INTRODUCTION:

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-thenyl-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 infections¹. Erdosteine (ERD) is a mucolytic agent, chemicaly, it is (+)-1S-(2-[N-3-(2-oxotetrahydrothienyl)- acetamido) thioglycolic acid, a thiol derivative developed for the treatment of chronic obstructive bronchitis, including acute infective exacerbation of chronic bronchitis². Clinical trial showed that combination therapy of 200 mg of CEF and 300 mg ERD significantly shows good effect when compared to either treatment alone.

Tablets containing 200 mg CEF and 300 mg ERD are formulated in the lab by using excipients like microcrystalline cellulose, poly-vinyl-pyrrolidone and magnesium stearate, which required for tablet formulation. Literature survey reveals that cefixime can be estimated by spectrophotometrically³, HPLC^4-8 and by HPTLC⁹ individually or with other drugs in bulk drugs and in human plasma, while Erdosteine is estimated individually by HPLC^10-11, HPTLC¹² and LC-MS/MS¹³.

However, there is no analytical method reported for the estimation of CEF and ERD in a combined dosage formulation. Present work describes two methods for simultaneous estimation of CEF and ERD in tablet formulation

MATERIAL AND METHODS:

Instrument: A double-beam Shimadzu UV- Visible spectrophotometer, 1700 Pharmaspec, with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of the resulting solution.

Materials: Glenmark Pharmaceuticals Ltd, Nashik, provided the standard gift sample of Cefixime and Erdosteine. Combined dose Cefixime and Erdosteine tablets were prepared in the lab.

Solvent: Methanol was used as a solvent.

Stock solution: Standard stock solutions of CEF (100 µg/ml) and ERD (100 µg/ml) were prepared in Methanol and used for the analysis.

Procedure:

Method A- Area Under Curve Method:

For the selection of analytical wavelength, solutions of CEF and ERD (20 µg/ml, each), were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm. From overlain spectra of both drugs (Fig. 1), the areas under the curve in the range of 294.5-284.5 nm (for CEF) and 240.0-230.0 nm (for ERD) were selected for the analysis. The calibration curves for CEF and ERD were prepared in the concentration range of 10-50 µg/ml at their respective AUC range. The ‘X’ values of the drugs were determined for both the drugs at the selected AUC range. The ‘X’ value is the ratio of area under the curve at selected wavelength ranges with the concentration of component in mg/ml. These ‘X’ values were the mean of six independent determinations. A set of two simultaneous equations obtained by using mean ‘X’ values are given below.

A1 = 364.11 C_CEF +144.45 C_ERD ---- (at λ_240.0-230.0 nm) ---(1)

A2 = 430.51 C_CEF----------- (at λ_294.5-284.5 nm)------- (2)

Where A1 and A2 are the area under the curve of sample at the wavelength range 240.0-230.0 nm and 294.5-284.5 nm, respectively, 364.11 and 430.51 are the ‘X’ values of CEF at the wavelength range 240.0-230.0 nm and 294.5-284.5 nm, respectively. Similarly 144.25 is the ‘X’ values of ERD at the wavelength range 240.0-230.0 nm. C_CEF is the concentration of CEF and C_ERD is the concentration of the ERD. The mixture concentration was determined by using the equation (1) and (2).

Fig.-1: Overlain Spectra of Cefixime (CEF) and Erdosteine (ERD) in Method- A

Method B- First Order Derivative Spectroscopy:

In this method solutions of CEF and ERD (20 µg/ml, each), were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained were derivatized from first to fourth order. First order derivative spectra were selected for analysis of both drugs. From the overlain spectra of both drugs (Fig. 2), 309.0 nm (for CEF) and 227.5 nm (for ERD) were selected for the analysis. The calibration curves for CEF and ERD were plotted in the concentration range of 10-50 µg/ml at their respective wavelength 309.0 nm and 227.5 nm, respectively. The absorptivity values of the drugs were determined at the selected wavelengths. These absorptrivity values are the mean of six determinations. Concentration of sample solution was determined by using following equations:

A1 = 0.34 C_CEF + 0.94 C_ERD (λmax at 227.5 nm)

A2 = -0.86 C_CEF -- (λmax at 309.0 nm)

Where, A1 and A2 are absorbance of the sample at 227.5 nm and 309.0 nm respectively, 0.34 and -0.086 are the absorptivity values of CEF at 227.5 nm and 309.0 nm respectively. Similarly 0.94 is the absorptivity value of ERD at 227.5 nm. C_CEF is the concentration of CEF and C_ERD is the concentration of the ERD.

