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Kashif et al. World Journal of Pharmacy and Pharmaceutical Sciences
DEVELOPMENT AND VALIDATION OF RP-HPLC-UV ANALYTICAL
METHOD FOR QUANTITATIVE DETERMINATION OF
METRONIDAZOLE
Kashif Ali Safdar1*, S. Baqir S. Naqvi2, Shahnaz Gauhar3, Abdullah Jabbar4
1*Research Scholar, Department of Pharmaceutics, Faculty of Pharmacy, University of
Karachi, Sindh Pakistan.
2Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi, Sindh Pakistan.
3RAK Medical & Health Sciences University, Ras Al Khaimah, UAE.
4Getz Pharma Private Limited, Karachi Pakistan.
ABSTRACT
The aim of the present study was to develop a simple, rapid and
sensitive reversed-phase high performance liquid chromatographic
(RH-HPLC) analytical method for the quantitative determination of
metronidazole in pharmaceutical dosage form. Metronidazole is a
nitroimidazole antibiotic medication used particularly for anaerobic
bacteria and protozoa.. The chromatographic separation was carried
out on Shimadzu prominence series 20-A apparatus equipped with
autosampler, degassing unit and built-in cooler by using Hibar® C18
RP column (250mm X 4.6mm, 5µm) as stationary phase and 0.01M
potassium dihydrogen phosphate (pH 3.0) and acetonitrile (83:17,
V/V) as mobile phase at 1.0 mL/minute flow rate. The UV detection
was carried out at 320nm. The method was validated as per the ICH
guideline for Validation of Analytical Procedures Q2(R1) and found to be specific without
interferences from mobile phase components and impurities. The method was linear over the
range of 10µg/mL to 30µg/mL (r2 = 0.9999) with adequate level of accuracy and precision.
The method has shown acceptable level of robustness with repeatability, reproducibility and
intermediate precision. The proposed method can be confidently employed with adequate
level of assurance for the routine testing of metronidazole in pharmaceutical dosage form.
KEYWORDS: Metronidazole, RP-HP Analytical Method, Validation by ICH.
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Article Received on
05 Oct 2014,
Revised on 30 Oct 2014,
Accepted on 26 Nov 2014
Nov 2014
*Correspondence for
Author
Kashif Ali Safdar
Research Scholar,
Department of
Pharmaceutics, Faculty of
Pharmacy, University of
Karachi, Sindh Pakistan.
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INTRODUCTION
Pharmacists in practice today work with drug products which are much more effective and
much more complex than the patent medicines of the 19th century. The major responsibility
of pharmacist have developed in safeguarding drug product quality through the selection of
source of supply and providing information to the public and other health professionals on
question of drug quality.
The choice of an analytical method is usually governed by the intrinsic analytical properties
of the drug molecule or its amenability to chemical derivation to render it compatible to
quantitation. The reliability of the quantitation depends on these analytical techniques.
Various chromatographic methods used for the analysis of pharmaceutical substances and
pharmaceutical dosage form. Currently, reversed phase high–performance liquid
chromatography with UV detection represents the analytical method of choice for the
quantitative determination of raw material, in-processes formulation, finished products as
well as in the physiological matrix likewise it is the method of choice for checking purity of
new drug candidates, monitoring changes during scale up or revision of synthetic procedures,
evaluating new formulations, and running control/assurance of the final drug product. It is a
very simple, accurate and robust technique for the analysis of pharmaceutical products.
Nowadays, Pharmacist pays their consideration on the development of HPLC method for the
identification, quantification and purification of individual components of the mixture thus
due to its high degree of accuracy, its utilization and popularity for the analysis of
pharmaceutical agents is continuously increasing.
Metronidazole is a nitroimidazole antibiotic medication used particularly for anaerobic
bacteria and protozoa. It is an antibiotic, amebicide, and antiprotozoal. [1] It is the drug of
choice for first episodes of mild-to-moderate Clostridium difficile infection. [2] In 1953
Maeda and its co-worker isolated an antibiotic named azomycin (2-nitro imidazole) from
streptomycete and later on in 1956; Horie established its affectivity against the
Trichomoniasis which ultimately resulted in synthesis and biological evaluation of number of
nitroimidazole compounds. One of them was 1-(β-hydroxyethyl)-2-methyl-5-nitroimidazole
now a day’s named as metronidazole showed a good result in vitro and in vivo against the T.
vaginalis and E. histolytica.[3]
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Its chemical structure and formula is as follows,
Chemical formula: C6H9N3O3
Molecular weight: 171.2
Chemical structure:
[4]
SCOPE
The objective of the study was to develop and validate a method which is applicable for
quantitative determination of metronidazole in pharmaceutical dosage forms. It will provide
simple and rapid analytical method for dissolution, assay and content uniformity analysis
during routine testing of metronidazole in different pharmaceutical dosage forms. It will also
be applicable for the identification and purity testing of Metronidazole in raw material with
high degree of accuracy, precision and sensitivity. It will also provide the cost effective
method for the determination of metronidazole during drug formulation development process
and for quality testing during stability studies.
