Determination of metronidazole from the solid dosage form

Metronidazole is determined or assayed spectrophotometrically as the present methods abbeys Beer’s Law in the concentration range of 100-150 µg at about 500nm. By using the absorbance of standard solution of Metronidazole, the unknown amount of this drug in the sample can be calculated. The unknown amount of metronidazole is generally calculated by drawing the standard curve.

1. DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY  
UNIVERSITY OF DHAKA 
MS PracticalMS PracticalMS PracticalMS Practical    

Name of Experiment : Determination of Metronidazole from the solid dosage form
Date : 15.02.11










Submitted bySubmitted bySubmitted bySubmitted by    
Md. Atai rabby
MS

2. Metronodazole is a 5-nitroimidazole derivative (2-methyl-5-nitroimidazole-1-ethanol),
C
5H9N3O2 , with activity against anaerobic bacteria and protozoa. The nitro group of
metronidazole undergoes reductive transformation to an active metabolite that ultimately
contributes to cell death. Metronidazole is indicated on the treatment of infection in
which anaerobic bacteria have been identified or are suspected to the cause.
Metronidazole is active against a wide range of pathogenic microorganisms, notably
species of bacteriodes, fusobacteria, Clostridia, eubacteria, anaerobic cocci and
gardnerella vaginalis. It is used in the prevention of postoperative infections due to
anaerobic bacteria, specially anaerobic streptococci.






Materials:
1. 20% w/v NaOH
2. Standard metronidazole
3. Sintered glass funnel
4. Volumetric flask
5. Filter Paper
6. Distilled water
7. Spectrophotometer

3. Assay Principle
Metronidazole is determined or assayed spectrophotometrically as the present methods
abbeys Beer’s Law in the concentration range of 100-150 µg at about 500nm. By using
the absorbance of standard solution of Metronidazole, the unknown amount of this drug
in the sample can be calculated. The unknown amount of metronidazole is generally
calculated by drawing the standard curve.

Preparation of standard solution:
Weigh out accurately 700 mg pure Metronidazole and dissolve in 140 ml distilled water.

Preparation of sample solution:
Dissolve 200 mg of tablet (powder) in 60-70 ml of water. Shake well and make the
volume upto 100 ml. Filter with sintered glass funnel, use for color development.
Procedure for color development:
1. To 20 ml of each standard and sample solution, 30 ml of 20% w/v NaOH was
added and diluted to 100 ml with water. Solution was Kept for 8-10 minutes at
room temperature.
2. Then diluted standard solution was taken 5.0, 5.5, 6.0, 6.5 and 7.0 to 10 ml with
water (standard curve).
3. the same procedure was followed to take 6.0 ml of sample solution (in duplicate)
and diluted each sample to 10.0 ml as mentioned above.
4. A blank was prepared by taking 2 ml of water, 3 ml of 20% w/v NaOH and diluted
it to 10 ml with water.
5. Absorbance was taken within 12-15 minutes at 500 nm.

Data : Standard Solution contain mg/ml

4. Table : Estimation of the absorbance of standard, supplied and unknown sample
solutions at 500 nm wavelength.

Tube no.
Volume
of
standard
(ml)

Volume
of
sample
(ml)

Volume of
distilled
water
(ml)
Volume
of total
solution
(ml)
Absorbance
at 500 nm
Amount of
Metronidazole
(mg)
Blank 0.0 7.0 10.0 0.0 0.000
Std 1 5.0 5.0 10.0 0.117 3.50
Std 02 5.5 4.5 10.0 0.120 3.85
Std 03 6.0 4.0 10.0 0.155 4.20
Std 04 6.5 3.5 10.0 0.163 4.55
Std 05 7.0 3.0 10.0 0.186 4.90
Unknown
sample-02
6.0 4.0 10.0 0.179
Given
sample -
01
6.0 4.0 10.0 0.139

Given
sample-
02
6.0 4.0 10.0 0.135

CALCULATION: 
For Standard solution,
Concentration of working standard solution is 0.7 mg/ ml
Test tube 1, amount of Metronidazole = 0.7 mg/ ml x 5.0 ml = 3.5 mg
Test tube 2, amount of Metronidazole = 0.7 mg/ ml x 5.5 ml = 3.85 mg
Test tube 3, amount of Metronidazole = 0.7 mg/ ml x 6.0 ml = 4.20 mg
Test tube 4, amount of Metronidazole = 0.7 mg/ ml x 6.5 ml = 4.55 mg
Test tube 5, amount of Metronidazole = 0.7 mg/ ml x 7.0 ml = 4.90 mg

