Int J Pharm Pharm Sci, Vol 7, Issue 2, 367-371Original Article
RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTITATIVE ESTIMATION OF METRONIDAZOLE AND NALIDIXIC ACID IN TABLETS
S. YASWANTH KUMAR1, P. SIVAPRASAD1, A. ASHOK KUMAR2*
1Chandra Labs, 5-5-35/173, Plot No-10, 1st Floor, IDA Prasanthi Nagar, Kukatpally, Hyderabad, India 500090, 2Department of Pharmaceutical analysis and Quality Assurance, Vijaya college of pharmacy, Munaganur (village), Hayathnagar (mandal), Hyderabad 501511, India.
Email: ashok576@gmail.com
Received: 08 Nov 2014 Revised and Accepted: 02 Dec 2014
ABSTRACT
Objective: To develop an accurate, precise and linear Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method for simultaneous quantitative estimation of Metronidazole and Nalidixic acid in tablets and validate as per ICH guidelines.
Methods: The optimized method uses a reverse phase C18 column Inertsil ODS C18 column (250X4.6 mm×5µ) mobile phase consisting of mixed phosphate buffer (pH 4.5; KH2PO4+K2HPO4):methanol: acetonitrile in the proportion of 30:50:20 v/v. The mobile phase was set at a flow rate of 1.2 ml/min and the volume injected was 20μl for every injection. The detection wavelength was set at 271 nm.
Results: The developed method resulted in Metronidazole eluting at 2.92 min and Nalidixic acid at 3.99 min. Metronidazole exhibited linearity in the range 30-70μg/ml, while Nalidixic acid exhibited linearity in the range 45-105μg/ml. The precision is exemplified by relative standard deviations of 0.714% for Metronidazole and 0.398% for Nalidixic acid. Percentage Mean recoveries were found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Metronidazole and Nalidixic acid were found to be 2.48µg/ml and 2.24µg/ml respectively, while limit of quantitiation (LOQ) for Metronidazole and Nalidixic acid were found to be 7.51µg/ml and 6.79µg/ml respectively.
Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of Metronidazole and Nalidixic acid in tablets and validated as per ICH guidelines. Hence it can be used for the routine analysis of Metronidazole and Nalidixic acid in tablets in various pharmaceutical industries.
Keywords: RP-HPLC, Metronidazole, Nalidixic acid, Method development, Validation.
INTRODUCTION
Metronidazole (fig. 1) chemically is 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethanol [1]. Metronidazole is a nitro imidazole antibiotic drug used against anaerobic organisms, amoebozoa infections and antiprotozoal. It is frequently used for mild-to-moderate Clostridium difficile infection [2]. Metronidazole is also used to treat bacterial vaginosis, pelvic inflammatory disease, pseudomembranous colitis, aspiration pneumonia, rosacea, fungating wounds, intra-abdominal infections, lung abscess, gingivitis, amoebiasis, giardiasis, trichomoniasis, and infections caused by susceptible anaerobic organisms such as Bacteroides fragilis spp, Fusobacterium spp, Clostridium spp, Peptostreptococcus spp and Prevotellaspp [3]. It has a molecular formula of C6H9N3O3 and a molecular weight of 171.15 g/mol.
Fig. 1: Structure of Metronidazole
Nalidixic acid (fig. 2) chemically is 1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid. It is a synthetic quinolone antibiotic effective against both gram-positive and gram-negative bacteria. At lower concentrations, it acts as a bacteriostatic and at higher concentrations; it is bactericidal [4]. Nalidixic acid is an inhibitor of the A subunit of bacterial DNA gyrase. Nalidixic acid is indicated for the treatment of urinary tract infections caused by susceptible gram-negative microorganisms, including the majority of E. Coli, Enterobacter species, Klebsiella species, and Proteus species. It works by killing the bacteria and preventing their growth tract by stopping the production of essential proteins needed by the bacteria to survive. It has a molecular formula of C12H12N2O3 and a molecular weight of 232.235 g/mol.