MATERIALS AND METHOD
Chemicals
The chemicals used during this research includes HPLC grade acetonitrile of Fischer
Chemical origin, analytical grade ortho-phosphoric acid of Merck origin, analytical grade
potassium dihydrogen phosphate of sigma Aldrich origin and Metronidazole were obtained as
gift sample from Sanofi-Aventis Pakistan limited. Flagyl 400mg tablets were purchased from
local market.
Mobile Phase Preparation
0.01M potassium dihydrogen phosphate buffer prepared and its pH adjusted to 3.0 with help
of dilute phosphoric acid. Then both 0.01M phosphate buffer and acetonitrile were mixed
together in a ratio of 83:17 (V/V), finally filtered through 0.45µm membrane filtered
Sample Preparation
Stock solution of Metronidazole (500µg/mL) was prepared in mobile phase and samples were
prepared by serial dilution method. The standard solution of Metronidazole (20µg/mL) was
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prepared by weighing 40.0mg reference standard powder and dissolving in 100mL mobile
phase and diluting it to 20µg/mL concentration. The sample of Flagyl 400mg tablets were
prepared by grinding the tablets to fine powder in a mortar and pestle and then weighing and
dissolving in mobile phase an amount equivalent to 40.0mg of metronidazole, finally diluting
to concentration of 20µg/mL
Chromatographic System
Shimadzu high pressure liquid chromatography (HPLC) prominence series model 20-A was
used which were equipped with autosampler, built-in cooler, degassing unit and injection
loop of 100µL with UV detector. The analysis was performed by using Isocratic reverse
phase technique with Hibar® C18, 5µm (250mm X 4.6mm) HPLC column of Merck origin.
The system also have Lab Solution software to control the chromatographic analysis and for
the data acquisition and integration. The samples were analyzed by auto injection of 25µL
solution at a flow rate of 1.0mL/minute with λmax of 320nm.
Validation Parameters
System Suitability
System suitability testing is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, electronics, analytical operations, and samples to be
analyzed constitute an integral system that can be evaluated as such. [5] The samples were run
on system to check the system suitability before starting the validation.
Specificity and Selectivity
Specificity or selectivity of the method is the ability to assess unequivocally the analyte in the
presence of components which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc.[6] The specificity and selectivity of the method was
established by analyzing the blank, spike amount (10µg/mL), flagyl 400mg tablets and
sample of flagyl 400mg tablets + spike amount of metronidazole (10µg/mL).
Linearity and Range
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the sample
and range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which it
has been demonstrated. [6] Linearity and range of the method was established by analyzing
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the samples of five different concentrations prepared by serial dilution from standard stock
solution.
Accuracy
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value and
the value found. [6] The accuracy of the method was established by triplicate injection of
50%, 100% and 150% concentration samples prepared by separate pipetting for each sample
from standard stock solution.
Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions. [6] The precision of the method was
demonstrated at two different levels i.e. repeatability and intermediate precision.
Limit of Detection (LOD)
The detection limit of an individual analytical procedure is the lowest amount of analyte in a
sample which can be detected but not necessarily quantitated as an exact value. The
capability of the method to detect the lowest amount of the metronidazole was determined by
using signal to noise ratio method described in the ICH guideline. [6]
Limit of Quantification (LOQ)
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in
a sample which can be quantitatively determined with suitable precision and accuracy. [6] The
limit of quantification of the method was also determined by signal to noise ratio method
described by the ICH guideline. Five samples of LOQ concentrations were prepared by serial
dilution method and analyzed on the system.
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides an indication of its
reliability during normal usage. [6]
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RESULTS AND DISCUSSION
The need to validate an analytical method is encountered by analysis in the pharmaceutical
industry on an almost daily basis, because adequately validated methods are a necessity for
approvable regulatory filings. The biggest advantage of method validation is that it builds a
degree of confidence, not only for the developer but also to the user that the results of the
analysis are accurate and precise secondly it results inexpensive by eliminating the frustrating
repetitions and leads to better time management in the end. Similarly the minor changes in
the conditions such as reagent supplier or grade, analytical setup are unavoidable due to
obvious reasons but the method validation absorbs the shock of such conditions and pays for
more than invested on the process.
