Development and Validation
of Ratio derivative Spectrophotometric Method for Estimation of Metronidazole
Benzoate and related Impurity in Bulk and Pharmaceutical Formulation
Tabassum
H. Aaraf1*, Hasumati A. Raj1, Vineet C. Jain1, Vishnu Sutariya2
1Shree Dhanvantary
College of Pharmacy, Kim, Surat, Gujarat
India.
2Shree Dhanvantary
Pharmaceutical Analysis & Research Centre, Kim, Surat,
Gujarat India.
*Corresponding
Author E-mail: tabassumaaraf@gmail.com
ABSTRACT:
A simple, accurate and precise spectroscopic method was developed for
simultaneous estimation of Metronidazole Benzoate and related impurity in bulk
and pharmaceutical formulation by ratio derivative simultaneous equation
method. The method is based on dividing the spectrum for a mixture into the
standard spectra for each of the analysis and to obtain a spectrum that is
independent of the analyte concentration used as a
divisor. In this method for MND, MNI and BA the linearity range 2.0-10 µg/ml
were divided by the divisor 2.0 µg/ml of MTZ. The use of standardized spectra
as divisors minimizes experimental errors. Ratio spectra derivative permits the
use of the wavelengths corresponding to maximum or minimum and also the use of
the distance between consecutive maximum and minimum. For that the divided
spectra was converted to second derivative using DL value 16 and SF value 100.
The Metronidazole shows max absorbance at 270.00 nm, 2-Methyl-5Nitroimidazole
show max absorbance at 257.42 nm and Benzoic Acid shows max. absorbance
at 245.61 nm. The method was found to be linear (r2>0.997) in the range of
2.0-10 μg/ml. The limit of determination was
0.049 μg/ml and 0.026 μg/ml
and0.038 μg/ml for Metronidazole,
2-Methyl-5-Nitroimidazole and Benzoic Acid, respectively. The limit of
quantification was 0.150 μg/ml and 0.079 μg/ml
and 0.115 μg/ml for Metronidazole,
2-Methyl-5-Nitroimidazole and Benzoic Acid, respectively. The accuracy of this
method was evaluated by recovery studies and good recovery result was obtained
100%. The method was successfully applied for simultaneous determination of
Metronidazole, 2-Methyl-5-Nitroimidazole and Benzoic Acid.
KEY WORDS: Metronidazole Benzoate, Metronidazole,
2-Methyl-5-Nitroimidazole, Benzoic Acid, Ratio derivative simultaneous equation
method.
INTRODUCTION:
Metronidazole
is an anti-protozoan and antibacterial agent belonging to the class of Nitroimidazoles. Metronidazole is active against a wide
range of pathogenic anaerobic Gram-negative micro-organisms notably species of Bacteroides fragilis and
spp., Fusobacterium spp, Gardnerella vaginalis,
and anaerobic Gram positives such as Peptococcus
spp., Peptostreptococcus spp., Clostridium spp. It is
also active against protozoaes such as Trichomonas vaginalis, Entamoeba histolytica, Giardia lamblia.
Metronidazole was first approved by Sanofi-Aventis
for marketing in France and its International Birth Date (IBD) is 29 July 1959.
Since that time it has been registered and marketed worldwide. In Europe, (Sanofi-Aventis) Metronidazole is currently approved by
national procedures and marketed in 19 countries in various formulations, such
as tablets, oral suspensions, suppositories, intravenous solution, vaginal pessaries/ tablets. The safety profile of metronidazole is well known, and adverse effects are
considered mainly to be mild to moderate in severity. The most common adverse
reactions reported involve the gastrointestinal tract. Rare serious adverse
reactions, including convulsive seizures and peripheral neuropathy,
characterized mainly by numbness or paresthesia of an
extremity, have been reported in patients receiving prolonged metronidazole treatment.[1]
ü A double beam UV/Visible spectrophotometer
(Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was
used to measure absorbance of all the solutions.
ü Spectra were automatically obtained by
UV-Probe system software.
ü An analytical balance (Sartorius CD2250,
Gottingen, Germany) was used for weighing the samples.