Fig.-1: Overlain Spectra of Cefixime (CEF) and Erdosteine (ERD) in Method- B

Application of the proposed method for the determination of CEF and ERD in its pharmaceutical formulation: Twenty tablets were weighed and crushed to obtained fine powder. Tablet powder equivalent to 100 mg of CEF was transferred to 100.0 ml volumetric flask and volume made-up to the mark with Methanol. The solution was then filtered through a Whatmann filter paper (No. 41). The filtrate was further diluted to the mark with same solvent to obtain 10 µg/ml of CEF and 15 µg/ml of ERD. For Method-A, the concentration of both CEF and ERD were determined by measuring the areas under the curve in the range of 294.5-284.5 nm and 240.0-230.0 nm and values were substituted in the respective formulae to obtain concentrations. In Method-B, the concentration of both CEF and ERD were determined by measuring the absorbance of the sample at 227.5 nm and 309.0 nm in first order spectrum mode. The results of the tablet analysis were calculated by solving simultaneous equation.

Validation:

The methods were validated with respect to linearity, accuracy, precision and selectivity.

Accuracy: To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery for CEF and ERD, by both the methods, was found in the range of 98.22 % to 101.38 %.

Linearity: The linearity of measurement was evaluated by analyzing different concentrations of the standard solution of CEF and ERD. For both the methods, the Beer-Lambert’s concentration range was found to be from 10-50 µg/ml for both CEF and ERD.

Precision: The reproducibility of the proposed method was determined by performing tablet assay at different time intervals (morning, afternoon and evening) on same day (Intraday assay precision) and on three different days (Interday precision). Result of intraday and interday precision is expressed in % RSD. Percent RSD for intraday assay precision was found to be 0.2380 (for CEF) and 0.25372 (for ERD) in area under the curve method; 0.6687 (for CEF) and 0.7501 (for ERD) in first order derivative spectroscopy method. Interday assay precision was found to be 0.6765 (for CEF) and 0.5641 (for ERD) in area under the curve method; 0.6687 (for CEF) and 0.6592 (for ERD) in first order derivative spectroscopy method.

RESULTS AND DISCUSSION:

The methods discussed in the present work provide a convenient and accurate way for simultaneous estimation of CEF and ERD in its pharmaceutical dosage form. In area under the curve method, the areas under the curve in the range of 294.5-284.5 nm (for CEF) and 240.0-230.0 nm (for ERD) were selected for the analysis. In first order derivative spectroscopy method, 227.5 nm (for CEF) and 309.0 nm (for ERD) were selected for the analysis In both the methods linearity for detector response was observed in the concentration range of 10-50 µg/ml for both CEF and ERD. ‘X’ values (Method-A) and absorptivity values (Method-B) were calculated for both the drugs at selected wavelengths and substituted in equations for determining concentration of CEF and ERD in its tablet dosage form. Percent label claim for CEF and ERD in tablet analysis, by area under the curve method was found to be 100.20 ±0.4901 and 99.64 ±0.5078, respectively.

Table 1. Optical Characteristics of Cefixime and Erdosteine

	Method-A		Method-B
	CEF	ERD	CEF	ERD
Working λ	294.5.5-284.5 nm	240.0-230.0 nm	309.0 nm	227.5 nm
Linearity range (µg/ml)	10-50	10-50	10-50	10-50
Regression value
1. Slope	0.4309	0.1462	0.0009	0.001
2. Intercept	-0.0175	0.0249	0.0002	-0.0003
3. Correlation-coefficient	0.9997	0.9999	0.9997	0.9998

Similarly percent label claim for CEF and ERD in tablet analysis, by first order derivative spectroscopy was found to be 100.19 ±0.8753 and 99.84 ±0.6292 respectively. Standard deviation and coefficient of variance for six determinations of tablet sample, by both the methods, was found to be less than ± 2.0 indicating the precision of both the methods. Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as % recovery. Percent recovery for CEF and ERD, by both the methods, was found in the range of 98.22 % to 101.38 %, values of standard deviation and coefficient of variation were satisfactorily low indicating the accuracy of both the methods. Based on the results obtained, it is found that the proposed methods are accurate, precise and reproducible and can be employed for routine quality control of Cefixime and Erdosteine in combined dose formulation.

ACKNOWLEDGEMENTS:

The authors are very thankful to Dr. Avinash D. Deshpande, Director of Pharmacy, Pad. Dr. D. Y. Patil Institute of Pharmaceutical Science and Research, Pimpri, Pune for providing necessary facilities. The authors are also very thankful to Glenmark Pharmaceuticals Ltd, Nashik, for providing gift samples of Cefixime and Erdosteine.

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Received on 13.04.2009 Modified on 08.06.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 404-406

Nanda RK*, Gaikwad J, Prakash A, Ghosh VK and Nagore DH