and dispositioned as
approved for manufacturing use by the quality unit.
b. Storage should prevent contamination.
4. Describe how raw materials are handled, sampled, and tested.
a. Representative samples are to be taken: the number of
containers sampled and the sample amount is to be statistically
appropriate.
b. Sampling technique should prevent contamination.
c. Samples are to be appropriately identified.
d. A minimum of at least one identity test is to be conducted.
5. Describe what the approval and rejection process is for raw materials.
• Each raw material should have a corresponding written specification
that was developed to ensure the appropriate quality of material is used
in the manufacturing process. In order to be released the materials must
meet those specifications.
• Once raw materials are approved for use, the materials management
department is responsible for using the oldest material first. This is
known as FIFO = first in, first out. There should be a procedure in place
that describes how the quality assurance unit will handle taw material
rejection.
• When raw materials are received, they should go directly in quarantine
until they have been tested and approved for manufacturing use. Raw
materials are brought in by the receiving department.
• This group should check the obvious damage to the shipping containers
and match up the type and quantity of the material to the purchase
order. If this information is correct, the material is moved to designated
quarantine area. When it has been determined that the material is
suitable for identification testing and any other testing requirements,
the lot is appropriately identified with a quarantine sticker.
• At this time, quality assurance or the incoming raw material inspectors
are notified of receipt and its quarantine status. The material will remain
in quarantine until it has been approved for use in manufacturing.
ITEMS NEEDED TO IDENTIFY MATERIALS
• The inspector uses raw material inspection sheet document which
consists of following information:
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ITEMS NEEDED TO IDENTIFY MATERIALS
Name of the manufacturer
Identity and quantity of each shipment of each batch of raw materials,
intermediates, or labeling and packaging materials for API's.
Name of supplier
Supplier's control number, if known, or other identification number. The
number allocated on receipt.
Date of receipt
The results of any test or examination performed and the conclusions
derived from this.
Records tracing the use of materials
Documentation of the examination and review of API labeling and packaging
materials for conformity with established specifications. The final decision
regarding rejected raw materials, intermediates, or API labeling and
packaging materials.
• After all the acceptance criteria have been met for a raw material, it is
marked as approved for manufacturing use.
• The entire lot is physically moved from the quarantine area to approved
area and if any acceptance criteria have not been met for the raw
materials then it is marked as rejected and returned to the suppliers.
5) PRODUCTION AND PROCESS CONTROL
WRITTEN PROCEDURE DEVIATION
• Written procedures are required for production and process controls to
ensure that the drug products have the correct identity, strength, quality
and purity.
• These procedures which include the charge-in of all components, use of
in-process controls, sample testing, and process and equipment
validation, must be followed for quality assurance. Any deviation from
the written procedures must be recorded and justified.
• In most instances, the operator records time and date of each key
operation and the supervisor signs off on it. When operations are
controlled by automated equipment, such equipment must be validated
regularly for precision.
EQUIPMENT IDENTIFICATION
• All product ingredients, equipment, and drums or other containers of
bulk finished product must be distinctively identified by labeling as to
content and or status.
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ITEMS NEEDED TO IDENTIFY MATERIALS
Name of the manufacturer
Identity and quantity of each shipment of each batch of raw materials,
intermediates, or labeling and packaging materials for API's.
Name of supplier
Supplier's control number, if known, or other identification number. The
number allocated on receipt.
Date of receipt
The results of any test or examination performed and the conclusions
derived from this.
Records tracing the use of materials
Documentation of the examination and review of API labeling and packaging
materials for conformity with established specifications. The final decision
regarding rejected raw materials, intermediates, or API labeling and
packaging materials.
• After all the acceptance criteria have been met for a raw material, it is
marked as approved for manufacturing use.
• The entire lot is physically moved from the quarantine area to approved
area and if any acceptance criteria have not been met for the raw
materials then it is marked as rejected and returned to the suppliers.
5) PRODUCTION AND PROCESS CONTROL
WRITTEN PROCEDURE DEVIATION
• Written procedures are required for production and process controls to
ensure that the drug products have the correct identity, strength, quality
and purity.
• These procedures which include the charge-in of all components, use of
in-process controls, sample testing, and process and equipment
validation, must be followed for quality assurance. Any deviation from
the written procedures must be recorded and justified.
• In most instances, the operator records time and date of each key
operation and the supervisor signs off on it. When operations are
controlled by automated equipment, such equipment must be validated
regularly for precision.
EQUIPMENT IDENTIFICATION
• All product ingredients, equipment, and drums or other containers of
bulk finished product must be distinctively identified by labeling as to
content and or status.
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SAMPLING AND TESTING OF IN PROCESS MATERIAL
• In-process samples are taken from the production batches periodically
for product control.
• In-process controls are of two general types:
Those performed by production personnel at the time of
operation to ensure that the machinery is producing output within
pre-established control limits. (tablet size, hardness)
Those performed by quality control laboratory personnel to
ensure compliance with all product specifications and batch to
batch consistency. (tablet content, dissolution)
6) PACKAGING AND LABELING CONTROL
• Written procedures are required for the receipt, identification, storage,
handling, sampling, and testing of drug product and issuance of labeling
and packaging materials.
• Labeling for each variation in drug product- strength, dosage form, or
quantity of contents- must be stored separately with suitable
identification.
• Obsolete and outdated labels and other packaging materials must be
destroyed.