ü Sonicator (D120/2H, Trans-O-Sonic)
ü All instruments and glass wares were
calibrated.
ü Metronidazole
Benzoate and 2-Methyl-5-Nitoimidazole raw material was received as gift sample
from Dano Pharmacham Pvt. Ltd.
Ankleswar.
ü Metronidazole
raw material was material was received as gift sample from Mc coy Drug
Pvt. Ltd. Sachin.
ü Benzoic acid LR
Grade.
ü Methanol
AR Grade, Distilled water, 0.1 N HCl, 0.1N NaOH were used for development purpose.
Accurately weighed quantity of Metronidazole Benzoate 10 mg
was transferred to 100ml volumetric flask, dissolved and diluted up to mark
with Methanol to give a stock solution having strength 100µg/ml.
Accurately weighed quantity of Metronidazole 10 mg was
transferred to 100ml volumetric flask, dissolved and diluted up to mark with
Methanol to give a stock solution having strength 100µg/ml.
Accurately weighed quantity of 2-Methyl-5-Nitroimidazole 10
mg was transferred into 100 ml volumetric flask, dissolved and diluted up to
mark with Methanol to give a stock solution having strength 100µg/ml.
Accurately weighed quantity of Benzoic acid 10 mg was
transferred to 100ml volumetric flask, dissolved and diluted up to mark with
Methanol to give a stock solution having strength 100µg/ml.
Pipette out accurately 1.0 ml
of Metronidazole Benzoate stock solution (100µg/ml), Metronidazole stock
solution (100µg/ml), 1.0 ml of 2-Methyl-5-Nitroimidazole stock solution
(100µg/ml) and 1.0 ml of Benzoic acid stock solution (100µg/ml) in 10 ml
volumetric flask and make up the volume up to the mark with Methanol. It gives
solution containing Metronidazole Benzoate 10µg/ml, 2-Methyl-5-Nitroimidazole
10µg/ml and Benzoic acid 10µg/ml.
Fig4.Ratio derivative
overlain division spectra of MND ,MNI and BA
2.0-10 µg/ml (MTZ 2.0 µg/ml
as divisor)
Table 1. Calibration data for MND, MNI and BA at
270.0nm, 257.42nm and 245.61
nm,
respectively. *(n=6)
|
Sr. No |
Concentration (μg/ml) |
Absorbance ±
SD* |
||||
|
|
MND |
MNI |
BA |
MND (270.0nm) |
MNI (257.42 nm) |
BA (245.61 nm) |
|
1 |
2.0 |
2.0 |
2.0 |
0.178±0.00041 |
0.096±0.00051 |
0.431±0.00089 |
|
2 |
4.0 |
4.0 |
4.0 |
0.286±0.00063 |
0.298±0.00040 |
0.823±0.00063 |
|
3 |
6.0 |
6.0 |
6.0 |
0.408±0.00075 |
0.543±0.00075 |
1.206±0.00089 |
|
4 |
8.0 |
8.0 |
8.0 |
0.536±0.00081 |
0.784±0.00042 |
1.628±0.00051 |
|
5 |
10 |
10 |
10 |
0.648±0.00089 |
1.021±0.00078 |
1.968±0.00043 |
Table 2. Intraday precision data for estimation of MND,
MNI and BA*(n=3)
|
Conc. (μg/ml) |
MND (270.00 nm) |
MNI (257.42 nm) |
BA (245.61nm) |
||
|
MND |
MNI |
BA |
Avg.±
RSD* |
Avg. ±
RSD* |
Avg. ±
RSD* |
|
2.0 |
2.0 |
2.0 |
0.181±0.84 |
0.097±0.57 |
0.460±0.36 |
|
4.0 |
4.0 |
4.0 |
0.288±0.65 |
0.301±0.82 |
0.839±0.86 |
|
6.0 |
6.0 |
6.0 |
0.425±0.24 |
0.559±0.49 |
1.487±0.54 |
RESULT AND DISCUSSION:
The standard solution of MTZ, MNI and BA were scanned
separately between 200-400nm. For MND, MNI and BA the linearity
range 2.0-10 µg/ml were divided by the divisor 2.0 µg/ml of MTZ. The divided
spectra were converted to second derivative using DL value 16 and SF value 100.
