INTRODUCTION:

Cefixime tri hydrate (CEF), is the third generation cephalosporin antibiotic. Chemically it is 5-thia-1-azabicyclo [4. 2. 0] oct-2- ene-2-corboxylic acid, 7-[[2-amino-4- thiazolyl) [(carboxymethoxy)imino] acetyl]amino]-3-ethenyl-8-oxo-, trihydrate, [6R-[6µ, 7b(Z)]]-(6R, 7R)-7-[2-(-amino-4-thiazolyl)glyoxylamido]-8-oxo-3-vinyl-5-thia-1-azabicyclo [4, 2, 0]oct- 2-ene-2-carboxylic acid, 72-(Z)-[O-carboxymethyl)oxime]¹. It is given orally in the treatment of susceptible infections including gonorrhoea, otitis media, pharyngitis, lower respiratory tract infections such as bronchitis, and urinary-tract infections^{2, 3, 4}.

Ofloxacin (OFL) is a fluoroquinolone derivative. Chemically, it is (±)-9-fluoro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido-[1, 2, 3-de]-1, 4-benzoxazine-6 carboxylic acid⁵. It is mainly used as antibacterial for the treatment of urinary tract infection and sexually transmitted diseases⁶. Structures of both the drugs (THC and DCF) are shown in figure 1, 2.

Ofloxacin is official in USP⁷ and BP⁸ whereas cefixime is official in USP¹, BP⁹ and Japanese Pharmacopoeia¹⁰. Both the drugs are marketed as combined tablet dosage form and the ratio is 1:1 CEF:OFL. Literature survey revealed that a number of analytical methods have been reported for estimation of CEF in combination with other drugs i.e. spectrophotometry^11-13. HPLC^14,15, RP-HPLC¹⁶. The analytical methods reported for estimation of OFL in combination with other drugs are spectrophotometry^17-
20, HPLC^{21, 22}, RP-HPLC²³. Present work describes simple, accurate, reproducible, rapid and economical method for simultaneous estimation of CEF and OFL in tablet formulation.

MATERIALS AND METHODS:

Apparatus:

Instrument used was an UV-Visible double beam spectrophotometer, maker: Shimadzu (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on Shimadzu analytical balance (Model AU-220).

Reagents and chemicals:

Pure drug samples of CEF and OFL were obtained from reputed pharmaceutical company.

Distilled water and methanol were used as solvent. Calibrated glassware was used throughout the work.

Marketed formulation:

Combined tablet formulation Zimnic-O 200 was procured from local market. Each tablet contains 200 mg Cefixime and 200 mg Ofloxacin.

Cefixime

Figure 1: Chemical structures of the cefixime.

Ofloxacin

Figure 2: Chemical structures of the ofloxacin.

Preparation of standard stock solution:

Accurately weighed quantity of CEF (100 mg) and OFL (100 mg) was transferred to two separate 100 ml volumetric flasks, dissolved in methanol and diluted to the mark with same. (Stock solutions: 1000 μg/ml of CEF and 1000 μg/ml of OFL).

Preparation of working standard solution:

100 mg/ml of CEF solution was prepared by diluting 10 ml of stock solution with distilled water in 100 ml volumetric flask up to the mark.

100 mg/ml of OFL solution was prepared by diluting 10 ml of stock solution with distilled water in 100 ml volumetric flask up to the mark.

Procedure for determination of wavelength for measurement:

2 ml of working standard stock solution of CEF (100 µg/ml) and 2 ml of working standard stock solution of OFL (100 µg/ml) were pipetted out into two separate 10 ml volumetric flask and volume was adjusted to the mark with water to get 20 µg/ml of CEF and 20 µg/ml of OFL. Each solution was scanned between 200-400 nm against water as a reagent blank and zero oder spectra shown in figure 3.

The first order derivative spectra of each solution were obtained. Smoothing of spectra done (Δ λ = 3 nm) The zero crossing points were found to be 358. 2 nm and 312. 0 nm for CEF and OFL respectively (figure 4 and 5).

Figure 3: Overlain zero order spectra of CEF (20 µg/ml) and OFL (20 µg/ml) in distilled water.

Figure 4. Overlain first order spectra of CEF (20 µg/ml) and OFL (20 µg/ml) in water.

Figure 5. 312 nm wavelength selected for estimation of Cefixime and 358. 2nm wavelength selected for estimation of Ofloxacin.

Figure 6: Calibration curve of standard CEF at 312. 0 nm by first order derivative spectrophotometry

Calibration curves for CEF and OFL:

Standard CEF solution from 5-40 µg/ml were prepared, by pipetting out 0. 5, 1. 0, 1. 5, 2. 0, 2. 5, 3. 0, 3. 5, and 4. 0 ml of the working standard stock solution of CEF (100 µg/ml) into series of 10 ml volumetric flasks and the volume was adjusted to mark with distilled water. Absorbance of each solution was measured at 312. 0 nm using first derivative spectrophotometry. A calibration curve was prepared by plotting absorbance against respective concentration (figure 6). Standard OFL solution from 5-40 µg/ml were prepared by pipetting out 0. 5, 1. 0, 1. 5, 2. 0, 2. 5, 3. 0, 3. 5, and 4. 0 ml of the working standard stock solution of OFL (100 µg/ml) into series of 10 ml volumetric flasks and the volume was adjusted to mark with distilled water. Absorbance of each solution was measured at 358. 2 nm using first order derivative spectrophotometry. A calibration curve was prepared by plotting absorbance against respective concentration (figure 7).