• Access to storage area must be limited to authorized personnel.
• All materials must be withheld for use in the packaging and labeling of
product until approved and released by the quality control unit.
• Control procedures must be followed, and record procedures must be
maintained for the issuance and use of product labeling.
• Before labeling operation commence, the labeling facilities must be
inspected to ensure that all drug products and labels have been
removed from the previous operations.
• During operation, the products are visually or electronically inspected
for correct labeling and packaging.
• All records of inspections and controls must be documented in the batch
production records.
7) HOLDING AND DISTRIBUTION
• Written procedures must be established and followed for the holding
and distribution of product.
• Finished pharmaceuticals must be quarantined in storage until released
by the quality control department.
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SAMPLING AND TESTING OF IN PROCESS MATERIAL
• In-process samples are taken from the production batches periodically
for product control.
• In-process controls are of two general types:
Those performed by production personnel at the time of
operation to ensure that the machinery is producing output within
pre-established control limits. (tablet size, hardness)
Those performed by quality control laboratory personnel to
ensure compliance with all product specifications and batch to
batch consistency. (tablet content, dissolution)
6) PACKAGING AND LABELING CONTROL
• Written procedures are required for the receipt, identification, storage,
handling, sampling, and testing of drug product and issuance of labeling
and packaging materials.
• Labeling for each variation in drug product- strength, dosage form, or
quantity of contents- must be stored separately with suitable
identification.
• Obsolete and outdated labels and other packaging materials must be
destroyed.
• Access to storage area must be limited to authorized personnel.
• All materials must be withheld for use in the packaging and labeling of
product until approved and released by the quality control unit.
• Control procedures must be followed, and record procedures must be
maintained for the issuance and use of product labeling.
• Before labeling operation commence, the labeling facilities must be
inspected to ensure that all drug products and labels have been
removed from the previous operations.
• During operation, the products are visually or electronically inspected
for correct labeling and packaging.
• All records of inspections and controls must be documented in the batch
production records.
7) HOLDING AND DISTRIBUTION
• Written procedures must be established and followed for the holding
and distribution of product.
• Finished pharmaceuticals must be quarantined in storage until released
by the quality control department.
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• Products must be stored and shipped under conditions that do not affect
product quality.
• Ordinarily the product oldest approved stock is distributed first.
8) LABORATORY CONTROLS
• Laboratory controls are requirements for the establishment of and
conformance to written specifications, standards, sampling plans, test
procedures and other such mechanisms.
• The specifications, which apply to each batch of drug product, include
provisions for sample size, test intervals, sample storage, stability and
special testing requirement for certain dosage forms.
• Reserve samples must be retained for distributed products for specified
periods depending on their category.
• Reserve samples must be maintained 1 to 3 years after the expiration
date of the last lot of the drug product.
9) RECORDS AND REPORTS
• Production, control, and distribution records must be maintained for at
least a year following the expiration date of a product batch.
• This includes equipment cleaning and maintenance logs; specifications;
and lot numbers of product components, including raw materials and
product containers and closures; and label records. Complete master
production and control records for each batch must be kept and must
include the following:
Name and strength of the product
Dosage form
Quantitative amounts of components and dosage units
Complete manufacturing and control procedures
Specifications
Special notations
Equipment used
In-process controls
Sampling and laboratory methods and assays results
Calibration of instruments
Distribution records
Dated and employee identified records
• These master records must document that each step in the production,
control, packaging, labeling, and distribution of the product was
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• Products must be stored and shipped under conditions that do not affect
product quality.
• Ordinarily the product oldest approved stock is distributed first.
8) LABORATORY CONTROLS
• Laboratory controls are requirements for the establishment of and
conformance to written specifications, standards, sampling plans, test
procedures and other such mechanisms.
• The specifications, which apply to each batch of drug product, include
provisions for sample size, test intervals, sample storage, stability and
special testing requirement for certain dosage forms.
• Reserve samples must be retained for distributed products for specified
periods depending on their category.
• Reserve samples must be maintained 1 to 3 years after the expiration
date of the last lot of the drug product.
9) RECORDS AND REPORTS
• Production, control, and distribution records must be maintained for at
least a year following the expiration date of a product batch.
• This includes equipment cleaning and maintenance logs; specifications;
and lot numbers of product components, including raw materials and
product containers and closures; and label records. Complete master
production and control records for each batch must be kept and must
include the following:
Name and strength of the product
Dosage form
Quantitative amounts of components and dosage units
Complete manufacturing and control procedures
Specifications
Special notations
Equipment used
In-process controls
Sampling and laboratory methods and assays results
Calibration of instruments
Distribution records
Dated and employee identified records
• These master records must document that each step in the production,
control, packaging, labeling, and distribution of the product was
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accomplished and approved be the quality control unit. Depending on
the operation, the operator’s and or supervisor’s full signatures, initials,
or written or electronic identification codes are required.
• Records of written and oral complaints regarding a drug product must
also be maintained, along with the information regarding the internal
disposition of each complaint. All records must be made available at the
time of inspection by FDA officials.
10) RETURNED AND SALVAGED DRUG PRODUCTS
• Returned drug products (e.g. from wholesalers) must be identified by lot
number and product quality determined through appropriate testing.
• Drug products that meet specifications may be salvaged or reprocessed.
