INTRODUCTION:

Cephalosporins are a major group of semi-synthetic β-lactam antibiotics used in clinical medicine for treatment of bacteria-related infections. They are closely related in fundamental structure and antibactericidal action mechanism to penicillins. They are used for the treatment of infections caused by both gram-negative and gram positive bacteria. Cephalosporin’s are among the oldest and most frequently prescribed naturally occurring antimicrobial agents¹. Cephalexin, 7-(D-amino-phenylacetamido)-3-methyl-3-cephem-4-carboxylic acid is a second-generation cephalosporin and one of the most commonly used cephalosporin antibiotics. Figure 1 shows the chemical structure of cephalexin.

Figure 1. Chemical structure of cephalexin

Few methods have been reported for the quantification of Cephalexin single drug and in Combination by HPTLC ^{2, 3,
4, 5}. The use of RP-HPLC procedures for determination in plasma, serum and urine has been reported^{6, 7, 8} some spectrophotometric and colorimetric methods also have been reported^{9, 10}. However till now, no simple UV spectrophotometric method for estimation of Cephalexin has been reported. The present work describes the development of two simple, precise and accurate methods for the estimation of Cephalexin in bulk drugs and marketed formulation. The method validation was carried out as per ICH guidelines.¹¹

MATERIALS AND METHODS:

Apparatus:

A Shimadzu version 1.12 - Double Beam UV-Visible spectrophotometer. UV spectra of standard and sample solutions were recorded in 1cm quartz cells at the wavelength ranges of 200-400 nm.

Chemicals and reagents:

Cephalexin (purity 99.5 %) was provided as a gift sample by Medrich Ceptech Ltd. Hyderabad, India. And was used without further purification. All the other reagents used were of analytical grade. HCl, Potassium di hydrogen phosphate, was obtained from SD Fine chem, Mumbai. (AR Grade), Distilled Water was prepared in house.

EXPERIMENTAL:

An UV–Visible double beam spectrophotometer with 1cm matched quartz cells was used for the spectral and absorbance measurements, an digital pH meter was used for pH measurements. All the chemicals and reagents used were of analytical grade and the aqueous solutions were freshly prepared with triple distilled water.

Preparation of the reagents:

All reagents were prepared using by Double distilled water and all chemicals are AR grade.

Method-A:

Preparation of Phosphate Buffer pH 2:

Dissolve 0.136grams of potassium dihydrogen phosphate in 800 ml of water, adjust the pH to 2.0 with HCL and add sufficient water to produce 1000 ml.

Preparation of Stock solution:

1 mg/ml stock solution of cephalexin was prepared by dissolving the appropriate weight in 100 mL of Phosphate buffer pH 2.0. Working standard solutions were daily prepared by diluting stock solutions at the concentrations of 5-40 μg/ml in Phosphate buffer pH 2.0. Then the absorbance of the solution was measured. Phosphate buffer pH 2.0 was used as a blank solution.

Method-B:

Preparation of 0.1 N Hydrochloric acid:

Measure accurately 36.46 ml of concentrated HCl and make up the volume to 1000 ml with distilled water.

Preparation of Stock solution:

1 mg/ml cephalexin solution was prepared by dissolving the appropriate weight in 100 mL of 0.1N HCl. Working standard solutions were daily prepared by diluting stock solutions at the concentrations of 5-50 μg/mL. Then the absorbance of the solution was measured. 0.1N HCl was used as a blank solution.

Experimental Procedure:

Method-A:

1mg/ml of stock solution was prepared by using phosphate buffer (pH 2.0) and the concentrations 5-40 μg/ml was prepared by diluting stock solutions 0.5 ml, 1 ml and upto 4 ml in a volumetric flask and finally make up the volume to 100 ml with phosphate buffer pH (2.0).

Selection of Wavelength:

Various concentrations of drug solution were scanned over the range of 200-400 nm. It was observed that the drug showed considerable absorbance at 261 nm. So, 261nm was selected as the wavelength for detection (Figure 2).