Fig. 2: Structure of Nalidixic acid
A detailed literature survey reveals that there exists literature on chromatographic methods for Nalidixic acid and Metronidazole individually and in combination with other drugs [4-24] in various matrices. There exist spectrophotometric [25], HPTLC [26] and supercritical fluid chromatographic method [27] for the simultaneous quantitative estimation of Metronidazole and Nalidixic acid in pharmaceutical dosage forms, while there is hardly any literature reported on RP-HPLC method development and validation for this combination. Hence we have explored in developing a new, accurate, precise, linear and a rapid isocratic RP-HPLC method for the simultaneous quantitative estimation of Metronidazole and Nalidixic acid in tablets and validate as per ICH guidelines.
MATERIALS AND METHODS
Chemicals and reagents
Analytically pure sample of Metronidazole and Nalidixic acid with purities greater than 99% were obtained as gift samples from Chandra Labs, Hyderabad, India and tablet formulation [Gramogyl Distab] was procured from Medplus pharmacy, Hyderabad, India with labelled amount 100mg and 150mg of Metronidazole and Nalidixic acid respectively. Acetonitrile (HPLC grade) and Methanol (HPLC Grade) were obtained from Sigma Aldrich (Hyderabad, India) and SD Fine chem. (Hyderabad) respectively, water (HPLC grade), potassium dihydrogen ortho phosphate (KH2PO4) and di potassium hydrogen ortho phosphate (K2HPO4) (AR grade), ortho phosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, India), 0.45μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.
Instrument
HPLC analysis was performed on Shimadzu LC-20AT VP Liquid Chromatograph comprising a LC-20AT pump, Shimadzu SPD-20A UV-VISIBLE detector and a reverse phase C18 column, Inertsil ODS 3V (250X4.6 mm; 5μ). A manually operating Rheodyne injector with 20 μL sample loop was equipped with the HPLC system. The HPLC system was controlled with “Spinchrom” software. A double beam UV-visible spectrophotometer Nicolet evolution 100 having two matched quartz cells with 1 cm light path was used for recording of spectra and measuring absorbance. An electronic analytical weighing balance (1mg sensitivity, Shimadzu BL220H), digital pH meter (Global digital) and sonicator (Citizen) were used in this study.
Methods
Selection of wavelength
Suitable wavelength for the HPLC analysis was determined by recording UV spectrums in the range of 200-400 nm for individual drug solutions of Nalidixic acid and Metronidazole. Suitable wavelength selected for simultaneous estimation is 271 nm (fig. 3-4).
Fig. 3: UV spectrum of standard Metronidazole
Fig. 4: UV spectrum of standard Nalidixic acid
Chromatographic conditions
The optimized method uses a reverse phase C18 column Inertsil ODS C18 column (250X4.6 mm×5µ) mobile phase consisting of mixed phosphate buffer (pH 4.5; KH2PO4+K2HPO4): methanol: acetonitrile in the proportion of 30:50:20 v/v. The mobile phase was set at a flow rate of 1.2 ml/min and the volume injected was 20μl for every injection. The detection wavelength was set at 271 nm.
Preparation of mixed buffer solution
1.625 gm of potassium dihydrogen orth phosphate (KH2PO4) and 0.3 gm of dipotassium hydrogen ortho phosphate (K2HPO4) was weighed and dissolved in 100 ml of water and volume was made up to 1000 ml with water. Adjust the pH to 4.5 using ortho phosphoric acid and potassium hydroxide solution. The buffer was filtered through 0.45µ filters to remove all fine particles and gases.
Mobile phase preparation
The mobile phase was prepared by mixing acetonitrile, methanol and mixed phosphate buffer in the ratio of 20:50:30 v/v and later it was sonicated for 10 minutes for the removal of air bubbles.
Diluent
Diluent used is the mobile phase itself.
Preparation of mixed standard solution
Weigh accurately 75mg of Nalidixic acid and 50 mg of Metronidazole in 100 ml of volumetric flask and dissolve in 10 ml of mobile phase and make up the volume with mobile phase. From above stock solution 75µg/ml of Nalidixic acid and 50µg/ml of Metronidazole is prepared by diluting 1 ml to 10 ml with mobile phase. This is treated as mixed working standards solution, 100% target concentration.