• Those that do not, along with those that have been subjected to
improper storage (e.g. extremes in temperature), shall not be returned
to marketplace.
• Records for all returned products must be maintained and must include
the date and reasons for the return; quantity and lot number of
products returned; procedures employed for holding, testing, and
reprocessing the product; and the product’s disposition.
THE INSPECTION FOR COMPLIANCE WITH GMP REGULATIONS
• Short description of the self-inspection system indicating whether an
outside, independent and experienced external export was involved in
evaluating the manufacturer’s compliance with Good manufacturing
Practices in all aspects of production.
• Periodic inspection of the garments shall be done by responsible staff.
CONTROLLED SUBSTANCES SAFEGUARDS
• Hazardous, toxic substances and flammable materials shall be stored in
suitably designed and segregated, enclosed areas in conformity with
Central and State Legislations.
• Highly hazardous, poisonous and explosive materials such as narcotics,
psychotropic drugs and substances presenting potential risks of abuse,
fire or explosion shall be stored in safe and secure areas. Adequate fire
protection measures shall be provided in conformity with the rules of
the concerned civic authority.
122
accomplished and approved be the quality control unit. Depending on
the operation, the operator’s and or supervisor’s full signatures, initials,
or written or electronic identification codes are required.
• Records of written and oral complaints regarding a drug product must
also be maintained, along with the information regarding the internal
disposition of each complaint. All records must be made available at the
time of inspection by FDA officials.
10) RETURNED AND SALVAGED DRUG PRODUCTS
• Returned drug products (e.g. from wholesalers) must be identified by lot
number and product quality determined through appropriate testing.
• Drug products that meet specifications may be salvaged or reprocessed.
• Those that do not, along with those that have been subjected to
improper storage (e.g. extremes in temperature), shall not be returned
to marketplace.
• Records for all returned products must be maintained and must include
the date and reasons for the return; quantity and lot number of
products returned; procedures employed for holding, testing, and
reprocessing the product; and the product’s disposition.
THE INSPECTION FOR COMPLIANCE WITH GMP REGULATIONS
• Short description of the self-inspection system indicating whether an
outside, independent and experienced external export was involved in
evaluating the manufacturer’s compliance with Good manufacturing
Practices in all aspects of production.
• Periodic inspection of the garments shall be done by responsible staff.
CONTROLLED SUBSTANCES SAFEGUARDS
• Hazardous, toxic substances and flammable materials shall be stored in
suitably designed and segregated, enclosed areas in conformity with
Central and State Legislations.
• Highly hazardous, poisonous and explosive materials such as narcotics,
psychotropic drugs and substances presenting potential risks of abuse,
fire or explosion shall be stored in safe and secure areas. Adequate fire
protection measures shall be provided in conformity with the rules of
the concerned civic authority.
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STATISTICAL QUALITY CONTROL
BASIC CONCEPTS
STATISTICS
• Planning, collecting, analyzing data for reporting certain conclusion
regarding data.
I. DESCRIPTIVE STATISTICS
• A presentation of facts, a survey or summary of population with data.
II. INFERENTIAL STATISTICS
• Drawing conclusion about whole population form a randomly selected
small sample.
• Random samples from a population truly represents the population
because every unit in a population has an equal chance of being selected
PARAMETERS OF DESCRIPTIVE STATISTICS
• Four parameters of descriptive statistics are important:
Mean or average (μ), measures central tendency and is estimated
by sample mean or xbar.
X̅=
∑x
i
n
i=1
n
Standard deviation (σ) measures spread of data.
√
∑(??????
??????−??????̅)
2�
??????=1
�−1
Range, difference between largest/smallest observations in a set
of data.
�=??????
�????????????−??????
�??????�
Distribution of Data shape:
▪ Normal or bell shaped – normally distributed with , and
or Skewed.
123
STATISTICAL QUALITY CONTROL
BASIC CONCEPTS
STATISTICS
• Planning, collecting, analyzing data for reporting certain conclusion
regarding data.
I. DESCRIPTIVE STATISTICS
• A presentation of facts, a survey or summary of population with data.
II. INFERENTIAL STATISTICS
• Drawing conclusion about whole population form a randomly selected
small sample.
• Random samples from a population truly represents the population
because every unit in a population has an equal chance of being selected
PARAMETERS OF DESCRIPTIVE STATISTICS
• Four parameters of descriptive statistics are important:
Mean or average (μ), measures central tendency and is estimated
by sample mean or xbar.
X̅=
∑x
i
n
i=1
n
Standard deviation (σ) measures spread of data.
√
∑(??????
??????−??????̅)
2�
??????=1
�−1
Range, difference between largest/smallest observations in a set
of data.
�=??????
�????????????−??????
�??????�
Distribution of Data shape:
▪ Normal or bell shaped – normally distributed with , and
or Skewed.
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VARIABLE
• Variable - a single quality characteristic that can be measured on a
numerical scale.
• Variables are characterized with Mean value of characteristic and
Variability (standard deviation, range, etc.) of characteristic.
DISTRIBUTION OR DATA SHAPE
• Distribution or shape of data is important to know for analytical
purposes. It must be bell shaped or normal distribution.
VARIATION
• Variation exists in all processes and may be:
I. COMMON OR RANDOM VARIATION
• Where causes cannot be identified and are unavoidable.
E.g. Differences in process variables, i.e. tablet diameter, weight,
etc.
• Common variation are also called as variation built into process.
