Metronidazole potecy is determined by measuring its absorbance at 277 nm in 0.1 N HCl solution in µg concentration.

1. DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
UNIVERSITY OF DHAKA
MS PracticalMS PracticalMS PracticalMS Practical
Name of Experiment : Determination of Potency Metronidazole.
Submitted bySubmitted bySubmitted bySubmitted by
Md. Atai rabby
MS

2. Metronodazole is a 5-nitroimidazole derivative (2-methyl-5-nitroimidazole-1-ethanol),
C5H9N3O2 , 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 HCl
2. Standard metronidazole
3. Sintered glass funnel
4. Volumetric flask
5. Filter Paper
6. Distilled water
7. Spectrophotometer
Assay principle of the potency of metronidazole:
Metronidazole potecy is determined by measuring its absorbance at 277 nm in 0.1 N HCl
solution in µg concentration.
Preparation of reference metronidazole solution:
Weigh out accurately a quantity of 0.1 gm of reference metronidazole and add about 80.0 ml of
0.1 N HCl. Shake for 15 min and add sufficient volume of 0.1 N HCl to make exactly 100 ml.
Filter by using sintered glass funnel and dilute 3.0 ml of filtrate to 100 ml with 0.1 N HCl (30
µg/ml).

3. From this solution transfer exactly 0.25, 0.5, 0.75 nand 1.0 ml in four different clean and dry test
tubes and add 0.75, 0.5, 0.25 and 0.0 ml of 0.1 N HCl solution respectively to make the final
volume 1.0 ml in each in each tube. The absorbance of four test tubes measured at 277 nm are
used to prepare the standard curve ( test tubes No 1-4).
Data : Standard Solution contain 15 µg/ml
Table: Estimation of the absorbance of standard and supplied sample solutions at 277 nm
wavelength
Tube No. Standard
sample
Taken (ml)
Supplied
sample taken
(ml)
0.1 N HCl
taken (ml)
Absorbance at
277nm
Amount of
metronidazole
(µg)
Blank 0.00 0.00 0.0 0.0
Std-01 0.250 0.750 0.594 3.75
Std-02 0.500 0.500 0.448 7.5
Std-03 0.750 0.250 0.302 11.25
Std-04 1.00 0.00 0.156 15
Sample -01 1.0 0.520
0.520
Sample -02 1.0 0.520
CALCULATION:
For Standard solution,
Concentration of working standard solution is 15 15 µg/ml
Test tube 1, amount of Metronidazole = 15 µg/ml x 0.250 ml = 3.75 µg
Test tube 2, amount of Metronidazole = 15 µg/ml x 0.500 ml = 7.5 µg
Test tube 3, amount of Metronidazole = 15 µg/ml x 0.750 ml = 11.25 µg
Test tube 4, amount of Metronidazole = 15 µg/ml x 1.0 ml = 15 µg

4. Sample
From standard curve 1 ml working sample contain = 5.63 µg
So, total amount of metronidazole = 5.63 x 4000 µg
= 22520 µg or 22.52 mg
Potency =
஺௠௢௨௡௧
ହ଴
x 100% =
ଶଶ.ହଶ
ହ଴
x 100% = 45.04%
RESULT
Potency of the given drug = 45.04 %
PRECAUSION
1. HCl was handled carefully.
2. The solution was filtered carefylly
3. The standard was weighted carefylly.
4. Time for reaction was maintained strictly.
DISCUSSION
Potency is the relationship between the the dose of a drug and the therapeutic effect. It refers to
the drug's strength. A drug is considered potent when a small amount of the drug achieves the
intended effect. Efficacy is the ability of a drug to produce the desired therapeutic effect.
Efficacy means that the drug is effective. When comparing two drugs that work equally, the one
with the lower dose has a higher potency. They have equal efficacy.
Potency : In the field of pharmacology, potency is a measure of drug activity expressed in terms
of the amount required to produce an effect of given intensity. A highly potent drug (e.g.,
morphine, alprazolam, chlorpromazine) evokes a larger response at low concentrations, while a
drug of lower potency (ibuprofen, acetylsalicylic acid) evokes a small response at low
concentrations. It is proportional to affinity and efficacy.
Affinity is the ability of the drug to bind to a receptor. Efficacy is the relationship between
receptor occupancy and the ability to initiate a response at the molecular, cellular, tissue or
system level. The response is equal to the effect, or (E), and depends on both the drug binding

