INTRODUCTION:

Multi-drug administration is often associated with clinically significant interaction, especially of narrow therapeutic index drugs, either at pre-absorption or post-absorption stage^.1.

Cefixime (CEF) is an oral third generation cephalosporin antibiotic. Chemically, it is (6R,7R) -7- {[2- (2-amino- 1,3- thiazol- 4-yl)-2-(carboxymethoxyimino) acetyl]amino}-3- ethenyl-8-oxo-5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2- carboxylic acid, clinically used in the treatment of susceptible infections including gonorrhoea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis, and urinary-tract infections^2. Ofloxacin,³ is an antimicrobial drug and chemically it is 9-fluro-2,3-dihydro -3- methyl -10- (4-methyl-1- piperizinyl) -7-oxo-7H –pyrido [1,2,3-de] -1,4-benzoxaine- 6-carboxylicacid.

Cefixime and Ofloxacin are markedly of combined tablet dosage form in the ratio of 200 mg each. Literature survey reveals that Cefixime can be estimated by spectrophotometrically⁴, HPLC^5-9 and by HPTLC¹⁰ individually or with other drugs in bulk drugs and in human plasma, literature survey reveals that various analytical methods have been reported for the estimation of OFLOX in single and in combination dosage form such as, Spectrophotometric^11-17 HPLC^18-27 and LC/MS/MS^28,29

Experiment:

Reagents and Solvents:

Cefixime and Ofloxacin were obtained from Sucram Pharmaceuticals Pvt Ltd, Theda, HP, India. All solvents used in the sample preparation and chromatographic separation were of the HPLC and AR grade Acetonitrile are supplied by Merck, Mumbai, India. Tetrabutyl ammonium hydrogen sulphate and Phosphoric acid were supplied by qualigens pharmaceutical, Mumbai, India. Ultra pure water was made using a Milli-Q ultra pure system (Millipore, Bedford, MA).

Instruments:

The apparatus used for HPLC was Shimadzu prominence, LC 20AT, Chromatography pump coupled with UV-VIS-SPD 20A detector (Shimadzu, Kyoto, Japan) and Rheudyne injector with 20 ml fixed loop. Chromatographic condition was Gemini, Phenomenex C18 (50X4.6 mm I.D, 5m) at ambient temperature. The mobile phase consisting of Acetonitrile : 0.05 M Tetrabutyl ammonium hydrogen sulphate (pH 6.0 with phosphoric acid)(35:65 v/v) was pumped at flow rate of 2.0 ml/min. The detection was monitored at 290 nm.

Preparation of stock solution:

Stock solution were prepared dissolving 200 mg of Cefixime and Ofloxacin in two 100 ml volumetric flask separately 80 ml of mobile phase and diluted to 100 ml with same. From this pipetted out 4.0, 4.5, 5.0, 5.5, 6.0 ml of stock solution transferred into 50 ml volumetric flask and made upto volume with mobile phase.

Preparation of sample solution:

20 Tablets were powdered finely. The quantity equivalent 200 mg of Cefixime and Ofloxacin was transferred to 100 ml volumetric flask and 80 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 filter paper. 5 ml of this solution was then diluted to 50 ml with mobile phase solution.

Method validation:

The method was validated for statistical parameters i.e. precision, accuracy, specificity, linearity, and robustness 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 intraday precision study of Cefixime and Ofloxacin was carried out by estimating the corresponding responses six times on the same day and the results are reported in terms of relative standard deviation (RSD, Table 2).

Table.1. Assay results of combined dosage form

Formulations	Labeled amount (mg/tablet)		Amount obtained (mg/tablet)		% assay
Formulations	CEF	OFX	CEF	OFX	CEF	OFX
Tablet	200	200	199.112	199.302	99.56	99.5

Table.2. Validation and System suitability Parameters

Parameter	Cefixime	Ofloxacin
Linearity range µg/ml	160-240	160-240
Correlation Coefficient (r²) ± S.D	0.9999	0.9999
Retention time (min.) ± S.D	7.443	4.277
Resolution	14.520
Tailing Factor	1.023	1.074
Theoretical Plate	12505	10133
Limit of detection (µg/ml)	0.23	0.47
Limit of Quantification (µg/ml)	0.7	1.43
Precision (RSD %) intraday (n=6)	0.39-0.27	0.45-0.49
Repeatability (RSD %) (n=6)	0.333	0.2

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.

Specificity:

The specificity of the RP-HPLC method was determined by complete separation of Cefixime and Ofloxacin as shown in Fig No.1 with parameters like retention time (t_R), resolution (Rs) and tailing factor (T). Here tailing factor for peaks of Cefixime and Ofloxacin was less than 2% and resolution was satisfactory. The peaks obtained for Cefixime and Ofloxacin were sharp and have clear base line separation.

Fig.1. Typical Chromatogram of Cefixime and Ofloxacin

Detection limit and quantification limit:

A calibration curve was prepared by using concentration in the expected detection limit range of 160-240 μg/ml for Cefixime and Ofloxacin. Detection limit of Cefixime and Ofloxacin was 0.23 μg/ml and 0.47 μg/ml respectively. quantification limit of Cefixime and Ofloxacin was 0.7 μg/ml and 1.43 μg/ml.

Robustness:

Robustness of the method was studied by deliberate variations of the analytical parameters such as pH of the mobile phase (pH 6± 2) concentration of Tetra butyl ammonium hydrogen sulphate (65 ±10 %).

RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both Cefixime and Ofloxacin. The mobile phase acetonitrile: tetra butyl ammonium hydrogen sulphate (pH:6.0 with phosphoric acid) (35:65 v/v) was found to be satisfactory and gave two symmetric and well resolved peaks for Cefixime and Ofloxacin. The resolution between Cefixime and Ofloxacin was found to be 14.52 which indicates good separation of both the compounds. The retention time for Cefixime and Ofloxacin were 7.43 min. and 4.27 min., respectively (Fig. no 1). The asymmetric factors for Cefixime and Ofloxacin were 1.023 and 1.074, respectively.

The calibration curve for Cefixime was obtained by plotting the peak area of Cefixime versus the concentrations of Cefixime over the range of 160-240 μg/ml, and it was found to be linear with r² = 0.9999. Similarly, the calibration curve for Ofloxacin was obtained over the range of 160-240 μg/ml and was found to be linear with r² = 0.9999. The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2. The quantitation limit for Cefixime and Ofloxacin were 0.7 μg/ml and 1.43 μg/ml respectively.

The recoveries of Cefixime and Ofloxacin 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 Cefixime and Ofloxacin in their combined dosage forms (Tablet formulation A ). The result for Cefixime and Ofloxacin were comparable with the corresponding labeled amounts (Table 1). Proposed study describes a new RP-HPLC method for estimation of Cefixime and Ofloxacin 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 Cefixime and Ofloxacin in their combined dosage form.

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Received on 16.12.2009 Modified on 18.01.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 367-369