• Standard deviation and Accuracy
II. CAUSE-ASSIGNABLE VARIATION
• Where causes of variation can be identified
and eliminated.
E.g. Poor training, worn tools, machine
needing repair.
• Cause-assignable variation are also called as
special or non-routine events.
QUALITY ASPECTS
• Two aspects of quality:
Normal distribution Skewed distribution
defective
raw
materials
poorly
trained
workers
poorly
calibrate
d
machines
worn or
loose
parts
tired or
indiffere
nt
workers
excessive
machine
vibration
124
VARIABLE
• Variable - a single quality characteristic that can be measured on a
numerical scale.
• Variables are characterized with Mean value of characteristic and
Variability (standard deviation, range, etc.) of characteristic.
DISTRIBUTION OR DATA SHAPE
• Distribution or shape of data is important to know for analytical
purposes. It must be bell shaped or normal distribution.
VARIATION
• Variation exists in all processes and may be:
I. COMMON OR RANDOM VARIATION
• Where causes cannot be identified and are unavoidable.
E.g. Differences in process variables, i.e. tablet diameter, weight,
etc.
• Common variation are also called as variation built into process.
• Standard deviation and Accuracy
II. CAUSE-ASSIGNABLE VARIATION
• Where causes of variation can be identified
and eliminated.
E.g. Poor training, worn tools, machine
needing repair.
• Cause-assignable variation are also called as
special or non-routine events.
QUALITY ASPECTS
• Two aspects of quality:
Normal distribution Skewed distribution
defective
raw
materials
poorly
trained
workers
poorly
calibrate
d
machines
worn or
loose
parts
tired or
indiffere
nt
workers
excessive
machine
vibration
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Features: More attributes meet desirability = high quality.
Freedom from defect = fewer defects = higher quality.
STATISTICAL QUALITY CONTROL
• SQC is set of statistical tools used in assessing quality characteristics or
parameters of a process, thus also called as Statistical Process Control
(SPQ).
• SQC or SPQ tool is a Control chart used in monitoring variation in the
characteristics of a product/process.
• Control chart is a graph, plotted in time order used to study how a
process changes (variates) over time.
CONTROL CHARTS
• Control Chart always shows sample data on a graph with central control
limit (CL), upper control limit (UCL), and lower control limit (LCL)
• Limits as lines on graph are determined from historical data.
• Upper and lower control limits represent 3 standard deviations above
and 3 standard deviations below mean line, respectfully.
125
Features: More attributes meet desirability = high quality.
Freedom from defect = fewer defects = higher quality.
STATISTICAL QUALITY CONTROL
• SQC is set of statistical tools used in assessing quality characteristics or
parameters of a process, thus also called as Statistical Process Control
(SPQ).
• SQC or SPQ tool is a Control chart used in monitoring variation in the
characteristics of a product/process.
• Control chart is a graph, plotted in time order used to study how a
process changes (variates) over time.
CONTROL CHARTS
• Control Chart always shows sample data on a graph with central control
limit (CL), upper control limit (UCL), and lower control limit (LCL)
• Limits as lines on graph are determined from historical data.
• Upper and lower control limits represent 3 standard deviations above
and 3 standard deviations below mean line, respectfully.
125
GM Hamad
• Reasons for taking 3 :
Probability that a random value of measurement falls in-between
3-σ limits is 0.9973, which is very high.
On other hand, probability that a random value of sample falls
outside of 3-σ limits is 0.0027, which is very low.
Thus, when values fall in-between 3-σ limits, variations are
attributed due to chance variation, then process is considered to
be statistically controlled.
But, when one or many values fall out of 3-σ limits, variations are
attributed to assignable variation and process is said to be not
under statistical control.
• By comparing current data to these lines, Conclusions can be drawn
about whether process variation is consistent (in control) or is
unpredictable (out of control, affected by special causes of variation).
WHEN TO USE CONTROL CHARTS
• When controlling ongoing processes by finding and correcting problems
as they occur.
• When predicting the expected range of outcomes from a process.
• When determining whether a process is stable (in statistical control)
• When analyzing patterns of process variation from special causes (non-
routine events) or common causes (built into the process)
• When determining whether your quality improvement project should
aim to prevent specific problems or to make fundamental changes to the
process.
CONTROL CHARTS – TYPES
• Types of control charts are presented below:
126
• Reasons for taking 3 :
Probability that a random value of measurement falls in-between
3-σ limits is 0.9973, which is very high.
On other hand, probability that a random value of sample falls
outside of 3-σ limits is 0.0027, which is very low.
Thus, when values fall in-between 3-σ limits, variations are
attributed due to chance variation, then process is considered to
be statistically controlled.
But, when one or many values fall out of 3-σ limits, variations are
attributed to assignable variation and process is said to be not
under statistical control.
• By comparing current data to these lines, Conclusions can be drawn
about whether process variation is consistent (in control) or is
unpredictable (out of control, affected by special causes of variation).
WHEN TO USE CONTROL CHARTS
• When controlling ongoing processes by finding and correcting problems
as they occur.
• When predicting the expected range of outcomes from a process.
• When determining whether a process is stable (in statistical control)
• When analyzing patterns of process variation from special causes (non-
routine events) or common causes (built into the process)
• When determining whether your quality improvement project should
aim to prevent specific problems or to make fundamental changes to the
process.