Here MND, MNI and BA shows peak at 270.0, 257.42 and 245.61 nm.
VALIDATION PARAMETERS:[4,5]
1. Linearity and Range
The second order derivative spectra of division spectra of
MND, MNI and BA (divisor
MTZ 2.0 µg/ml) showed linear absorbance at 270.0 nm for MND (2.0-10µg/ml),
257 . 41 nm for MNI (2.0-10 µg/ml) and 245.61 nm for BA.
This method obeyed beer’s law in the concentration range 2.0 -10 µg/ml, for MTZ, MND and BA respectively. Correlation
coefficient (r2) for calibration curve of MND, MNI and BA were found
to be 0.999, 0.998 and 0.997, respectively
Fig.5 Calibration curve for MND
at 270.0nm
ig.6 Calibration curve for MNI
at 257.42 nm
Fig.7 Calibration curve for BA
at 245.61 nm
The regression line equation for MND, MNI and BA are as
following,
y = 0.116x - 0.152 for MND
y = 0.059x + 0.054 for MNI
y = 0.194x + 0.047 for BA
I. Intraday precision
The data for intraday precision for combined standard
solution of MND, MNI and BA is presented in Table. The % R.S.D was found to be
0.24 -0.84 % for MND, 0.49-0.82 % for MNI and 0.36-0.86 % for BA. These %RSD
values were found to be less than ± 2.0 indicated that the method is precise.
II. Interday precision
The data for interday precision for combined standard
solution of MND, MNI and BA is presented in Table. The % R.S.D was found to be
0.38-0.91 % for MND, 0.54-0.96 % for MNI and 0.39-0.99 % for BA. These %RSD
values were found to be less than ± 2.0 indicated that the method is precise.
III. Accuracy
Accuracy of the method was determined by recovery study
from dosage form at three levels (80%, 100%, and 120%) by spiking method. The %
recovery values are tabulated in Table. Percentage recovery for MTZ, MNI and BA
by this method was found in the range of 100.21 to 100.16 %, 100.32 to 100.65 %
and 100.31 to 100.75 %, respectively. The value of %RSD within the limit
indicated that the method is accurate and percentage recovery shows that there
is no interference from the excipients.
IV. Limit of Detection and Quantification
The LOD for MND, MNI and BA was conformed to be 0.049
µg/ml, 0.029 µg/ml and 0.039 µg/ml, respectively. The
LOQ for MND, MNI and BA was conformed to be 0.150µg/ ml, 0.079
µg/ml and 0.115 µg/m, respectively. The obtained LOD and LOQ results are presented
in Table 6.
V. Robustness and Ruggedness
The % R.S.D was
found to be 0.24 - 0.86 % for MTZ,
0.19 – 0.96 % for MNI and 0.19 -
0.89 % for BA.
These %RSD value was found to be less than ± 2.0 indicated that the method
is precise. No significant
changes
in
the
spectra were
observed, proving that the
developed
method is rugged and
robust. For different stock
solutions for MTZ stock-1 is 100 µg/ml and stock-2 is 50 µg/ml, for MNI stock-1 is 100 µg/ml
and stock-2 is 50 µg/ml.
Where for BA stock-1 is 100 µg/ml
and stock-2 is 50 µg/ml.(Table-7).