Figure 2: λmax of cephalexin in phosphate buffer (pH 2.0)

Procedure for formulations:

Twenty capsule contents of cephalexin were accurately weighed and mixed. A portion of the powder equivalent to the average weight of one capsule was transferred into a 100 mL volumetric flask and 30 mL of Phosphate buffer pH2.0 was added. The content of the flask was sonicated for 5 min and diluted to volume with Phosphate buffer pH2.0.This solution was centrifuged for 15 min at 3000 rpm to separate out the insoluble excipients. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant and diluting them with Phosphate buffer pH 2.0 to give final concentration (20 μg mL^-1). Then the absorbance of these solutions was measured. The amount of cephalexin per capsule was calculated using the calibration curve.

Method-B:

1mg/ml of stock solution was prepared by using 0.1N HCl and the concentrations 5 to 50μg/ml were prepared by diluting stock solutions 0.5ml to 50ml in a volumetric flask and finally make up the volume to 100 ml with 0.1N HCl.

Selection of Wavelength:

Various concentrations of drug solutions were scanned over the range of 200-400 nm. It was observed that the drug showed considerable absorbance at 257 nm. So, 257 nm was selected as the wavelength for detection (Figure 3).

Figure 3: λmax of cephalexin im 0.1N HCl

Procedure for formulations:

Twenty capsules contents of cephalexin were accurately weighed and mixed. A portion of the powder equivalent to the average weight of one capsule was transferred into a 100 mL volumetric flask and 30 mL of 0.1 N HCl was added. The content of the flask was sonicated for 5 min and diluted to volume with 01N HCl. This solution was centrifuged for 15 min at 3000 rpm to separate out the insoluble excipients. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant and diluting them with 0.1 N HCl to give final concentration (20 μg mL^-1). Then the absorbance of these solutions was measured. The amount of cephalexin per capsule was calculated using the calibration curve.

RESULTS:

Method-A:

Method validation:

Validation is one of the most important steps in method development for analytical determinations. The main validation parameters^11,12such as stability, linearity, sensitivity, precision, accuracy, recovery, specificity, robustness and ruggedness were evaluated in developed method ¹¹.

Linearity range:

Under the experimental conditions, the calibration graphs of the absorbance versus concentration were found to be linear over the range of 5-40 μg mL for proposed method. The calibration graph (Figure 4) was constructed after analysis of 5 different concentrations with each concentration was measured six times. Each point of the calibration graph corresponded to the mean value obtained from 5 independent measurements. The regression equations (with standard error of intercept and slope) and correlation coefficients of the mean of 5 consecutive calibration curves are given in figure 4. The regression equation was y = 0.020x+0.021 where y is the absorbance and x is the concentration in μg mL-1 (R²= 0.999).

Figure 4: calibration graphs of Cephalexin

Detection and Quantification limit:

The limit of quantification (LOQ) is the lowest concentration of cephalexin on the calibration curve that can be quantified with acceptable precision and accuracy. The LOQ was found as 2.85μg/mL for proposed method. And the limit of detection =0.855 μg/mL.

Precision:

The precision of a method is defined as the closeness of agreement between independent test results obtained under optimum conditions. Three different concentrations of cephalexin in the linear range (5, 10 and 15 μg mL−1) were analyzed in 6 independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision) from three measurements of every sample in each series. The precision of the analysis was determined by calculating the relative standard deviation (RSD %). The RSD values of intra-day and inter-day studies varied from 0.250 to 1.29% showed that the intermediate precision of the method was satisfactory.

Accuracy:

The accuracy of the method was determined by calculating the percentage relative error (Bias %) between the measured mean concentrations and added concentrations at the same concentration of cephalexin. The results obtained for intra and inter-day accuracy were between 0.263 - 1.185 %. Observed concentration values are in good agreement with the expected ones.