CONTROL CHARTS – TYPES
• Types of control charts are presented below:
126
GM Hamad
CONTROL CHARTS – SHEWHART
• Control chart for variables are used to monitor a measurable continuous
characteristics, e.g. weight, diameter, etc.
CONTROL CHARTS FOR VARIABLES
• Control charts for variable are:
X-bar Chart
R-Chart
• Both, x-bar chart and R-chart are used as pair.
x
Chart
Control Charts
R
Chart
c
Chart
p
Chart
Shewhart
Control Charts
Process
Acceptance
Control
Charts
Variables Attributes
127
CONTROL CHARTS – SHEWHART
• Control chart for variables are used to monitor a measurable continuous
characteristics, e.g. weight, diameter, etc.
CONTROL CHARTS FOR VARIABLES
• Control charts for variable are:
X-bar Chart
R-Chart
• Both, x-bar chart and R-chart are used as pair.
x
Chart
Control Charts
R
Chart
c
Chart
p
Chart
Shewhart
Control Charts
Process
Acceptance
Control
Charts
Variables Attributes
127
GM Hamad
• This type of chart graphs the means (or averages) of a set of samples,
plotted in order to monitor the mean of a variable, for example the
length of steel rods, the weight of bags of compound, the intensity of
laser beams, etc.
• In constructing this chart, samples of process outputs are taken at
regular intervals, the means of each set of samples are calculated and
graphed onto the X bar control chart.
• This chart can then be utilized to determine the actual process mean,
versus a nominal process mean and will demonstrate if the mean output
of the process is changing over time. Mean of the process changing over
time.
• This type of chart demonstrates the variability within a process. It is
suited to processes where the sample sizes are relatively small, for
example <10. Sets of sample data are recorded from a process for the
particular quality characteristic being monitored.
• For each set of date, the difference between the smallest and largest
readings are recorded. This is the range “R” of the set of data. The
ranges are now recorded onto a control chart. The center line is the
averages of all the ranges.
• x-bar and Range charts are SQC tools used to monitor variables data
when samples are collected at regular intervals from a process.
• Assume process is normally distributed, and fraction nonconforming can
be found by: P(x < LCL) + P(x > UCL), i.e., beyond limits.
• x-bar chart detects changes in mean value of a quality characteristics
across all units or a process
• x-bar monitor central tendency.
128
• This type of chart graphs the means (or averages) of a set of samples,
plotted in order to monitor the mean of a variable, for example the
length of steel rods, the weight of bags of compound, the intensity of
laser beams, etc.
• In constructing this chart, samples of process outputs are taken at
regular intervals, the means of each set of samples are calculated and
graphed onto the X bar control chart.
• This chart can then be utilized to determine the actual process mean,
versus a nominal process mean and will demonstrate if the mean output
of the process is changing over time. Mean of the process changing over
time.
• This type of chart demonstrates the variability within a process. It is
suited to processes where the sample sizes are relatively small, for
example <10. Sets of sample data are recorded from a process for the
particular quality characteristic being monitored.
• For each set of date, the difference between the smallest and largest
readings are recorded. This is the range “R” of the set of data. The
ranges are now recorded onto a control chart. The center line is the
averages of all the ranges.
• x-bar and Range charts are SQC tools used to monitor variables data
when samples are collected at regular intervals from a process.
• Assume process is normally distributed, and fraction nonconforming can
be found by: P(x < LCL) + P(x > UCL), i.e., beyond limits.
• x-bar chart detects changes in mean value of a quality characteristics
across all units or a process
• x-bar monitor central tendency.
128
GM Hamad
• R-chart detects dispersion or variability, changes in range (standard
deviation of quality characteristics across all units of a process.
• R-charts monitor the dispersion.
• In general, xbar chart is insensitive to small departures from normality.
• R chart is more sensitive to non-normality than the xbar chart
• Process can show acceptable central tendencies on x-bar chart but
unacceptable variability on R-chart OR acceptable variability on R-chart
but unacceptable central tendencies on x-bar
• Thus, R-chart is interpreted first:
If R-chart is in control, interpret the X-bar chart (i) if in control,
process is in control; (ii) if out of control: process average is out of
control.
If R-chart is out of control, process variation is out of control then
investigate cause; no need to interpret the X-bar chart
• X-bar and R-bar Charts reveal different problems:
Shift in mean in x-bar chart but not be R-chart
Shift in dispersion in R-chart but not be x-bar chart
CONTROL CHARTS FOR ATTRIBUTES
• Control charts for attributes are used to monitor feature that have
discrete values and can be counted, e.g. % defective, number of flaws in
a syrups, number of chipped tablets
129
• R-chart detects dispersion or variability, changes in range (standard
deviation of quality characteristics across all units of a process.
• R-charts monitor the dispersion.
• In general, xbar chart is insensitive to small departures from normality.
• R chart is more sensitive to non-normality than the xbar chart
• Process can show acceptable central tendencies on x-bar chart but
unacceptable variability on R-chart OR acceptable variability on R-chart
but unacceptable central tendencies on x-bar
• Thus, R-chart is interpreted first:
If R-chart is in control, interpret the X-bar chart (i) if in control,
process is in control; (ii) if out of control: process average is out of
control.