5. Given Sample
From Standard curve, The Given sample contain 4.28 mg Metronidazole
So, the total amount of Metronidazole in given sample =
Md.Ataiitr

mg
= 356.67 mg
Unknown Sample
The amount of Metronidazole in unknown sample is 5.4 mg

RESULT 
Amount of given sample = 356 mg
Amount of Unknown Sample = 5.4 mg

PRECAUTION 
1. NaOH is Corrosive so it was handled carefully
2. Metronidazole was measured carefully
3. Absorbance was taken within 12-15 minutes
 
DISCUSSION 
When atoms or molecules absorb light, the incoming energy excites a quantized structure
to a higher energy level. The type of excitation depends on the wavelength of the light.
Electrons are promoted to higher orbitals by ultraviolet or visible light, vibrations are
excited by infrared light, and rotations are excited by microwaves.
An absorption spectrum is the absorption of light as a function of wavelength. The
spectrum of an atom or molecule depends on its energy level structure, and absorption
spectra are useful for identifying of compounds.

6. Measuring the concentration of an absorbing species in a sample is accomplished by
applying the Beer-Lambert Law. 5-Nitroimadazoles Such as Metronidazole are
extensively used as antiamoebic, antiprotozoal and antibacterial drugs. The discovery of
the antibacterial and antitrichomonal properties of the antibiotic azomycin led to the
investigation of nitroimidazoles as antiparasitic agents1, 2 .Nitroimidazole derivatives
Presents biological activity against anaerobic micro-organisms, being largely used as
active ingredient of antihelminthic medicine3. The discovery of the antitrichromal
properties of metronidazole revolutionized the treatment of disease. The properties of
metronidazole were studied; it was not clinically tested until some years later. In
laboratory tests, Metronidazole is effective against intestinal amoebiasis in rats and
hepatic amoebiasis in hamsters and also active against Entamoeba histolytica in
vitro4.The initial clinical tests of metronidazole indicated that it was capable of Curing
invasive amoebic dysentery and amoebic liver abscess5. Subsequent clinical tests have
established metronidazole as the drug of choice in the treatment of all forms of
amoebiasis in humans.
Metronidazole is officially determined by titrimetry, potentiometry and HPLC methods.
IndianPharmacopoeia8 describes the non-aqueous titration method using Perchloric acid
as titrant and malachitegreen as indicator for the assay of metronidazole. British
Pharmacopoeia9 describes potentiometric andnon-aqueous methods using perchloric acid
as titrant.United states Pharmacopoeia 10 describes HPLC andnon aqueous titration
methods for the assay of metronidazole. Several methods have been reported for
thedetermination of metronidazole including Spectrophotometry, Polarograpy. Most of
Spectrophotometric methods found in the literature for the determination of etronidazole
in the visible region involve initial reduction by treatment with Zn Powder and HCl15,22
followed by the diazotizationand coupling of the resulting amine.
All these methods are less sensitive21 involve tedious Procedures such as heating and
extractionutilize costly reagents.
In present Study, two Spectrophotometric methods for the quantitative estimation of
metronidazole have been developed after converting it to its reduced form by using Zn
powder and HCl as well as the reaction of its reduced Product with 1, 10- Phenanthrolin
and 4-Choloro3-Nitro Aniline followed by diazotization was studied to establish the
optimum reaction Conditions, optical characterictics, Precision and accuracy of the
Proposed methods.