Table 3. Interday precision data for estimation of MND,
MNI and BA*(n=3)
|
Conc. (μg/ml) |
MND(270.0
nm) |
MNI(257.42
nm) |
BA(245.61
nm) |
||
|
MND |
MNI |
BA |
Avg.±
RSD* |
Avg.±RSD* |
Avg. ±
RSD* |
|
2.0 |
2.0 |
2.0 |
0.182±0.91 |
0.099±0.63 |
0.461±0.39 |
|
4.0 |
4.0 |
4.0 |
0.289±0.63 |
0.302±0.96 |
0.846±0.99 |
|
6.0 |
6.0 |
6.0 |
0.426±0.38 |
0.561±0.54 |
1.513±0.58 |
Table 4. Recovery data
of MND *(n=3)
|
Conc. of MND from formulation (µg/ml) |
Amount of Std.MND added (µg/ml) |
Total amount of MND (µg/ml) |
Total amount of
MND found (µg/ml)
Mean ± SD* |
% Recovery (n=3) |
% RSD MTZ |
|
5.0 |
4.0 |
9.0 |
9.00±0.0038 |
100.13 |
0.12 |
|
5.0 |
5.0 |
10 |
10.02±0.0459 |
100.16 |
0.18 |
|
5.0 |
6.0 |
12 |
12.02±0.0322 |
100.21 |
0.24 |
Table 5. Recovery data
of MNI *(n=3)
|
Conc. of MNI from formulation (µg/ml) |
Amount of Std. MNI added (µg/ml) |
Total amount of MNI (µg/ml) |
Total amount of
MNI found (µg/ml)
Mean ± SD* |
% Recovery (n=3) |
% RSD
MNI |
|
0.0 |
0.4 |
0.4 |
0.40± 0.00418 |
100.43 |
0.19 |
|
0.0 |
0.5 |
0.5 |
0.50± 0.00413 |
100.32 |
0.12 |
|
0.0 |
0.6 |
0.6 |
0.62±0.000839 |
100.65 |
0.28 |
Table 6. Recovery
data of BA*(n=3)
|
Conc. of BA from formulation (µg/ml) |
Amount of Std.BA added (µg/ml) |
Total amount of BA (µg/ml) |
Total amount of
BA found (µg/ml)
Mean ± SD* |
% Recovery (n=3) |
% RSD
BA |
|
0.0 |
0.4 |
0.4 |
0.410±0.0052 |
100.31 |
0.20 |
|
0.0 |
0.5 |
0.5 |
0.513 ±
0.0075 |
100.53 |
0.23 |
|
0.0 |
0.6 |
0.6 |
0.60±0.0011 |
100.75 |
0.19 |
Table 6. LOD and LOQ data of MTZ,
MNI and BA*(n=10)
|
Conc. (μg/ml) |
MND(270.0 nm) |
MNI(257.42 nm) |
BA(245.61 nm) |
|||||
|
MND |
MNI |
BA |
Avg. ± SD* |
% RSD |
Avg. ± SD* |
% RSD |
Avg. ± SD* |
% RSD |
|
2.0 |
2.0 |
2.0 |
0.178±0.0042 |
0.23 |
0.096±0.0082 |
0.32 |
0.431±0.0031 |
0.19 |
|
LOD(μg/ml) |
0.049 |
0.026 |
0.038 |
|||||
|
LOQ(μg/ml) |
0.150 |
0.079 |
0.115 |
|||||
Table 7. Robustness
and
Ruggedness MND, MNI and BA*(n=3)
|
Drugs |
Concentration (ppm) |
Mean ±% RSD(n=3) |
Mean ±% RSD(n=3) |
||
|
Stock
Solution I |
Stock
Solution II |
Instrument I |
Instrument II |
||
|
Metronidazole |
02 |
0.182±0.86 |
0.183±0.81 |
0.181± 0.86 |
0.184±0.84 |
|
04 |
0.288±0.65 |
0.289±0.64 |
0.287±0.65 |
0.289±0.63 |
|
|
06 |
0.431±0.25 |
0.434±0.47 |
0.436±0.47 |
0.435±0.24 |
|
|
2-Methyl-5-Nitroimidazole |
02 |
0.097±0.34 |
0.098±0.34 |
0.099±0.34 |
0.097±0.34 |
|
04 |
0.301±0.96 |
0.302±0.19 |
0.304±0.93 |
0.305±0.19 |
|
|
06 |
0.559±0.65 |
0.558±0.15 |
0.556±0.68 |
0.557±0.15 |
|
|
Benzoic
Acid |
02 |
0.460±0.48 |
0.462±0.29 |
0.463±0.19 |
0.463±0.20 |
|
04 |
0.839±0.89 |
0.838±0.67 |
0.837±0.59 |
0.838±0.43 |
|
|
06 |
1.487±0.54 |
1.486±0.55 |
1.488±0.73 |
1.489±0.68 |
|
Metronidazole
Benzoate oral suspension contains 200mg/5 ml and Pipette out accurately 1.0 ml of suspension in to 100 ml
volumetric flask make up the volume with Methanol, sonicate
for 15 min. Filter it with Nylon membrane filters (0.22 µm, 20 mm D) Pipette
out 1.0 ml dillute solution into 100 ml volumetric
flask make up the volume using Methanol Measure absorbance at 270.0 nm, 257.42
nm and 245.61 nm Determine the
concentration of MND using equation.