If R-chart is out of control, process variation is out of control then
investigate cause; no need to interpret the X-bar chart
• X-bar and R-bar Charts reveal different problems:
Shift in mean in x-bar chart but not be R-chart
Shift in dispersion in R-chart but not be x-bar chart
CONTROL CHARTS FOR ATTRIBUTES
• Control charts for attributes are used to monitor feature that have
discrete values and can be counted, e.g. % defective, number of flaws in
a syrups, number of chipped tablets
129
GM Hamad
• Control charts for attributes are:
p-Chart
c-Chart
• P-Charts are used for quality characteristics that are discrete and involve
yes/no or good/bad decisions.
E.g. Number of leaking tubes in a lot of 48
• C-Charts are used for discrete defects when there can be more than one
defect per unit.
E.g. Number of flaws on a tablets
P-CHARTS
• A statistical control chart that plots proportion for defective sample (p)
over time Involves taking of sample and each item is inspected for
determining sample proportion defective by dividing the number of
defective items by sample size.
• P charts are utilized where there is a pass / fail determination on a unit
inspected. The p chart will show if the proportion defective within a
process changes over the sampling period (the p indicates the portion of
successes).
• In the p chart the sample size can vary over time. A similar chart to the p
chart is the np chart. However, with the np chart the sample size needs
to stay constant over the sampling period.
• An advantage of the np chart is that the number non-conforming is
recorded onto the control rather than the fraction non-conforming.
Some process operators are more comfortable plotting the number
rather than the fraction of non-conformances.
130
• Control charts for attributes are:
p-Chart
c-Chart
• P-Charts are used for quality characteristics that are discrete and involve
yes/no or good/bad decisions.
E.g. Number of leaking tubes in a lot of 48
• C-Charts are used for discrete defects when there can be more than one
defect per unit.
E.g. Number of flaws on a tablets
P-CHARTS
• A statistical control chart that plots proportion for defective sample (p)
over time Involves taking of sample and each item is inspected for
determining sample proportion defective by dividing the number of
defective items by sample size.
• P charts are utilized where there is a pass / fail determination on a unit
inspected. The p chart will show if the proportion defective within a
process changes over the sampling period (the p indicates the portion of
successes).
• In the p chart the sample size can vary over time. A similar chart to the p
chart is the np chart. However, with the np chart the sample size needs
to stay constant over the sampling period.
• An advantage of the np chart is that the number non-conforming is
recorded onto the control rather than the fraction non-conforming.
Some process operators are more comfortable plotting the number
rather than the fraction of non-conformances.
130
GM Hamad
• Plot the sample proportion defective on the control chart and compare
with UCL and LCL to determine if process is out of control
• Mean = p, Sd = Sigma
??????=���(??????
1−??????
�
)
• Where, p = proportion defective item and n is the sample size
??????L=P
????????????�=??????+3√(??????
1−??????
�
)
�??????�=??????−3√(??????
1−??????
�
)
C-CHART
• C-chart for number (count) of defects per unit plots movement in the
number of defects per unit.
• Randomly selected one item and number of defects are counted in that
item.
• Number of defects are plotted on control chart which are compared
with UCL and LCL to determine if process is out of control.
▪ Sd=SQR of mean
▪ UCL= C + 3.SQR of mean
▪ LCL=C – 3. SQR of mean
• The c chart is used where there can be a number of defects per sample
unit and the number of samples per sampling period remains constant.
• In the u chart, again similar to the c chart, the number of defects per
sample unit can be recorded, however, with the u chart, the number of
samples per sampling period may vary.
CONTROL CHARTS – PROCESS ACCEPTANCE CONTROL CHARTS
• Charts that give information about capability of process relative to its
specification limits (not mean).
• These charts estimates fraction of nonconforming items deviating
specification limit for any process.
• In these charts, CL is Specification limit (SL), LCL is LSL and USL
131
• Plot the sample proportion defective on the control chart and compare
with UCL and LCL to determine if process is out of control
• Mean = p, Sd = Sigma
??????=���(??????
1−??????
�
)
• Where, p = proportion defective item and n is the sample size
??????L=P
????????????�=??????+3√(??????
1−??????
�
)
�??????�=??????−3√(??????
1−??????
�
)
C-CHART
• C-chart for number (count) of defects per unit plots movement in the
number of defects per unit.
• Randomly selected one item and number of defects are counted in that
item.
• Number of defects are plotted on control chart which are compared
with UCL and LCL to determine if process is out of control.
▪ Sd=SQR of mean
▪ UCL= C + 3.SQR of mean
▪ LCL=C – 3. SQR of mean
• The c chart is used where there can be a number of defects per sample
unit and the number of samples per sampling period remains constant.
• In the u chart, again similar to the c chart, the number of defects per
sample unit can be recorded, however, with the u chart, the number of
samples per sampling period may vary.
CONTROL CHARTS – PROCESS ACCEPTANCE CONTROL CHARTS
• Charts that give information about capability of process relative to its
specification limits (not mean).
• These charts estimates fraction of nonconforming items deviating
specification limit for any process.
• In these charts, CL is Specification limit (SL), LCL is LSL and USL
131
GM Hamad
• Specification for assay of Active ingredient is 95-105 % and an ideal
target specification is 100%.
• In the real world, results vary 90% , 98.5%, 104% , 110 % etc. and mean
may be different from the target specification, i.e. 108% vs 100%.