7. Beer–Lambert law : In optics
Lambert–Beer law or the Beer
properties of the material through which the light is traveling.
Diagram of Beer–Lambert absorption of a beam of light as it travels through a
width σ.
The law states that there is a logarithmic dependence between the transmission (or
transmissivity), T, of light through a substance and the product of the
the substance, α, and the distance the light travels through the material (i.e. th
The absorption coefficient can, in turn, be written as a product of either a
(extinction coefficient) of the absorber,
material, or an absorption cross section
For liquids, these relations are usually written as:

where I
0 and I are the
intensity
respectively; σ is cross section of light absorption by a single particle and N is the density
(number per unit volume) of absorbing particles

Ultraviolet and Visible Absorption Spectroscopy (uv
UV-vis spectroscopy is the measurement of the wavelength and intensity of
near-ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic
enough to promote outer electrons to higher energy levels. UV
usually applied to molecules and inorganic ions or complexes in solution. The
spectra have broad features that are of limited use for sample identification but are very
useful for quantitative measurements. The concentration of an analyte in solution can be
determined by measuring the absorbance at some wavelength and applyin
Lambert Law.
optics, the Beer–Lambert law, also known as Beer's law
Beer–Lambert–Bouguer law relates the absorption
properties of the material through which the light is traveling.

Lambert absorption of a beam of light as it travels through a
The law states that there is a logarithmic dependence between the transmission (or
, of light through a substance and the product of the absorption coefficient
, and the distance the light travels through the material (i.e. the path length),
can, in turn, be written as a product of either a mola
(extinction coefficient) of the absorber, ε, and the concentration c of absorbing species in the
orption cross section, σ, and the (number) density N' of absorbers.
For liquids, these relations are usually written as:

intensity (or power) of the incident light and the transmitted light,
is cross section of light absorption by a single particle and N is the density
of absorbing particles
Ultraviolet and Visible Absorption Spectroscopy (uv-vis)
vis spectroscopy is the measurement of the wavelength and intensity of
ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic
enough to promote outer electrons to higher energy levels. UV-vis spectroscopy is
usually applied to molecules and inorganic ions or complexes in solution. The
spectra have broad features that are of limited use for sample identification but are very
useful for quantitative measurements. The concentration of an analyte in solution can be
determined by measuring the absorbance at some wavelength and applyin
Beer's law or the
absorption of light to the
Lambert absorption of a beam of light as it travels through a cuvette of
The law states that there is a logarithmic dependence between the transmission (or
absorption coefficient of
e path length), σ.
molar absorptivity
of absorbing species in the
N' of absorbers.
) of the incident light and the transmitted light,
is cross section of light absorption by a single particle and N is the density
vis spectroscopy is the measurement of the wavelength and intensity of absorption of
ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic
vis spectroscopy is
usually applied to molecules and inorganic ions or complexes in solution. The uv-vis
spectra have broad features that are of limited use for sample identification but are very
useful for quantitative measurements. The concentration of an analyte in solution can be
determined by measuring the absorbance at some wavelength and applying the Beer-

8. The light source is usually a hydrogen or deuterium lamp for uv measurements and a
tungsten lamp for visible measurements. The wavelengths of these continuous light
sources are selected with a wavelength separator such as a prism or grating
monochromator. Spectra are obtained by scanning the wavelength separator and
quantitative measurements can be made from a spectrum or at a single wavelength.

Metronidazole : Metronidazole (INN) (pronounced /mtrnadzol/) is a nitroimidazole
antibiotic medication used particularly for anaerobic bacteria and protozoa.
Metronidazole is an antibiotic, amebicide, and antiprotozoal. It is the drug of choice for
first episodes of mild-to-moderate Clostridium difficile infection. It is marketed by Pfizer
under the trade name Flagyl in the US, by Sanofi-Aventis globally under the same
tradename Flagyl, in Pakistan and Bangladesh it is also available with the brand name of
Nidagyl manufactured and marketed by Star Laboratories. In Thailand it is marketed as
Mepagyl by Thai Nakhorn Patana. They are also marketed in UK by Milpharm Limited
and Almus Pharmaceuticals. Metronidazole was developed in 1960.
Metronidazole is also used as a gel preparation in the treatment of the dermatological
conditions such as rosacea (Rozex and MetroGel by Galderma) and fungating tumours
(Anabact, Cambridge Healthcare Supplies).
Mechanism of action
Metronidazole, taken up by diffusion, is selectively absorbed by anaerobic bacteria and
sensitive protozoa. Once taken up by anaerobes, it is non-enzymatically reduced by
reacting with reduced ferredoxin, which is generated by pyruvate oxido-reductase. This
reduction causes the production of toxic products to anaerobic cells, and allows for
selective accumulation in anaerobes.
The metronidazole metabolites are taken up into bacterial DNA, and form unstable
molecules. This function only occurs when metronidazole is partially reduced, and
because this reduction usually happens only in anaerobic cells, it has relatively little
effect upon human cells or aerobic bacteria.
Indications
Bacterial
• Bacterial vaginosis, commonly associated with overgrowth of Gardnerella species
and coinfective anaerobes (Mobiluncus, Bacteroides), in symptomatic patients

