validation

analytical and bio analytical method validation

1. ANALYTICAL & BIO-
ANALYTICAL METHOD
VALIDATION
1

2. 2
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

3. Validation is a documented evidence which high degree of
assurance that specific process will produce a product meeting it’s
pre determined specifications and quality attributes..
Drug substances and drug product manufacturers must perform
validations, it is very important that this understanding be shared
throughout the organization.
The term validation generally to cover the entire spectrum of
cGMP.
3
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

4. ANALYTICAL METHOD VALIDATION
Validation of an analytical method is the process by which it is
established, by laboratory studies, that the performance
characteristics of the method meet the requirements for the
intended analytical applications.
The process to Confirm that the analytical procedure employed
for a specific test is suitable for intended use and that they
support the identity, quality, purity and potency of the drug
substances and drug products.
4
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

5. 5
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

6. CONCEPT OF REVALIDATION
When we make any changes inWhen we make any changes in
Analytical procedureAnalytical procedure
Drug substance (e.g. synthetic route)Drug substance (e.g. synthetic route)
Drug product (e.g. composition)Drug product (e.g. composition)

6
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

7. PARAMETERS PARAMETERS
Specificity
LOD & LOQ (if applicable)
Linearity
Precision
i.System Suitability
ii. Method Repeatability
iii. Intermediate Precision (or) Ruggedness
iv.Method Reproducibility
Accuracy (or) Recovery
Robustness
7
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

8. DEFINITION
Specificity Specificity of analytical method as its ability to measure
accurately an analyte in the presence of interference,
such as synthetic precursors, excipients, enantiomers
and known (or likely) degradation product that may be
expected to be present in the sample matrix.
1. SPECIFICITY
8
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

9. peyye efftyeeyye e
y ytyeeee 2. LIMIT OF DETECTION AND LIMIT OF
QUANTIFICATION (LOD & LOQ)
DEFINITIONDEFINITION
LOD: LOD: Lowest amount of analyte in a sample which can be detected Lowest amount of analyte in a sample which can be detected
but not necessarily quantitated, under the stated experimental but not necessarily quantitated, under the stated experimental
conditions (LOD) conditions (LOD)
LOQ: LOQ: Lowest amount of analyte in a sample which can be Lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy quantitatively determined with suitable precision and accuracy
(LOQ)(LOQ)
9
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

10. 10
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

11. 3. LINEARITY
DEFINITIONDEFINITION
The The LinearityLinearity of an analytical procedure is its ability (within a given range) to of an analytical procedure is its ability (within a given range) to
obtain test results that are directly proportional to the concentration (amount) of obtain test results that are directly proportional to the concentration (amount) of
analyte in the sample analyte in the sample
Range: The interval between the upper and lower level( Including these level) Range: The interval between the upper and lower level( Including these level)
that have been demonstrated to be determined with precision, accuracy and that have been demonstrated to be determined with precision, accuracy and
linearity.linearity.
11
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

12. 4. PRECISION
DEFINITIONDEFINITION
Closeness of agreement (degree of scatter) between a series of Closeness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the same measurements obtained from multiple sampling of the same
homogenous sample under the prescribed conditions.homogenous sample under the prescribed conditions.
Precision may be considered at …. Precision may be considered at ….
1.1.System precision (System suitability)System precision (System suitability)
2.2.Method RepeatabilityMethod Repeatability
3.3.Intermediate precisionIntermediate precision
4.4.ReproducibilityReproducibility
12
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

13. Repeatability: Repeatability: precision under same operating conditions (with-in a laboratory over precision under same operating conditions (with-in a laboratory over
a short period of time using the same analyst with the same equipment)a short period of time using the same analyst with the same equipment)
Measurement / Injection repeatability (System Precision)Measurement / Injection repeatability (System Precision)
Method repeatability (Method Precision)Method repeatability (Method Precision)
Intermediate precisionIntermediate precision: precision under different laboratory conditions (within-: precision under different laboratory conditions (within-
laboratory variation, as on different days, or with different analysts, or equipments laboratory variation, as on different days, or with different analysts, or equipments
within the same laboratory)within the same laboratory)
ReproducibilityReproducibility: precision between laboratories / intermediate precision can be : precision between laboratories / intermediate precision can be
considered during the standardization of a procedure before it is submitted to the considered during the standardization of a procedure before it is submitted to the
pharmacopoeiapharmacopoeia
13
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

14. 5. ACCURACY
DEFINITIONDEFINITION
The accuracy of an analytical procedure expresses the closeness of The accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as a agreement between the value, which is accepted either as a
conventional true value or an accepted reference value and the value conventional true value or an accepted reference value and the value
found found
 It is some times termed as truenessIt is some times termed as trueness
14
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

15. 6. ROBUSTNESS
DEFINTIONDEFINTION
Measure of its capacity to remain unaffected by small, but deliberate Measure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides indication of its reliability variations in method parameters and provides indication of its reliability
during its normal usageduring its normal usage
varying method parameters within a realistic range and the quantitative varying method parameters within a realistic range and the quantitative
influence of the variables is determined, and, if the influence of the influence of the variables is determined, and, if the influence of the
parameter is within a previously specified tolerance, then, the parameter is parameter is within a previously specified tolerance, then, the parameter is
said to be within the method’s robustness range.said to be within the method’s robustness range.
15
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

16. Typical variations include under validation Typical variations include under validation
programmeprogramme
Flow rateFlow rate
WavelengthWavelength
Mobile phase composition, generally, Organic compositionMobile phase composition, generally, Organic composition
TemperatureTemperature
pH of the mobile phasepH of the mobile phase
 Stability of analytical solutionsStability of analytical solutions
 Different columns ( Different lots and suppliers ) Different columns ( Different lots and suppliers )
16
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