Preparation of stock and working sample solution
10 tablets were weighed and taken into a mortar, crushed and then uniformly mixed. Test stock solutions of Nalidixic acid (1500μg/ml) and Metronidazole (1000μg/ml) were prepared by dissolving average of 10 tablets, equivalent to 150mg of Nalidixic acid and 100mg of Metronidazole and made up to 100 ml with mobile phase. Sonicated for 5 min and later filtered the solution using 0.45micron syringe filter. 0.5 ml of the above stock solution was pipetted out and made up to 10 ml to get working sample solution equivalent to a concentration of 75µg/ml for Nalidixic acid and 50µg/ml for Metronidazole, concentrations equal to 100% target concentration.
RESULTS AND DISCUSSION
Method development
A reverse phase HPLC method was developed keeping in mind the system suitability parameters i. e. Resolution factor (Rs) between peaks, Asymmetric factor (A), number of theoretical plates (N), runtime and the cost effectiveness. The optimized method developed resulted in the elution of Nalidixic acid at 3.99 min and Metronidazole at 2.92 min. Fig. 5 and 6 represent chromatograms of blank solution and mixture of standard solutions respectively. The total run time is 6 minutes.
System suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (Rt), number of theoretical plates (N), peak resolution (Rs) and Asymmetric factor (A) was evaluated for six replicate injections of the standards at working concentration. The results given in table 1 were within acceptable limits.
Fig. 5: Typical chromatogram of blank solution
Fig. 6: Typical chromatogram of mixture of standards solution
Fig. 7: Typical chromatogram of sample solution
Table 1: System suitability studies results
| Parameters | Acceptance Limits | Nalidixic acid | Metronidazole |
| Retention time (min) | - | 3.994 | 2.921 |
| Resolution factor (Rs) | Not less Than 2 | 3.577 | |
| Number Of Theoretical plates (N) | Not less Than 2000 | 4492 | 3749 |
| Tailing factor (T) | Not More Than 2 | 1.9 | 1.41 |
In order to test the applicability of the developed method to a commercial formulation, ‘Gramogyl Distab’ tablets were chromatographed at working concentration and it is shown in fig. 7. The sample peaks were identified by comparing the relative retention times with the mixture of standards solution (fig. 6-7). System suitability parameters were within the acceptance limits, ideal for the chromatographed sample. Integration of separated peak area was done and each drug concentration was determined by using the peak area concentration relationship obtained in the standardization step. The protocol affords reproducible quantification of the two drugs with error less than 10%, which is the standard level in any pharmaceutical quality control.
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. HPLC method developed was validated according to International Conference on Harmonization (ICH) guidelines [28] for validation of analytical procedures. The method was validated for the parameters like linearity, accuracy, system precision, intra-day precision, limit of detection (LOD) and limit of quantitiation (LOQ).
Specificity
Fig. 5-7 for blank, mixture of standards drug solution and sample chromatogram reveal that the peaks obtained in the standards solution and sample solution at working concentrations are only because of the drugs as blank has no peak at the retention time of Nalidixic acid and Metronidazole standards. Accordingly it can be concluded that, the method developed is said to be specific.
Precision
System precision
Six replicate injections of the mixture of standards solution at working concentration showed % RSD (Relative Standard Deviation) less than 2 concerning peak area for both the drugs, which indicate the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in Tables 2-3.
Method precision
Method precision was determined by performing assay of the sample under the test of repeatability (Intraday precision) at working concentrations.
Repeatability (Intraday precision)
Six consecutive injections of the sample from the same homogeneous mixture at working concentration showed % RSD less than 2 concerning % assay for both the drugs which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results (Table 4).