9. • Pelvic inflammatory disease in conjunction with other antibiotics such as
ofloxacin, levofloxacin, or ceftriaxone
• Anaerobic infections such as Bacteroides fragilis, spp, Fusobacterium spp,
Clostridium spp, Peptostreptococcus spp, Prevotella spp, or any other anaerobes
in intra-abdominal abscess, peritonitis, diverticulitis, empyema, pneumonia,
aspiration pneumonia, lung abscess, diabetic foot ulcer, meningitis and brain
abscesses, bone and joint infections, septicemia, endometritis, or endocarditis
• Pseudomembranous colitis due to Clostridium difficile
• Helicobacter pylori eradication therapy, as part of a multi-drug regimen in peptic
ulcer disease
Protozoal
Amoebiasis: Infections caused by Entamoeba histolytica.
Giardiasis: infection of the small intestine caused by the ingestion of infective cysts of
protozoan Giardia lamblia. Giardiasis occurs worldwide with a prevalence of 20–30% in
developing countries. The Centers for Disease Control and Prevention reports that, in the
US, Giardia infects over 2.5 million people annually. There are multiple modes of
transmission, including person-to-person, water-borne, and venereal. Person-to-person
transmission accounts for a majority of Giardia infections, and is usually associated with
poor hygiene and sanitation. Water-borne transmission is common in United States.
Giardia epidemics are often associated with the ingestion of unfiltered contaminated
water. Sexual transmission happens through fecal-oral contamination. Additionally,
diaper changing and inadequate hand washing are risk factors for transmission.
Epidemics of Giardia have developed through the contamination of food by infected food
handlers.
A small number of infected individuals experience an abrupt onset of abdominal cramps,
explosive, watery diarrhea, vomiting, foul flatus, and fever which may last for 3–4 days
before proceeding into a more sub-acute phase. The majority of infected persons develop
gradual symptoms that become recurrent or resistant.
Trichomoniasis: infection caused by Trichomonas vaginalis, which is a common cause of
vaginitis and is the most frequently presenting new infection of the common sexually
transmitted diseases.
Treatment with metronidazole
Adult dosage: 250 mg three times a day for 5 days, or 400 mg 3 times a day for 7 days.
Alternative: 2000 mg one time only.
Metronidazole should always be taken with a large glass of water and with or after food.

10. Pediatric dosage: 15 mg per kilogram of body weight per dose, 3 times per day, for 5
days
Nonspecific
• Prophylaxis for those undergoing potentially contaminated colorectal surgery or
appendectomies and may be combined with neomycin
• Acute gingivitis and other dental infections (TGA approved, non-U.S. Food and
Drug Administration (FDA) approved)
• Crohn's disease with colonic or perianal involvement (non-FDA approved) –
believed to be more effective in combination with ciprofloxacin
• Topical metronidazole is indicated for the treatment of rosacea, and in the
treatment of malodorous fungating wounds.
Prevention of preterm births
A 2005 study found "Metronidazole therapy before 32 weeks was associated with an
increased risk of preterm birth", possibly as a result of "changes in the vaginal flora…
seen with vaginal clindamycin or oral metronidazole therapy."
Metronidazole has also been used in women to prevent preterm birth associated with
bacterial vaginosis, amongst other risk factors including the presence of cervicovaginal
fetal fibronectin (fFN). A randomised controlled trial demonstrated that metronidazole
was ineffective in preventing preterm delivery in high-risk pregnant women and,
conversely, the incidence of preterm delivery was actually higher in women treated with
metronidazole.
Lamont has argued that Metronidazole is not the right antibiotic to administer in these
circumstances and was often administered too late to be of use. Clindamycin
administered early in the second trimester to women who test positive for bacterial
vaginosis seems to be more effective.
Veterinary Use
Metronidazole is not labeled for animal use but is widely used to treat infections of
Giardia in dogs, cats, and other companion animals. It is also used to treat enteric (gi) and
systemic infections. In the US, the FDA prohibits the use of metronidazole in food
animals.
Adverse effects
Common adverse drug reactions (≥1% of patients) associated with systemic
metronidazole therapy include: nausea, diarrhea, and/or metallic taste in the mouth.

