Asian J. Research Chem. 2(1): Jan.

-March, 2009 ,

ISSN 0974-4169 RESEARCH ARTICLE

www.ajrconline.org

Simultaneous RP-HPLC Estimation of Nitazoxanide and Ofloxacin in Tablet Dosage Forms

Dept. of Pharmaceutical Analysis, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore- 641 402. Tamilnadu, India. *Corresponding Author E-mail: andrilan@rediffmail.com

R Siva Kumar*, P Kumar Nallasivan, S Saravanakumar, CS Kandasamy and R Venkatnarayanan

ABSTRACT

Reverse Phase HPLC method for the determination of nitazoxanide and ofloxacin in bulk and tablet formulations. The determination was carried out by using Phenomenex C18 column with 0.24% sodium lauryl sulphate: acetonitrile: acetic acid (pH-4.0) (58:40:02) as the mobile phase. The flow rate was 1.5 ml/ min. and the eluent was monitored at 295 nm. The Retention time of nitazoxanide and ofloxacin were 2.2 and 5.4 respectively. Linearity for the nitazoxanide and ofloxacin were found in the range of 400-600 g/ml and 160 - 240 g/ml respectively. The method was reproducible, with good resolution between nitazoxanide and ofloxacin and can be use for routine analysis.

KEY WORDS RP-HPLC, Nitazoxanide and Ofloxacin

Nitazoxanide (NTX) is chemically [2-[(5-nitro-1, 3-thiazol-2yl) carbamoyl] phenyl] ethanoate1. It is a synthetic antiprotozoal agent for oral administration. Ofloxacin (OFX) is chemically ()-9-Fluoro-2,3-dihydro-3-methyl-iO-(4-methyl1-piperazinly)-7-oxo-7H-pyrido[1,2,3-de]1,4 benzoxazine-6carboxilic acid2. It is used for the treatment of gonorrhoea and is an alternative treatment to ciprofloxacin for anthrax. Few methods such as spectrophotometric method 1-6, HPLC method 7-10 have been reported for individual drugs or in combination with other drugs in formulation. So far no simultaneous method has been reported for estimation of NTX and OFX. The aim of this study was to develop a precise, specific, accurate and sensitive method for simultaneous determination of NTX and OFX combination by RP-HPLC method. NTX was obtained from Alembic pharmaceuticals Ltd, Mumbai, India and OFX from Pharmafabricon Ltd Madurai. Acetonitrile HPLC grade of Rankem, sodium lauryl sulphate and acetic acid AR grade of S.D. Fine chemicals were used for the study. A Shimadzu Binary Gradient system was used coupled with SPD 10A UV detector and Rheodyne injector with 20 l fixed loop. Chromatographic analysis was performed using spinchrome software on a phenomenex C18 column (250x4.5mm, 5 particle size) at ambient temperature. The mobile phase consisting of 0.24% sodium lauryl sulphate: acetonitrile: acetic acid (pH-4.0) (58:40:02) was pumped at a flow rate 1.0 ml per min, the detection was monitored at 295 nm. Received on 22.12.2008 Accepted on 15.02.2009 Modified on 30.01.2009 AJRC All right reserved

INTRODUCTION:

Preparation of stock solution of Nitazoxanide and Ofloxacin: Standard stock solutions were prepared by dissolving 100 of NTX and 40 mg of OFX in two 100 ml flask separately in 80 ml of mobile phase and diluted to 100 ml with the same. In case of NTX varying amounts of (4.0, 4.5, 5.0, 5.5 and 6.0) of the above stock solution of NTX (1000 g/ml) was taken in five different 10 ml volumetric flasks and the volume was made up to the mark with the mobile phase. An aliquot of 20 l of the solution from each flask was injected two times. In case of OFX, varying amounts (4.0, 4.5, 5.0, 5.5, and 6.0 ml) of the above stock solution of OFX (400 g/ml) was taken in five different 10 ml volumetric flasks and the volume was made up to the mark with the mobile phase. Chromatographic Conditions: Calibration curve: An aliquot of 20 l of the solution from each flask was injected two times. Calibration curves were constructed by plotting mean peak areas against the corresponding drug concentrations. NTX and OFX were found to be linear in the range of 400-600 g/ml and 160-240 g/ml with coefficient of correlation (r2) 0.9995 and 0.9996 for NTX and OFX, respectively. Determination of NTX and OFX in their combined dosage forms: Twenty tablets were powdered finely. A quantity equivalent to 250 and 100 mg of NTX and OFX was transferred to a 100 ml volumetric flask and 30 ml of mobile phase was added. The flask was shaken for 15 min and then contents were diluted to 100 ml and filtered through Whatman No 41

MATERIALS AND METHODS:

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TABLE 1: ASSAY RESULTS OF COMBINED DOSAGE FORM Labeled amount (mg/tablet) Amount obtained (mg/tablet RSD*) Formulations NTX OFX NTX OFX Brand A 500 200 501.50.044 203.10.021 Brand B 500 200 500.90.073 199.70.056 * denotes average of five determinations. NTX and OFX denote, Nitazoxanide and Ofloxacin respectively. TABLE 2: VALIDATION AND SYSTEM SUITABILITY PARAMETERS Parameter Linearity Range (g/ml) Correlation Coefficient (r2) S.D* Retention time (min.) S.D* Resolution Tailing factor Theoretical plates NITAZOXANIDE 400-600 0.99990.202 2.20.2 34.28 1.11 13537 1.90 6568 0.06 0.36 0.36-0.74 0.355 OFLOXACIN 160-240 0.99840.142 5.49 0.05