The Internal Conference on Harmonization (ICH) guidelines have been mutually accepted as
law in the Europe, Japan and in the United States, but in reality, besides these other countries
are also using them. As these guidelines reflect the current inspectional tendencies, they carry
the de facto force of regulation. [7] Similarly United States Pharmacopeia, Food and Drug
Administration of United States also outlines the requirement and procedure of method
validation but after the International Conference on Harmonization (ICH) guidelines
“Validation of Analytical Procedures: Text and Methodology” Q2(R1) is now accepted and
followed worldwide and is also used as basis for the validation of this RP-HPLC analytical
method.[6, 8-10]
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(A) (B)
(C) (D)
Figure: 1. Chromatograms of specificity
(A) Chromatogram of blank run
(B) Chromatogram of spike amount of standard
(C) Chromatogram of Flagyl 400mg Tablets
(D) Chromatogram of Spike amount of standard + Flagyl 400mg tablets
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Figure No. 2. Calibration curve (linearity) of metronidazole
Table 01: System Suitability Parameters & Results Data
Parameter
Value
Retention Time (minutes)
5.333 (%RSD - 0.01%)
Theoretical Plates
9496
Tailing Factor
1.215
Peak Area %RSD
0.03%
Table 02: Regression Analysis of Calibration Curve
Parameter
Result
Linearity
10µg/mL - 30µg/mL
Regression Equation
y = 80656x - 18911
Correlation Coefficient
1.0000
R2 (Residual Sum of squares)
0.9999
Slope
80656
Y-Intercept
-18911
X-Intercept
0.2358
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Table 03: Accuracy Data
Concentration
% Recovery
Result
Statistical Tool
50%
100.52
Mean
100.43%
101.56
SD
1.18
99.21
%RSD
1.18%
100%
100.42
Mean
100.12%
100.02
SD
0.27
99.91
%RSD
0.27%
150%
100.56
Mean
100.59%
100.71
SD
0.11
100.49
%RSD
0.11%
Over all Mean
100.38 %
Over all SD
0.64
Over all %RSD
0.64 %
Table 04: Precision Repeatability Data
Concentration
Sample No.
%Result
100 %
(20µg/mL)
1
99.61
2
100.31
3
100.62
4
100.80
5
99.98
6
98.38
Mean
99.95%
SD
0.88
%RSD
0.88%
Table 05: Intermediate Precision Data
Concentration
Sample No.
%Result
100 %
(20µg/mL)
1
100.77
2
99.33
3
98.83
4
99.45
5
99.85
6
100.47
Mean
99.78%
SD
0.73
%RSD
0.73%
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Table 06: Robustness Parameters & Results Data
Change Type
%Result
Statistical Tool
Flow Rate
0.8 mL
100.16
Mean
100.10%
100.41
SD
0.35
99.72
%RSD
0.35%
Flow Rate
1.2 mL
100.02
Mean
99.99%
100.36
SD
0.38
99.60
%RSD
0.38%
Detector Wavelength
318nm
100.12
Mean
100.00%
100.23
SD
0.31
99.65
%RSD
0.31%
Detector Wavelength
322 nm
100.09
Mean
99.96%
100.16
SD
0.29
99.62
%RSD
0.29%
Mobile Phase
Buffer:ACN,
820:180
100.09
Mean
99.94%
100.17
SD
0.33
99.56
%RSD
0.33%
Mobile Phase
Buffer:ACN,
840:160
100.26
Mean
99.99%
100.15
SD
0.37
99.56
%RSD
0.37%
Mobile Phase Buffer pH
at pH 2.9
100.08
Mean
99.86%
100.01
SD
0.32
99.49
%RSD
0.32%
Mobile Phase Buffer pH
at pH 3.1
100.12
Mean
99.87%
100.03
SD
0.35
99.47
%RSD
0.35%
Sample Stability
(Fresh, after 24Hrs, after 48
Hrs.)
99.77
Mean
99.70%
99.74
SD
0.09
99.60
%RSD
0.09%
Validation
System suitability was achieved by injecting six replicated injection of standard
metronidazole solution. The percentage Relative standard deviation (% RSD) of the retention
times and the peak areas of metronidazole were 0.01% and 0.03% respectively. The Mean
theoretical plates (mean) count, based on USP tangent calculations (USP-2014) for
metronidazole peak was 9496 (Table 1).
Specificity and selectivity of the method was established by running the different sample
such as blank, spike amount (10µg/mL), flagyl 400mg tablets and sample of flagyl 400mg
tablets + spike amount of metronidazole (10µg/mL). The resultant chromatogram did not
show any interference from excipients, mobile phase components, impurities and degradation
products as shown in Fig:1.
