Innovare International Journal of Pharmacy and Pharmaceutical Sciences Academic Sciences ISSN- 0975-1491 Vol 7, Issue 2, 2015 Original 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: [email protected] 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; KH 2PO4+K2HPO4):methanol: acetonitrile in the proportion of 30:50:20 v/v. The mobile phase was set at a flow rate of 1.2 271 nm. Resultml/mins: and The the developed volume injectedmethod wasresulted 20μl forin Metronidazole every injection. eluting The detection at 2.92 minwavelength and Nalidixic was set acid at at 3.99 min. Metronidazole the range 30- - deviations of 0.714% for Metronidazole and 0.398% for Nalidixic acid. Percentage Mean recoveries were found to be in the rangeexhibited of 98- 102,linearity during in 70μg/ml, The limit while of detection Nalidixic (LOD) acid exhibitedfor Metronidazole linearity andin theNalidixic range acid 45 105μg/ml.were found The to be precision 2.48µg/ml is andexemplified 2.24µ by relative standard while limit of quantitiation (LOQ) for Metronidazole and Nalidixic acid were found to be 7.51µg/ml and 6.79µ accuracy studies. g/ml respectively, Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed forg/ml simultaneous respectively. quantitative estimation of Metronidazole and Nalidixic acid in tablets and validated as per ICH guidelines. Hence it Metronidazole and Nalidixic acid in tablets in various pharmaceutical industries. can be used for the routine analysis of Keywords: RP-HPLC, Metronidazole, Nalidixic acid, Method development, Validation. INTRODUCTION species. It wor nting their growth tract Metronidazole (fig. is 2-(2- -5-nitro-1H- bacteria to survive.ks by Itkilling has a the molecular bacteria formula and preve of C 12H12N2O3 and a imidazol-1- ethanol [1]. Metronidazole is a nitro imidazole molecularby stopping weight theof 2 production32.235 g/mol. of essential proteins needed by the antibiotic drug used 1)against chemically anaerobic organisms,methyl amoebozoa infections anyl) mild-to- CH3 moderate Clostridium difficile infection [2]. Metronidazole is also used to treatd bacterialantiprotozoal. vaginosis It is frequently used for N N CH3 pseudomembranous colitis, aspiration pneumonia, rosacea, fungating wounds, intra-abdominal infections,, pelvic lung inflammatory abscess, gingivitis, disease, amoebiasis, giardiasis, t HOOC susceptible anaerobic organisms such as Bacteroides , fragilis spp O Fusobacterium spp, Clostridiumrichomoniasis, spp, Peptostreptococcus and infections caused spp andby Prevotellaspp [3]. It has a molecular formula of C 6H9N3O3 and a Fig. 2: Structure of Nalidixic acid molecular weight of 171.15 g/mol. N re exists literature on chromatographic methods for Nalidixic acid and Metronidazole H C NO A detailed literaturein combination survey reveals with that other the drugs [4-24] in various 3 N 2 matrices. There exist spectrophotometric [25], HPTLC [26] and supercriticalindividually andfluid chromatographic method [27] for the simultaneous quantitative estimation of Metronidazole and Nalidixic acid in pharmaceutical dosage forms, while there is OH literature reported on RP-HPLC method development and validation Fig. 1: Structure of Metronidazole for this combination. Hence we have explored in developihardlyng a new,any accurate, precise, linear and a rapid isocratic RP-HPLC method for the simultaneous quantitative estimation of Metronidazole and Nalidixic acid (fig. is 1- -1,4- -7- -4- Nalidixic acid in tablets and validate as per ICH guidelines. oxo-1,8- -3- antibiotic effective 2)against chemically both gramethyl-positivedihydro and grammethyl-negative MATERIALS AND METHODS bacteria.naphthyridine At lower concentrations,carboxylic it acid acts. Itas is a a bacteriostatic synthetic quinolone and at Chemicals and reagents higher concentrations; it is bactericidal [4]. Nalidixic acid is an inhibitor of the A sub Nalidixic acid is Metronidazole and Nalidixic acid with indicated for the treatment of purities greater than 99% were obtained as gift samples from susceptible gram- unit of bacterial DNA gyrase. Analytically pure sample of of E. Coli, Enterobacter species,urinary Klebsiella tract species,infections and caused Proteus