Table 2: System precision results of Nalidixic acid
| Injection | Retention time (min) |
Peak area |
| 1 | 3.993 | 2012.58 |
| 2 | 3.993 | 2009.765 |
| 3 | 3.990 | 2023.634 |
| 4 | 3.987 | 2028.946 |
| 5 | 3.990 | 2031.51 |
| 6 | 4.010 | 2035.765 |
| Mean | 3.994 | 2023.700 |
| SD | 0.008 | 10.506 |
| %RSD | 0.21 | 0.52 |
Table 3: System precision results of Metronidazole
| Injection | Retention time (min) |
Peak area |
| 1 | 2.917 | 765.269 |
| 2 | 2.920 | 760.464 |
| 3 | 2.923 | 768.530 |
| 4 | 2.923 | 763.825 |
| 5 | 2.917 | 762.172 |
| 6 | 2.930 | 775.496 |
| Mean | 2.921 | 765.959 |
| SD | 0.0049 | 5.425 |
| %RSD | 0.17 | 0.71 |
Table 4: Intraday precision results of Metronidazole and Nalidixic acid
| Injection | % Assay (Nalidixic acid) | % Assay (Metronidazole) |
| 1 | 100.05 | 99.08 |
| 2 | 99.75 | 100.61 |
| 3 | 99.59 | 99.28 |
| 4 | 100.75 | 99.5 |
| 5 | 99.94 | 98.43 |
| 6 | 100.02 | 99.38 |
| Mean | 100.022 | 99.38 |
| SD | 0.399 | 0.71 |
| %RSD | 0.3989 | 0.714 |
Linearity
Standards solutions of Metronidazole and Nalidixic acid at different concentrations were prepared. Calibration curves (fig. 8-9) were constructed by plotting the concentration level versus corresponding peak area for both the drugs. The results show an excellent correlation between peak areas and concentration within the concentration range of 45-105µg/ml for Nalidixic acid and 30-70µg/ml for Metronidazole (Tables 5-6). The correlation coefficients were greater than 0.99 for both the drugs, which meet the method validation acceptance criteria and hence the method is said to be linear for both the drugs.
Table 5: Calibration data for Nalidixic acid
| Concentration (µg/ml) | Peak Area |
| 45 | 1131.154 |
| 60 | 1589.595 |
| 75 | 2210.885 |
| 90 | 2728.440 |
| 105 | 3181.713 |
| Regression equation | y=34.93x-451.6 |
| Regression coefficient | 0.997 |
Fig. 8: Linearity graph of Nalidixic acid
Table 6: Calibration data for Metronidazole
Concentration (µg/ml) |
Peak Area |
| 30 | 455.279 |
| 40 | 637.105 |
| 50 | 898.173 |
| 60 | 1062.233 |
| 70 | 1295.814 |
| Regression equation | y=21.06x-183.3 |
| Regression coefficient | 0.996 |
Fig. 9: Linearity graph of Metronidazole
Accuracy
Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of both the drugs at three different levels (80-120%). At each level, three determinations were performed. Percent mean recovery is calculated as showed in table 7. The accepted limits of mean recovery are 98% -102% and all observed data were within the required range, which indicates good recovery values and hence the accuracy of the method developed.
Table 7: Results of Accuracy studies for Metronidazole and Nalidixic acid
| Concentration level (%) | % Mean recovery metronidazole | % Mean recovery nalidixic acid |
| 80 | 98.20 | 101.43 |
| 100 | 98.11 | 101.96 |
| 120 | 100.47 | 99.75 |
Sensitivity
The sensitivity of measurement of Metronidazole and Nalidixic acid by use of the proposed method was estimated in terms of the limit of quantitation (LOQ) and limit of detection (LOD). LOQ and LOD were calculated by the use of the equations LOD = 3.3σ/S and LOQ = 10σ/S where σ is the standard deviation of response of calibration plots and S is the slope of the corresponding calibration plot. The limit of detection (LOD) for Metronidazole and Nalidixic acid were found to be 2.48µg/ml and 2.24µg/ml respectively, while limit of quantitiation (LOQ) for Metronidazole and Nalidixic acid were found to be 7.51µg/ml and 6.79µg/ml respectively.
CONCLUSION
A reverse phase HPLC isocratic method developed has been validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, limit of detection and limit of quantitation, for for simultaneous quantitative estimation of Metronidazole and Nalidixic acid in tablets. The developed method resulted in Metronidazole eluting at 2.92 min and Nalidixic acid at 3.99 min. Metronidazole exhibited linearity in the range 30-70μg/ml, while Nalidixic acid exhibited linearity in the range 45-105μg/ml. The precision is exemplified by relative standard deviations of 0.714% for Metronidazole and 0.398% for Nalidixic acid. Percentage Mean recoveries were found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Metronidazole and Nalidixic acid were found to be 2.48µg/ml and 2.24µg/ml respectively, while limit of quantitiation (LOQ) for Metronidazole and Nalidixic acid were found to be 7.51µg/ml and 6.79µg/ml respectively.
ACKNOWLEDGEMENT
The authors would like to thank the management of Chandra labs, Hyderabad, for providing the necessary facilities to carry out of this research work and also for providing drugs in form of gift samples.
CONFLICT OF INTERESTS
Declared None
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