5. and the drug-bound receptor then producing a response; thu
and efficacy. The agonist, the ligand, drug or hormone that binds to the receptor and initiates the
response is usually abbreviated A
low to measure but at higher concentrations it becomes appreciable and rises with increasing
agonist concentration [A] until at sufficiently high concentrations it can no longer be increased
by raising [A] and asymptotes to a maximum
Semi-log plots of two agonists with different K
The Emax is the maximum possible effect for the agonist. The concentration of
50% of Emax is termed the half maximal effective concentration
commonly EC50. The term "potency" refers to the
concentration of a drug is required to produce 50% of
potency.
Higher potency does not necessarily mean more
What is the difference between absorbance and transmittance?
Absorbance is the measure of the quantity of light that a sample neither transmits nor reflects and
is proportional to the concentration of a substance in a solution. On the meter of the Spectronic
20™, the absorbance is displayed on the bottom scale, whi
The transmittance of a sample is the ratio of the intensity of the light that has passed through the
sample to the intensity of the light when it entered the sample ( T = I
is displayed as a percentage on the top scale of the meter of the Spectronic 20™.
To convert between the absorbance and transmittance scales, use the equation:
Absorbance = -log (percent transmittance/100)
bound receptor then producing a response; thus, potency depends on both affinity
and efficacy. The agonist, the ligand, drug or hormone that binds to the receptor and initiates the
A or D. Below a certain concentration of agonist (
igher concentrations it becomes appreciable and rises with increasing
until at sufficiently high concentrations it can no longer be increased
and asymptotes to a maximum Emax.
log plots of two agonists with different Kd.
is the maximum possible effect for the agonist. The concentration of
half maximal effective concentration and is abbreviated
. The term "potency" refers to the [A]50 value. The lower the [A]
concentration of a drug is required to produce 50% of maximum effect and the higher the
Higher potency does not necessarily mean more side effects.
What is the difference between absorbance and transmittance?
Absorbance is the measure of the quantity of light that a sample neither transmits nor reflects and
is proportional to the concentration of a substance in a solution. On the meter of the Spectronic
20™, the absorbance is displayed on the bottom scale, which is logarithmic.
The transmittance of a sample is the ratio of the intensity of the light that has passed through the
sample to the intensity of the light when it entered the sample ( T = Iout / Iin ). The transmittance
he top scale of the meter of the Spectronic 20™.
To convert between the absorbance and transmittance scales, use the equation:
log (percent transmittance/100)
s, potency depends on both affinity
and efficacy. The agonist, the ligand, drug or hormone that binds to the receptor and initiates the
. Below a certain concentration of agonist ([A]), E is too
igher concentrations it becomes appreciable and rises with increasing
until at sufficiently high concentrations it can no longer be increased
is the maximum possible effect for the agonist. The concentration of A at which E is
and is abbreviated [A]50, or more
[A]50, the less the
maximum effect and the higher the
Absorbance is the measure of the quantity of light that a sample neither transmits nor reflects and
is proportional to the concentration of a substance in a solution. On the meter of the Spectronic
The transmittance of a sample is the ratio of the intensity of the light that has passed through the
). The transmittance
he top scale of the meter of the Spectronic 20™.

6. In optics and spectroscopy, transmittance is the fraction of incident light at a specified
wavelength that passes through a sample. A related term is absorptance, or absorption factor,
which is the fraction of light absorbed by a sample at a specified wavelength. Occasionally one
also hears the terms visible transmittance (VT) and visible absorptance (VA), which are the
respective fractions for the spectrum of visible light. In equation form,
where I0 is the intensity of the incident light and I is the intensity of the light coming out of the
sample and and are transmittance and absorptance respectively. In these equations,
scattering and reflection are considered to be close to zero or otherwise accounted for. The
transmittance of a sample is sometimes given as a percentage.
Accuracy
The accuracy of an analytical measurement is how close a result comes to the true value.
Determining the accuracy of a measurement usually requires calibration of the analytical method
with a known standard.
Precision
Precision is the reproducibility of multiple measurements and is usually described by the
standard deviation, standard error, or confidence interval.
Working Curve
A working curve is a plot of the analytical signal (the instrument or detector response) as a
function of analyte concentration. These working curves are obtained by measuring the signal
from a series of standards of known concentration. The working curves are then used to
determine the concentration of an unknown sample or to calibrate the linearity of an analytical
instrument.
Q. Why blank was used ?
In order to effectively use a spectrophotometer we must first zero the machine, we do this using
"the blank." The blank contains everything except the compound of interest which absorbs light.
Thus, by zeroing the machine using "the blank," any measured absorbance is due to the presence
of the solute of interest.