DIFFERENCE BETWEEN 2 TYPES OF CHARTS
Shewhart Control Charts Acceptance Control charts
Central Line is practical
average/mean deduced from data,
101%
Central line is the target specification
100 %
Indicates how process is behaving
from practical calculated mean?
Indicates how process is behaving
from theoretical set target
specification?
A process consistency – may not be
within regulatory spec, for example
mean is 108 % , SD 1.2, YES, the
process is consistent, but it is not
meeting Specification.
Tells that process consistency with
respect to the Regulatory Registered
Specification.
Used during development setting of
specifications.
Tells, once specification is set, how
the process is behaving with respect
to the set specification.
Parameters include UCL, CL, LCL. Parameters include USL, SL and LSL
and the Process Capability ratio (Cp)
and Process Capability Index (CPk).
CONTROL CHARTS – PROCESS
1. Setting limits and Zones
2. Process procedure
3. Pattern Analysis
4. Interpretation
USL
SL
LSL
132
• Specification for assay of Active ingredient is 95-105 % and an ideal
target specification is 100%.
• In the real world, results vary 90% , 98.5%, 104% , 110 % etc. and mean
may be different from the target specification, i.e. 108% vs 100%.
DIFFERENCE BETWEEN 2 TYPES OF CHARTS
Shewhart Control Charts Acceptance Control charts
Central Line is practical
average/mean deduced from data,
101%
Central line is the target specification
100 %
Indicates how process is behaving
from practical calculated mean?
Indicates how process is behaving
from theoretical set target
specification?
A process consistency – may not be
within regulatory spec, for example
mean is 108 % , SD 1.2, YES, the
process is consistent, but it is not
meeting Specification.
Tells that process consistency with
respect to the Regulatory Registered
Specification.
Used during development setting of
specifications.
Tells, once specification is set, how
the process is behaving with respect
to the set specification.
Parameters include UCL, CL, LCL. Parameters include USL, SL and LSL
and the Process Capability ratio (Cp)
and Process Capability Index (CPk).
CONTROL CHARTS – PROCESS
1. Setting limits and Zones
2. Process procedure
3. Pattern Analysis
4. Interpretation
USL
SL
LSL
132
GM Hamad
1. A. SETTING LIMITS
• Control limits are functions of natural variability of process
• Natural tolerance limits represent natural variability of process (usually
set at 3-sigma from mean)
• Specification limits are determined by regulatory bodies, e.g., FDA.
• Specification width and process variability
• Depends on Nature of a process or industry norms
1. B. Assigning different Zones
• For interpretation of pattern on charts, data is represented over 6
standard deviations, 3 standard deviations from mean line to UCL and 3
from mean to the LCL.
• To help analyze charts, the chart area is divided into 6 sections A, B, and
C representing the standard deviations.
EXAMPLE OF R-CHART WITH ASSIGNED ZONES
• Example of R chart with assigned zones is given below:
133
1. A. SETTING LIMITS
• Control limits are functions of natural variability of process
• Natural tolerance limits represent natural variability of process (usually
set at 3-sigma from mean)
• Specification limits are determined by regulatory bodies, e.g., FDA.
• Specification width and process variability
• Depends on Nature of a process or industry norms
1. B. Assigning different Zones
• For interpretation of pattern on charts, data is represented over 6
standard deviations, 3 standard deviations from mean line to UCL and 3
from mean to the LCL.
• To help analyze charts, the chart area is divided into 6 sections A, B, and
C representing the standard deviations.
EXAMPLE OF R-CHART WITH ASSIGNED ZONES
• Example of R chart with assigned zones is given below:
133
GM Hamad
2. PROCESS PROCEDURE
• Random sampling of output of a process
• Inspecting output from a process for measuring the quality
characteristics
• Charting as graph with set CL, UCL and LCL
• Helpful in identifying in-process variation
• Acceptance sampling used to randomly inspect a batch of products to
determine acceptance/rejection
3. PATTERNS OF POINTS
• Position of points on control chart makes patterns of data distribution.
• Pattern of point distribution with reference to UCL and LCL and 6 zones
on the graphs are observed.
• An unusual or nonrandom pattern in the data:
Number of points outside of control limits
Cyclic Patterns
Shift in process level (all above/below CL)
Trend (increasing/decreasing)
Stratification (3 up and 3 down)
• Patterns of points provide useful diagnostic information on process
which is used to make process modifications that reduce variability.
• Pattern tell when there is a problem in process.
4. INTERPRETATION
• Sample observation (points) consistently below CL
134
2. PROCESS PROCEDURE
• Random sampling of output of a process
• Inspecting output from a process for measuring the quality
characteristics
• Charting as graph with set CL, UCL and LCL
• Helpful in identifying in-process variation
• Acceptance sampling used to randomly inspect a batch of products to
determine acceptance/rejection
3. PATTERNS OF POINTS
• Position of points on control chart makes patterns of data distribution.
• Pattern of point distribution with reference to UCL and LCL and 6 zones
on the graphs are observed.
• An unusual or nonrandom pattern in the data:
Number of points outside of control limits
Cyclic Patterns
Shift in process level (all above/below CL)
Trend (increasing/decreasing)
Stratification (3 up and 3 down)
• Patterns of points provide useful diagnostic information on process
which is used to make process modifications that reduce variability.
• Pattern tell when there is a problem in process.