17. VALIDATION REPORT
Generally method validation report should have
Objective and scope of the method
Molecule details (IUPAC name, CAS No. Molecular Formulae, Molecular
weight and its Molecular Structure etc.,)
Detailed list of chemicals, reagents, reference standards
Listing of equipment and its functional and performance requirements
Methodology followed
Validation data (parameter wise – procedure, results, conclusion etc.,)
17
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

18. Instrument out puts, which should represent critical method parameters
Specificity and LOD – for Identification Test (Generally, photographs)
Selectivity / Specificity data (discriminating chromatogram, peak purity data,
blank and placebo chromatograms and stressed samples chromatograms)
Linearity graphs
Resolution and related system suitability chromatograms .
18
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

19. BIO ANALYTICAL METHOD
VALIDATION
19
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

20. INTRODUCTION OF BIO ANLAYTICAL METHOD
VALIDATION.
BASIC STEPS IN BIO ANALYTICAL METHOD
VALIDATION.
VALIDATION PARAMETERS.
CONCLUSION.
REFERENCE.
20
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

21. INTRODUCTION
It involves the quantitative determination of drug and its
metabolites in biological fluids .
It plays a vital role in the evaluation & interpretation of the
bioavailability, bioequivalence, pharmacokinetic and
toxicokinetic study data.
21
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

22. BASIC STEPS OF BIOANALYTICAL
PROCESS
22
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

23. 1)SELECTIVITY
It is the ability of an analytical method to identify and
quantify the analyte in the presence of other
components in the sample.
LLOQ – It is the lowest amount of analyte in the sample
that can be quantified with accuracy and precision.
2)MATRIX EFFECT
It is the direct or indirect alteration or interference in
response due to the presence of unintended analytes in
the biological sample.

VALIDATION PARAMETERS
23

24. If MF = 1 ( No matrix effect)
If MF < 1 ( Suppression)
If MF > 1 ( Enhancement)
3) SENSITIVITY
Sensitivity test should be conducted to prove the
reproducibility for samples at LOQ.
ACCEPTANCE CRITERIA FOR LOQ :
For accuracy : ± 20%
For precision : ≤ 20%
For S/N ratio: 5: 1
24
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

25. 4) CALIBRATION AND QC STANDARDS
Calibration standard is the biological matrix to which a known amount
of analyte is added.
QC standard is a spiked sample used to monitor the performance of a
bioanalytical method and is used to assess the integrity and validate
the results of unknown samples.
5) RECOVERY (OR) EXTRACTION EFFICIENCY
Determined by comparing the detector response of the analyte (or) IS
from an extracted sample to the detector response of the analyte from
an unextracted sample representing 100% recovery.
25
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

26. 6) DILUTION INTEGRITY
Dilution of the study samples is performed when the
obtained concentration is exceeding the ULOQ, or when
there is less sample availability compared to the method
requirement.( can be tested on dilutions from 1:2 to 1:10
& dilution integrity will be evaluated at 1:2to 1:4)
ACCEPTANCE CRITERIA :
QC samples , Accuracy = ±15%
Precision = ≤15%
26
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

27. 7) CARRYOVER
It is the appearance of an analyte signal in
blank sample peaks after the analysis of
samples with a high analyte concentration.
To evaluate carryover a blank sample will
be placed ULQ standard in a sequence.
It is insignificant when blank sample
response is ≤ 20% of LOQ sample.
27
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

28. It is evaluated when different anticoagulants in plasma
are used in the preparation of the QC and Calibration
control standards.
Anticoagulant effect is nullified ,when QC standard
shows,
 Accuracy = ±15% and Precision = ≤15%
9) STABILITY EVALUATION
The stability of analyte in the matrix during collection,
storage of samples should be assessed
8) ANTICOAGULANT EFFECT
28
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

29. Short term stability of analyte and IS In solvent
29

30. MATRIX STABILITY
AUTO SAMPLER STABILITY
It is evaluated to cover the anticipated run time for the
analytical batch and to handle the situations like system
malfunctioning.
ALL THE QC SAMPLE
CONC. ARE BACK
CALCULATED USING
CALIBRATION CURVE
30

31. It is performed to evaluate the stability of the samples,
which are kept on bench during the extraction process.
The anticipated time is usually 4 to 24 hrs
BENCH TOP STABILITY
31
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

32. LONG TERM STABILITY
It is performed to demonstrate the stability of the
analyte in the matrix for longer duration of time.
ACCEPTENCE CRITERIA :
 The relative means of back calculated
concentration (test/reference) for both levels must
be within 85-115%.
32
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

33. FREEZE THAW STABILITY
It is performed , to evaluate the stability of the
analyte in the matrix after multiple cycles of freezing
and thawing.

33
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

34. CONCLUSION
Validation is done according to standard protocol and
used it always produce a product which meets its
predetermined specification and quality control.
Bioanalytical methods must be validated to objectively
demonstrate the fitness for their intended use.
Bioanalysis and the production of pharmacokinetic, toxicokinetic
and metabolic data plays a fundamental role in pharmaceutical
research and development involved in the drug discovery and
development process.
34
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

35. REFERENCE
Bioanalytical Method Validation and Its
Pharmaceutical Application- A Review article
from Pharmaceutica Analytica
 James agalloco,frederick J. Carleton. Validation
of pharmaceutical process, third edition, Page no
5.
The United State Pharmacopoeia 24; The National
Formulary 19; 2000: [1225] VALIDATION OF
COMPENDIAL METHODS.
35
VIGNAN PHARMACY COLLEGE,VADLAMUDI.

36. 36
VIGNAN PHARMACY COLLEGE,VADLAMUDI.