11. Intravenous administration is commonly associated with thrombophlebitis. Infrequent
adverse effects include: hypersensitivity reactions (rash, itch, flushing, fever), headache,
dizziness, vomiting, glossitis, stomatitis, dark urine, and/or paraesthesia.
High doses and/or long-term systemic treatment with metronidazole is associated with
the development of leukopenia, neutropenia, increased risk of peripheral neuropathy
and/or CNS toxicity.
Metronidazole is listed by the US National Toxicology Program (NTP) as reasonably
anticipated to be a human carcinogen. Although some of the testing methods have been
questioned, it has been shown to cause cancer in experimental animals. Yet,
metronidazole was shown to be safe in humans.It appears to have a fairly low potential
for cancer risk and under most circumstances the benefits of treatment outweigh the risk.
Metronidazole is listed as a possible carcinogen according to the WHO International
Agency for Research on Cancer (IARC).
Due to its potential carcinogenic properties, metronidazole is banned in the EU and the
USA for veterinary use in the feed of animals and is banned for use in any food animals
in the USA. In the USA, this type of restriction is covered under the Delaney clause.
Earlier studies suggested a relation between metronidazole and various birth defects.
Those studies are nowadays considered flawed and more recent studies "do not support a
significant increased risk for birth defects or other adverse effects on the fetus."
Common adverse drug reactions associated with topical metronidazole therapy include
local redness, dryness, and/or skin irritation; and eye watering (if applied near eyes).
Interaction with alcohol
Consuming ethanol (alcohol) while using metronidazole has long been thought to have a
disulfiram-like reaction with effects that can include nausea, vomiting, flushing of the
skin, tachycardia (accelerated heart rate), and shortness of breath, however there are
studies calling that notion into question. Consumption of alcohol should be avoided by
patients during systemic metronidazole therapy and for at least 48 hours after completion
of treatment. However, the mechanism of this reaction in the clinical setting has recently
been questioned by some authors, and a possible central toxic serotonin reaction for the
alcohol intolerance suggested.
Stevens-Johnson syndrome with mebendazole
Metronidazole alone rarely causes Stevens-Johnson syndrome but is reported to occur at
high rates when combined with mebendazole.

12. Potentially fatal serotonin syndrome
It is important to note that Serotonin Syndrome is not fully understood. The complex
drug interaction can happen after a couple days or take up to months. The exact
mechanism is not known, a theory of serotonin dysfunction helps explain how the
syndrome presents and how it is to be treated. Signs and symptoms are muscle rigidity,
headache, elevated blood pressure, and changes in blood chemistry. The only direct
treatment is to discontinue the offending drugs. Recently, there have been reported cases
of SSRI/SNRI antidepressant drugs and metronidazole induced serotonin syndrome, this
information is not included on the metronidazole patient information leaflet. SSRI and
SNRI antidepressants include Prozac, Lexapro, Celexa, Zoloft, Effexor, Cymbalta, etc.
Shape and color
Metronidazole is available with a prescription under the brand names Flagyl and
Protostat. Other brand or generic formulations may also be available.
• Almus 200 mg - circular, white tablets
• Flagyl 250 mg - blue tablets
• Flagyl 375 mg - light-green/grey capsules
• Flagyl 500 mg - oblong, blue tablets
• Flagyl ER 750 mg - oval, blue film-coated tablets
• Protostat 250 mg - capsule-shaped, white tablets
• Protostat 500 mg - capsule-shaped, white tablets
• APO-Metronidazole 250 mg - circular, white tablets, marked APO 250
Question : Why do we prefer to express the Beer-Lambert law using absorbance as a measure of
the absorption rather than %T ?
Answer : The linear relationship between concentration and absorbance is both simple and
straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as a
measure of the absorption rather than %T.

Conclusion : The proposed spectrophotometric method for the determination of
secnidazole is simple accurate, precise and cheap. The statistical analyses show that the
data from the proposed method are in good agreement with those of the Concentration
(µg ml-1) reported method. The color reaction does not require stringent conditions nor
any specific reagent or buffer. The color is stable up to 20 min, which is sufficient time
for the analyst to perform the analysis.