Asian J. Research Chem. 2(1): Jan.-March, 2009 ,

% Assay* NTX OFX 100.30 101.55 100.18 99.85

Specificity: The specificity of the RP-HPLC method was determined by complete separation of NTX and OFX as shown in Fig No. 1 with parameters like retention time (tR), resolution (Rs) and tailing factor (T). Here tailing factor for peaks of NTX and OFX was less than 2% and resolution was satisfactory. The peaks obtained for NTX and OFX were sharp and have clear base line separation. Detection limit and quantification limit: A calibration curve was prepared by using concentration in the expected detection limit range of 0.2-4 g/ml for NTX and OFX. The standard deviation of y-intercepts of regression lines were determined and kept in the following equation for the determination of detection limit and quantification limit. Detection limit = 3.3 /s; quantification limit = 10 /s; Where is the standard deviation of yintercepts of regression lines and s is the slope of the calibration curve. Robustness: Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1.5 0.2 ml/min), pH of the mobile phase (pH 4 2) concentration of sodium lauryl sulphate (0.24 0.2 %) and also by observing the stability of the drugs for 24 h at 350 temperature in the mobile phase.

Limit of detection 0.2 (g/ml) Limit of 5.1 quantification (g/ml) Precision (RSD*, 0.20-0.85 %) Intraday (n=5) Repeatability 0.205 (RSD*, %) n=5 * Mean of five determinations (n=5).

filter paper. 2 ml of this solution was then diluted to 10 ml with mobile phase solution. Results of the triplicate analysis are given in Table 1. The method was validated for statistical parameters i.e. precision, accuracy, specificity, linearity, stability, of analytical solutions and ruggedness criteria. Results of the method validation experiments are given in Table 2. The precision of the method was determined by knowing percentage RSD of means of three replicate solutions of all the three independent samples. Precision: The intra day precision study of NTX and OFX was carried out by estimating the corresponding responses five times on the same day and the results are reported in terms of relative standard deviation (RSD, Table 2). Accuracy: The accuracy of method is determined by adding known amount of standard to that of sample (above and below the normal level) at 3 different levels to cover both above and below (80% to 120%) the normal levels expected in the sample.

RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both NTX and OFX. The mobile phase 0.24% sodium lauryl sulphate: acetonitrile: acetic acid (58:40:02 v/v) was found to be satisfactory and gave two symmetric and well resolved peaks for NTX and OFX. The resolution between NTX and OFX was found to be 34.28 which indicates good separation of both the compounds. The retention time for NTX and OFX were 2.2 min and 5.45 min, respectively (Fig. no 1). The asymmetric factors for NTX and OFX were 1.24 and 1.03, respectively.
Fig. 1 A Typical Chromatogram of Nitazoxanide (NTX) and Ofloxacin (OFX).

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TABLE 3: RECOVERY STUDIES OF NTX AND OFX IN COMBINED DOSAGE FORM Nitazoxanide Ofloxacin Formulations % added % recovered % RSD* % recovery % added % recovered % RSD* Brand A 80 80.240.016 100.30 80 80.680.29 100 100.020.28 100.02 100 100.330.38 120 120.310.075 100.25 120 120.140.31 80 80.570.250 100.71 80 79.520.030 Brand B 100 99.940.315 99.94 100 100.970.230 120 121.970.356 101.64 120 120.890.071 *Mean of five determinations (n=5). NTX and OFX denote, Nitazoxanide and Ofloxacin respectively.

Asian J. Research Chem. 2(1): Jan.-March, 2009 ,

% recovery 100.85 100.33 100.11 99.40 100.97 100.74

The calibration curve for NTX was obtained by plotting the peak area of NTX versus the concentrations of NTX over the range of 400-600 g/ml, and it was found to be linear with r2 = 0.9999. Similarly, the calibration curve for OFX was obtained over the range of 160-240 g/ml and was found to be linear with r2 = 0.9984. The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2. The quantitation limit for NTX and OFX were 5.1 and 0.36 g/ml respectively. The recoveries of NTX and OFX were found to be in the range of 99.94-101.64% and 99.12-100.90%, respectively. The system suitability test parameters are shown in Table 2. The chromatographic method was applied to the determination of NTX and OFX in their combined dosage forms (Tablet formulation A and B). The result for NTX and OFX were comparable with the corresponding labeled amounts (Table 1). Proposed study describes a new RP-HPLC method for estimation of NTX and OFX combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time. The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of NTX and OFX in their combined dosage form. The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, for providing the required facilities and also to Alembic pharmaceuticals Ltd, Mumbai and Pharmafabricon Ltd Madurai, for providing the gift sample of nitazoxanide and ofloxacin.

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ACKNOWLEDGEMENTS:

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