Table 8. Analysis data of commercial formulation *(n=3)
|
DRUGS |
% Assay ± % RSD(n=3) |
IP LIMIT |
|
Metronidazole |
100.41±0.39
|
99.0-101.0%
|
All the parameters for all the three substances met the
criteria of the ICH guidelines for the method validation and found to be
suitable for routine quantitative analysis in pharmaceutical dosage forms. The
result of linearity, accuracy, precision proved to be within limits with lower
limits of detection and quantification. Ruggedness and Robustness of method was
confirmed as no significant were observed on analysis by subjecting the method
to slight change in the method condition.
Assay results obtained by proposed method are in fair agreement.
Table 9. Summary
of validation parameters
|
Parameter |
Metronidazole |
2-Methyl-5-Nitroimidazole |
Benzoic Acid |
|
λmax |
270.0 nm |
257.42 nm |
245.61 nm |
|
Concentration range (µg/ml) |
2.0 – 10 |
2.0 – 10 |
2.0– 10 |
|
Regression
equation |
y = 0.059x + 0.054 |
y = 0.116x - 0.152 |
y = 0.194x + 0.047 |
|
Correlation
coefficient (r2) |
0.999 |
0.998 |
0.997 |
|
Accuracy(%Recovery) (n=3) |
100.16 |
100.45 |
100.53 |
|
Intra-day Precision (%RSD) (n=3) |
0.24-0.84 |
0.49-0.82 |
0.36-0.86 |
|
Inter-day precision (%RSD) (n=3) |
0.38-0.91 |
0.54-0.96 |
0.39-0.99 |
|
LOD (µg/ml) |
0.049 |
0.026 |
0.038 |
|
LOQ (µg/ml) |
0.150 |
0.079 |
0.115 |
|
Ruggedness and Robustness |
0.24–0.86 |
0.15–0.96 |
0.19– 0.89 |
|
% Assay |
100.41 |
– |
– |
CONCLUSION:
The developed UV-spectroscopy
method was proved to be simple, rapid & reproducible. The validation data
indicate good specificity, precision, accuracy & reliability of the method.
The developed method offers several advantages in terms simultaneous
determination of Metronidazole Benzoate and related impurity in bulk and
pharmaceutical formulation.
ACKNOWLEDGEMENT:
We are
sincerely thankful to Shree Dhanvantary Pharmacy
College, Kim, Surat, for providing us infrastructure
facilities and moral support to carry out this research work. We are also
thankful to SDPARC for giving us their special time and guidance for this
research work. We also thank our colleagues for their helping hand.
REFERENCE:
1.
Introduction
Available on: 2010 www.ebookbrowse.com/ metronidazole-
metronidazole-spiramycin
2.
Rao N R, Mani SS,
and Prasanthi N.L, “Pharmaceutical Impurities: An
Overview.”Indian J. Pharm. Educ. Res.
2010, 44(3), 301-309.
3.
International
Conference on Harmonization, Harmonized Tripartite Guideline, Validation of
Analytical Procedures Text and Methodology, ICH Q2(R1),
2005.
4.
Davidson AG, Beckett AH and Stenlake JB; Practical Pharmaceutical Chemistry; 4th
edition, part-2; CBS Publishers, New Delhi, 2002, pp 275-300.
5.
Engineering Mathematics; Open Learning
Unit Level 1: “Cramer’s Rule for Solving Simultaneous Linear Equations”,
www.3.ul.ie/chap 8/8.1.pdf.
Received on 25.03.2015 Accepted on 11.04.2015
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Tech. 2015; Vol. 5: Issue 2, Pg
66-70
DOI: 10.5958/2231-5713.2015.00011.2