Analytical Method Development and Validation of Cefixime and Dicloxacillin Tablet by RP-HPLC

 

Kathiravan S.*, Anbarasi B. and Mathankumar S.

Department of Pharmaceutical Analysis, J.K.K.Munirajah Medical Research Foundation College of Pharmacy, Komarapalayam, Namakkal-Dt, Tamilnadu, India-638183.

*Corresponding Author E-mail: kathir85shiva@gmail.com

 

ABSTRACT:

A simple and sensitive high-performance liquid chromatography method with ultraviolet (UV) detection has been developed and validated procedure was developed for simultaneous determination of Cefixime and Dicloxacillin in tablet dosage form by reverse phase C18 column (water symmetry C18 5µ, 250mm x 4.6mm). The mobile phase used as combination of Acetonitrile: Potassium dihydrogen Phosphate in the ratio of 60:40 (pH adjusted to 6.0 with potassium hydroxide). The detection of the combined dosage form was carried out at 220 nm and flow rate of 1.0 ml/min. The retention time for Cefixime and Dicloxacillin was found to be 2.027 and 3.249 min respectively, and recoveries from combined dosage form were between 98% to 102%. Linearity obtained in the concentration ranges from 60, 80, 100, 120, 140% Cefixime and Dicloxacillin with correlation coefficient of 0.9991 and 0.9996 respectively. The method herein described was successfully applied for the evaluation of Cefixime and Dicloxacillin combined dosage form.

 

KEYWORDS: Cefixime, Dicloxacillin and HPLC.

 


 

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..

 

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 gonorrhea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis, and urinary-tract infections(2,3,4). Dicloxacillin is known chemically (2S, 5R, 6R)-6-{[3-(2, 6-dichlorophenyl)-5-methyloxazole-4-carbonyl] amino}-3, 3-dimethyl-7-oxo-4-thia-1azabicyclo [3.2.0] heptane-2-carboxylic acid. It has an empirical formula of C19H17N3O5S. Dicloxacillin is isoxazolyl penicillin used primarily for the treatment of infections due to staphylococci resistant to benzyl penicillin.

 

These include bone and joint infections, endocarditic, pneumonia, skin infections (including soft-tissue infections), and toxic shock syndrome1,2.

 

Both the drugs are marketed as combined dose tablet formulation in the ratio of 200:500 mg Cefixime trihydrate: Dicloxacillin sodium. Literature survey reveals that cefixime can be estimated by spectrophotometrically3,4, HPLC5-12 and by HPTLC13 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 Dicloxacillin single and in combination dosage form such as, Spectrophotometric,  HPLC14-17.

 

MATERIAL AND METHODS:

Reagents and Solvents:

Cefixime and Dicloxacillin were obtained from Sucram 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. Potassium dihydrogen phosphate and potassium hydroxide 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 Agilent 1100 series , Chromatography pump coupled with UV-VIS-SPD 20A detector (Shimadzu, Kyoto, Japan) and Rheudyne injector with 20 ml fixed loop. Chromatographic condition was Gemini, C18 (250X4.6 mm I.D, 5m) at ambient temperature. The mobile phase consisting of Acetonitrile: Potassium di hydrogen phosphate in the ratio of 60:40 v/v (pH adjusted to 6.0 with potassium hydroxide) was pumped at flow rate of 1.0 ml/min. The detection was monitored at 220 nm.

 

Preparation of Standard solution:

Stock solutions were prepared dissolving 55 mg of cefixime and 125mg Dicloxacillin in 100 ml volumetric flask. And add 80 ml of mobile phase shake and diluted to 100 ml with same. From this pipette out 5ml of above solution to 50ml.

 

Preparation of sample solution:

20tablets were powdered finely. The quantity equivalent one tablet weight 1.0142gm of cefixime and Dicloxacillin was transferred to 200 ml volumetric flask and 80 ml of mobile phase was added. The flask was shaken for 15 min. and then contents were diluted to 200 ml and filtered through whatmann No. 41 filter paper. 5 ml of this solution was then diluted to 100 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 Dicloxacillin 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).

 

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 (tR), resolution (Rs) and Asymmetry. Here tailing factor for peaks of cefixime and Dicloxacillin was less than 2% and resolution was satisfactory. The peaks obtained for Cefixime and Dicloxacillin were sharp and have clear base line separation.

 

Fig.1. Typical Chromatogram of Cefixime  and  Dicloxacillin

 

Linearity of Cefixime

Sl. No

Conc. in (%)

Area

Average Area

1

60

1415581

1463452

1439516

2

80

1887432

1920135

1903783

3

100

2347070

2371535

2359302

 

4

120

2831162

2881922

2856542

5

140

3303023

3258371

3280697

 

Robustness:

Robustness of the method was studied by deliberate variations of the analytical parameters such as pH of the mobile phase (pH 6± 0.2) and flow rate of change ± 0.2ml.and wavelength changed.

 

Table.1. Assay results of combined dosage form

Formulations

Labeled amount (mg/tablet)

Amount obtained (mg/tablet)

% assay

CEF

Diclox

CEF

Diclox

CEF

Diclox

Tablet

200

500

193.33

499.37

96.67

99.91

 

 


RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both Cefixime and Dicloxacillin. The mobile phase acetonitrile: potassium dihydrogen phosphate (pH adjusted to 6.0 with potassium hydroxide) (40:60 v/v) was found to be satisfactory and gave two symmetric and well resolved peaks for Cefixime and Dicloxacillin. The resolution between cefixime and ofloxacin was found to be 8.34 which indicate good separation of both the compounds. The retention time for cefixime and ofloxacin were 2.044 min and 3.27 min, respectively (Fig. no 1). The asymmetric factors for cefixime and Dicloxacillin. 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 33-77 μg/ml, and it was found to be linear with r2=  0.9991. Similarly, the calibration curve for Dicloxacillin was obtained over the range of 75-150 μg/ml and was found to be linear with r2= 0.9996. The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2.

 

Linearity of Dicloxacillin

Sl. No

Conc. in (%)

Standard weight in mg

Sample Area

1

60

3404212

3413754

3408983

 

2

80

4574741

4542831

4558786

3

100

5673687

5634298

5653992

4

120

6892662

6919020

6905841

5

140

8036193

8015920

8026050

 

 

The recoveries of cefixime and Dicloxacillil were found to be in the range of 98.44-99.39% and 99.83-100.23%, respectively. The system suitability test parameters are shown in Table 2. The chromatographic method was applied to the determination of cefixime and Dicloxacillin in their combined dosage forms (Tablet formulation A ).

 

Table.2. Validation and System suitability Parameters

Parameter

Cefixime

Ofloxacin

Linearity range µg/ml

33-77

75-175

Correlation Coefficient (r2) ± S.D

0.9991

0.9996

Retention time (min.) ± S.D

0.3008

0.3645

Resolution

8.91

Tailing Factor

1.002

1.008

Theoretical Plate

4692

7167

Precision (RSD %) (n=5)

0.6762

0.7752

Repeatability (RSD %) (n=5)

0.902

0.8323

 

The result for cefixime and Dicloxacillin were comparable with the corresponding labeled amounts (Table 1).

 

The ruggedness was performed. All parameters including flow rate, temperature, detection, wavelength and sensitivity are maintained constant through the procedure except for robustness studies

 

DISCUSSION:

Proposed study describes a new RP-HPLC method for estimation of cefixime and Dicloxacillin 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 Dicloxacillin in their combined dosage form.

 

REFERENCES:

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Received on 11.02.2010        Modified on 10.03.2010

Accepted on 25.03.2010        © AJRC All right reserved

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 865-868