4. INTERPRETATION
• Sample observation (points) consistently below CL
134
GM Hamad
• Points consistently above CL
• Points consistently increasing
• Points consistently decreasing
• A cyclic trend
• One point outside of the 3 sigma control limits (beyond zone A)
• Two out of any three successive points fall in zone A of the same side
135
• Points consistently above CL
• Points consistently increasing
• Points consistently decreasing
• A cyclic trend
• One point outside of the 3 sigma control limits (beyond zone A)
• Two out of any three successive points fall in zone A of the same side
135
GM Hamad
• Four out of any five successive points fall in or beyond zone B of the
same side
• The Process” behaviour”
• The process is considered in control for n samples collected if all points
are inside control limits and no systematic behavior is identified
• Above indicates that trial control limits are suitable for controlling
current or future production.
• If points plot out of control, then control limits must be revised.
• Before revising, identify out of control points and look for assignable
causes.
If assignable causes can be found, then recalculate control limits.
136
• Four out of any five successive points fall in or beyond zone B of the
same side
• The Process” behaviour”
• The process is considered in control for n samples collected if all points
are inside control limits and no systematic behavior is identified
• Above indicates that trial control limits are suitable for controlling
current or future production.
• If points plot out of control, then control limits must be revised.
• Before revising, identify out of control points and look for assignable
causes.
If assignable causes can be found, then recalculate control limits.
136
GM Hamad
If no assignable causes can be found then consider control limits
as appropriate for current control.
CONTROL CHARTS APPLICATIONS
• Applied where a process has tendency to go out of control which may be
harmful and costly
• May be used to study past performance and or to evaluate present
conditions.
• Helps controlling ongoing processes by finding (and correcting) problems
as they occur.
• Data collected from control chart may form basis for process
improvement.
• Helps determine what process adjustments need to be made.
• Help predicting expected range of outcomes in a process.
• Help determining whether a process is stable or in control.
• Help making defects visible.
• Help analyzing patterns of process variation from special causes or
common causes.
• Target quality improvement aim to prevent specific problems or to make
fundamental changes to process.
• Tool to:
Improve process
Improve productivity
Make defects visible
Zero defects
PROCESS CAPABILITY – AN APPLICATION OF PROCESS ACCEPTANCE
CONTROL CHART
• Acceptance charts help in determining capability/feasibility of a process
• Two parameters of process capability are:
Process Capability Ratio (Cp)
Process Capability Index (Cpk)
PROCESS CAPABILITY RATIO
• Process-Capability Ratio (Cp) is used to express process capability.
• A way to deal with quality of product by devising quantifiable
measurements of quality element
137
If no assignable causes can be found then consider control limits
as appropriate for current control.
CONTROL CHARTS APPLICATIONS
• Applied where a process has tendency to go out of control which may be
harmful and costly
• May be used to study past performance and or to evaluate present
conditions.
• Helps controlling ongoing processes by finding (and correcting) problems
as they occur.
• Data collected from control chart may form basis for process
improvement.
• Helps determine what process adjustments need to be made.
• Help predicting expected range of outcomes in a process.
• Help determining whether a process is stable or in control.
• Help making defects visible.
• Help analyzing patterns of process variation from special causes or
common causes.
• Target quality improvement aim to prevent specific problems or to make
fundamental changes to process.
• Tool to:
Improve process
Improve productivity
Make defects visible
Zero defects
PROCESS CAPABILITY – AN APPLICATION OF PROCESS ACCEPTANCE
CONTROL CHART
• Acceptance charts help in determining capability/feasibility of a process
• Two parameters of process capability are:
Process Capability Ratio (Cp)
Process Capability Index (Cpk)
PROCESS CAPABILITY RATIO
• Process-Capability Ratio (Cp) is used to express process capability.
• A way to deal with quality of product by devising quantifiable
measurements of quality element
137
GM Hamad
• Involves assessing process variability relative to preset product
specifications
• Cp assumes that the process is centered in the specification range.
??????
??????=
??????�� −���
6??????
• Cp > 1 indicates that process exceeds minimal specifications (a low
number of nonconforming items are being produced).
• Cp = 1 reflects process variability just meets the specifications (about
0.27% nonconforming units are being produced).
• Cp < 1 reveals that process is not capable of producing within
specifications (a large number of nonconforming items are being
produced.
PROCESS CAPABILITY INDEX
• Process Capability Index (Cpk) helps to address a possible lack of
centering of the process.
??????
??????�=�??????�(
??????��−??????
3??????
,
??????−���
3??????
)
Where, min is a function which outputs the minimum value of the
given value from above 2 resultant values.
• Cpk is less than 1, revealing that the process is not capable.
138
• Involves assessing process variability relative to preset product
specifications
• Cp assumes that the process is centered in the specification range.
??????
??????=
??????�� −���
6??????
• Cp > 1 indicates that process exceeds minimal specifications (a low
number of nonconforming items are being produced).
• Cp = 1 reflects process variability just meets the specifications (about
0.27% nonconforming units are being produced).
• Cp < 1 reveals that process is not capable of producing within
specifications (a large number of nonconforming items are being
produced.
PROCESS CAPABILITY INDEX
• Process Capability Index (Cpk) helps to address a possible lack of
centering of the process.
??????
??????�=�??????�(
??????��−??????
3??????
,
??????−���
3??????
)
Where, min is a function which outputs the minimum value of the
given value from above 2 resultant values.
• Cpk is less than 1, revealing that the process is not capable.
138
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