by Distab negative microorganisms, including the majority Chandra Labs, Hyderabad, India and tablet formulation [Gramogyl ] was procured from Medplus pharmacy, Hyderabad, India Ashok et al. Int J Pharm Pharm Sci, Vol 7, Issue 2, 367-371 with labelled amount 100mg and 150mg of Metronidazole and Nalidixic acid and Methanol injection. The detection wavelength was set at 271 nm. (HPLC Grade) were obtained from Sigma Aldrich flow rate of 1.2 ml/min and the volume injected was 20μl for every respectively. Acetonitrile (HPLC, grade)water (HPLC grade), Preparation of mixed buffer solution 2 4(Hyderabad, India) potassium ortho phosphate (KH PO ) and di potassium 1.625 gm of potassium orth phosphate (KH 2PO4) and anddrogen SD Fine ortho chem. phosphate (Hyderabad) (K2HPO respectively4) (AR grade), ortho phosphoric 0.3 gm of dipotassium ortho phosphate (K2HPO 4) was dihydrogen weighed and dissolved indihydrogen 100 ml of water and volume was made up hy to 1000 ml with water. Adjusthydrogen the pH to 4.5 using ortho phosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, acid . The buffer was filtered India), 0.45μm Nylon membrane filters were obtained from Instrument through 0.45µ filters to remove all fine particles and gases. Spincotech Private Limited, Hyderabad, India. and potassium hydroxide solution was performed on Shimadzu LC-20AT VP Liquid Mobile phase preparation Chromatograph comprising a LC-20AT pump, Shimadzu SPD-20A acetonitrile, methanol UVHPLC-VISIBLE analysis detector and a reverse phase C18 column, Inertsil ODS and mixed phosphate buffer in the ratio of 20:50:30 v/v and later it 3V (250X4.6 mm; 5 ). Thewas sonicatedmobile phase for 10 was minutes prepared for the by removal mixing of air bubbles. μ A manuallySpinchromoperating” software. Rheodyne A doubleinjector beam with Diluent 20UV -μLvisible sample spectrophotometer loop was equipped Nicolet with theevolution HPLC system.100 having The HPLC two Diluent used is the mobile phase itself. systemmatched was quartz controlled cells with with 1 cm“ light path was used for recording of spectra and measuring absorbance. An Preparation of mixed standard solution weighing BL220H), digital pH meter (Global digital) and sonicator (Citizenelectronic) were used analytical in this 75mg of Nalidixic acid and 50 mg of Metronidazole balance (1mg sensitivity, Shimadzu in 100 ml of volumetric flask and dissolve in 10 ml of mobile phase Weighand make accurately up the volume with mobile phase. From above stock study.Method s solution 75µg/ml of Nalidixic acid and 50µg/ml of Metronidazole is 1 ml to 10 ml with mobile phase. This is treated Selection of wavelength as mixed working standards solution, 100% target concentration. prepared by diluting recording UV spectrums in the range of 200-400 nm for individual Preparation of stock and working sample solution drugSuitable solutions wavelength of Nalidixicfor the HPLCacid andanalysis Metronidazole was determined. Suita bleby 10 tablets were weighed and taken into a mortar, crushed and wavelength selected for simultaneous estimation is 271 nm (fig. 3-4). Nalidixic acid (15 Metronidazole (100 dissolvingthen uniformly average mixed. of 10 tablets,Test stock equivalent solutions to 15of0mg of Nalidixic acid00μg/ml) and 100 mgand of Metronidazole and0μg/ml) made upwere to 10prepared0 ml with by mobile phase. Sonicated for 5 min and later filtered the solution using 0.45 0.5 ml of the above stock solution was pipetted out and made up to 10 ml to get working sample solution equmicronivalent syringe to a concentrationfilter. 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 . e. Resolution factor (Rs) between peaks, factor (A), number of theoretical plates (N), runtimesystem suitability and the cost parameters effectiveness. i The optimized method developed Fig. 3: UV spectrum of standard Metronidazole resultedAsymmetric 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 The total run time is 6 minutes. respectively. and are used to ensure adequate performance of the chromatographic System suitability tests areRt), an number integral of part theoretical of method plates development (N), peak resolution (Rs) and factor (A) was evaluated for six replicatesystem.