Figure 7: Calibration curve of standard OFL at 358. 2 nm by first order derivative spectrophotometry

Assay of tablet formulation:

Twenty tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 100 mg of CEF and 100 mg of OFL was transferred to 100 ml volumetric flask and the volume was made up to the mark using methanol as solvent. The resulting solution was filtered through Whatmann filter paper and 10 ml of this filtrate was appropriately diluted with distilled water to get concentration of 20 μg/ml of CEF and 20 μg/ml of CEF. Absorbance of sample solutions was measured at 312 nm and 358. 2 nm for CEF and OFL respectively.

Method validation:

Linearity and range:

Aliquots of standard stock solutions of CEF and OFL were taken in volumetric flasks and diluted with distilled water to get final concentrations in range of 5-40 μg/ml for CEF and 5-40 μg/ml for OFL. This calibration range was prepared five times and absorbances were measured at respective wavelengths for each drug separately.

Precision:

Precision of the method was determined by performing interday variation, intraday variation and method repeatability studies. In interday precision was determined by analyzing CEF and OFL in combination for five consecutive days and intraday precision was determined by analyzing CEF and OFL in combination five times in same day. In repeatability study, three concentrations of both the drugs were measured seven times.

Recovery studies:

To study the accuracy of the proposed method, recovery studies were carried out by standard addition method at four different levels. A known amount of drug was added to preanalyzed tablet powder and percentage recoveries were calculated.

Ruggedness:

Analysis by proposed method was done by two analyst and ruggedness was determined.

RESULTS AND DISCUSSION:

The proposed method was validated as per ICH guideline. The plot of absorbances versus respective concentrations were found to be linear in the concentration range of 5-40 μg/ml and for both drugs with correlation coefficient 0. 9997 at 312 nm and 0. 9996 at 358. 2 nm.

Precision was calculated as repeatability, intraday and interday variations and % RSD was found to be in the range of 0. 93-4. 5 % shown in Table 1.

Table 1: Validation Parameters

PARAMETERS	CEF	OFL
Linearity range	5-40 µg/ml	5-40 µg/ml
Correlation Coefficient	0. 9997	0. 9996
Precision (% RSD)
Repeatability Intraday Interday	2. 1 % 1. 01-3. 8 % 1. 05-2. 9%	2. 3% 0. 93-4. 5 % 1. 06-3. 02 %
% Recovery	100. 1-101. 8 %	97. 2-99.0%
Ruggedness	1. 99 %	2. 77 %

*CEF- Cefixime; OFL-Ofloxacin; RSD-Relative Standard Deviation.

_{Table 2:}_{Recovery studies}

_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_%Recovery*	_SD
CEF	1	100. 1	0. 404
OFL	1	97. 2	0. 00
CEF	1. 5	101. 8	0. 00
OFL	1. 5	97. 3	0. 346
CEF	2	101. 1	0. 410
OFL	2	99. 3	1. 098
CEF OFL	2. 5 2. 5	101. 8 98. 6	0. 404 1. 096

*Mean of Three estimations CEF-cefixime;

OFL ofloxacin; SD-Standard Deviation.

The accuracy of method was determined at 50, 75, 100 and 125 % level. The % recovery ranges for CEF100.1-101.8% and OFL 97.2-99.0% shown in Table 2. The % RSD of ruggedness for CEF was 1. 99 %, while for OFL it was found to be 2. 77 %. Results for all validation parameters are presented in table 1 and 2. The derivative spectroscopic method can be successfully _used for simultaneous estimation of CEF and _{OFL in
their combined tablet dosage form.}Marketed tablets were analyzed and results obtained were within the range of 98-102 % (Table 3).

Table 3: Results of simultaneous estimation of CEF and OFL in marketed Formulation.

Brand	mg/tablet		% Label claim*
Brand	CEF	OFL	CEF	OFL
ZIMNIC-O200	200	200	102. 8	100. 4

*Average of five determinations;

CONCLUSION:

The proposed method gives accurate and precise results for determination of CEF and OFL in marketed tablet formulation and is easily applied for routine analysis. The method is simple, accurate, and rapid. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and ruggedness. The developed method has several advantages, as it is simple, accurate and precise. The proposed method was successfully applied for the determination of these drugs in commercial tablets.

ACKNOWLEDGEMENT:

The authors are thankful to the Principal, Maliba Pharmacy College for providing necessary facilities.

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Received on 16.11.2010 Modified on 27.11.2010

Asian J. Research Chem. 4(3): March 2011; Page 415-418