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Linearity
The method was linear over the range of 10µg/mL to 30µg/mL with adequate level of
accuracy and precision. Calibration curve was constructed (Fig: 2); r2 value was calculated
and found to be 0.9999. Regression analysis is summarized in Table. 2.
Accuracy
The percent recoveries was calculated by samples solution prepared in triplicate at three
levels over a range of 50-150% and analyzed according to the procedure. The percentage
recovery ranged from 99.21 % to 101.56 % with overall mean of 100.38 % and % RSD value
of 0.64% (Table. 3).
Repeatability expresses the precision under the same operating conditions over a short
interval of time. It is also termed intra-assay precision [6]. The repeatability of the method
was established by injecting six samples of 100% concentration (20µg/mL) prepared by
weighing the sample amount each time. The result of all the samples is tabulated in Table No.
4. % RSD calculated and found to be 0.88% which is well within the acceptance limit of ≤
2%.
Intermediate precision of the method was demonstrated by different analyst on different day,
using different HPLC column and system. The result of samples is tabulated in Table No. 5;
the calculated %RSD of 0.73% is well within the acceptance limit of ≤2%.
The calculations for Limit of detection (LOD) and limit of quantification (LOQ) were done
on the basis of peak responses of metronidazole standard solution and it was found LOD =
8.14ng/mL and LOQ = 32.56ng/mL respectively. Accuracy and % RSD calculated and found
to be of 100.80% and 0.61% respectively.
The robustness of the purposed method was checked by making slight variations in flow rate
(0.8-1.2ml/min), change in ratio of mobile phase (820: 180 vs 840: 160), change in pH (from
2.9 to 3.1) and also made the changes in detector wavelength that was 318 and 322nm (Table.
No. 6). All the results show that the method is robust and rugged with no significant changes
in the results.
The stability of the drug in mobile phase was evaluated, in order to verify that no degradation
has taken place between the time of sample preparation and the analyses. The percentage
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RSD values obtained were 0.09% after the 48 hours of sample preparation (Table No. 6).
That shows the drug is stable in the composition of purposed mobile phase
CONCLUSION
The results of all the validation parameters are well within the acceptance criteria defined by
the ICH guideline for the validation of analytical procedures hence it lead to conclude that the
proposed RP-HPLC-UV analytical method is simple, rapid, sensitive, accurate, precise and
robust for the quantitative determination of metronidazole and can be employed for the
routine testing of metronidazole in oral solid dosage forms. Since the method is sensitive,
cost effective and utilizes the most simple and common technique of HPLC that makes it
suitable for the purity testing of raw materials, testing of dissolution samples, testing during
drug formulation development and testing during stability studies.
ACKNOWLEDGEMENTS
I would like to thank Sanofi-Aventis Pakistan for providing the gift sample of metronidazole
standard drug. I would also like to thank Dr. S. Baqir S. Naqvi, Dr. Shahnaz Gauhar &
Abdullah Jabbar for their continuous support and guidance throughout the project.
The authors would like to thank Department of Pharmaceutics, Faculty of Pharmacy,
University of Karachi, Karachi, Pakistan, for all financial and moral supports
REFERENCES
1. Merck Manual for Professionals (2011) archived from
http://www.merckmanuals.com/professional/lexicomp/metronidazole.html.
2. http://www.reboundhealth.com/cms/images/pdf/metronidazole%20id%2014724.pdf.
3. James W. Tracy and Leslie T. Webster, Jr. (2001) drugs used in the chemotherapy of
protozoal infections in Goodman and Gilman’s the pharmacological basis of therapeutics,
Tenth Edition, McGraw-Hill, chapter 41; pp1105-1108.
4. British Pharmacopoeia 2009 volume I&II Monographs Medicinal and Pharmaceutical
substances-Metronidazole pp 3952-3955.
5. Guidance for industry Q2B Validation of Analytical Procedures: Methodology FDA US,
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidanc
es/ucm073384.pdf.
6. ICH Q2(R1), Validation of Analytical Procedures: Text and Methodology.
7. http://shodhganga.inflibnet.ac.in/bitstream/10603/8513/9/09_chapter%202.pdf.
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8. Guidance for Industry Analytical Procedures and Methods Validation FDA US,
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidanc
es/ucm386366.pdf.
9. Guidance for Industry Analytical Procedures and Methods Validation for Drugs and
Biologics
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidanc
es/ucm386366.pdf
10. Validation of compendia methods USP-36, Chapter number 1225.