Specificity:

Analysis of pharmaceutical formulations:

The optimized spectrophotometric method was applied to the direct determination of cephalexin in capsules using calibration curve method without any sample extraction or filtration. The average amount present was determined by taking average of six replicate analysis and the amount present were found to be 255 mg/capsule. The percentage purity was found to be 100.23%. The results (table 1) show that the proposed method was successfully applied for the assay of cephalexin in its pharmaceutical formulations. Table 2 shows the optical characteristics of the proposed method (method A)

Table 1: Assay of Capsule dosage form (method A):

S. No	Absorbance		% purity	Mean % purity
S. No	Standard	Test	% purity	Mean % purity
1	462	440	99.69%	100.23
2	463	448	101.01
3	466	443	100.01

Table 2: Optical and regression characteristics, precession and accuracy of the proposed method (method A)

Parameter	Results
Analytical wavelength (nm)	261
Beer’s law limits (µg/ml)	0.5-50
Limits of detection (µg/ml)	0.855
Limits of quantization (µg/ml)	2.85
Molar absorptivity (lmol^-1 cm^-1)	2.2x10^-2
Slope (b)	0.020
Intercept (a)	0.021
Correlation coefficient (γ)	0.999
Standard Deviation (SD)	0.0057

Method B:

Method validation:

Validation is one of the most important steps in method development for analytical determinations. The main validation parameters ^{11, 12}such as stability, linearity, sensitivity, precision, accuracy, recovery, specificity, robustness and ruggedness were evaluated in developed method ¹¹.

Linearity range:

Under the experimental conditions, the calibration graphs of the absorbance versus concentration were found to be linear over the range of 5-50 μg mL for proposed method. The calibration graph (Figure 5) was constructed after analysis of 5 different concentrations with each concentration was measured six times. Each point of the calibration graph corresponded to the mean value obtained from 5 independent measurements. The regression equations (with standard error of intercept and slope) and correlation coefficients of the mean of 5 consecutive calibration curves are given in figure 3. The regression equation was y = 0.014x+0.102 where y is the absorbance and x is the concentration in μg mL-1 (R²= 0.998).

Detection and Quantification limit:

The limit of quantification (LOQ) is the lowest concentration of cephalexin on the calibration curve that can be quantified with acceptable precision and accuracy. The LOQ was found as 7.857μg/mL for proposed method. And the limit of detection =2.357 μg/mL

Figure 3: calibration graphs of Cephalexin

Precision:

The precision of a method is defined as the closeness of agreement between independent test results obtained under optimum conditions. Three different concentrations of cephalexin in the linear range (5, 10 and 15 μg mL−1) were analyzed in 6 independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision) from three measurements of every sample in each series. The precision of the analysis was determined by calculating the relative standard deviation (RSD %). The RSD values of intra-day and inter-day studies varied from 0.258%. to -0.589 % showed that the intermediate precision of the method was satisfactory.

Accuracy:

The accuracy of the method was determined by calculating the percentage relative error (Bias %) between the measured mean concentrations and added concentrations at the same concentration of cephalexin. The results obtained for intra and inter-day accuracy were between 0.662-1.185 % Observed concentrations values are in good agreement with the expected ones.

Specificity:

Analysis of pharmaceutical formulations:

The optimized spectrophotometric method was applied to the direct determination of cephalexin in capsules using calibration curve method without any sample extraction or filtration. The average amount present was determined by taking average of six replicate analysis and the amount present were found to be 255 mg/capsule. The percentage purity was found to be 99.88%. The results (table 3) show that the proposed method was successfully applied for the assay of cephalexin in its pharmaceutical formulations. Table 4 shows the optical characteristics of the proposed method (method B)

Table 3: Assay of Capsule dosage form (method B)

S. No	Absorbance		% purity	Mean % purity
S. No	Standard	Test	% purity	Mean % purity
1	576	611	99.89	99.88
2	575	608	100.22
3	579	614	99.53

Table 4: Optical and regression characteristics, precession and accuracy of the proposed method (method B)

Parameter	Results
Analytical wavelength (nm)	257
Beer’s law limits (µg/ml)	1.25-60
Limits of detection (µg/ml)	2.357
Limits of quantization (µg/ml)	7.857
Molar absorptivity (lmol^-1 cm^-1)	2.03x10^-2
Slope (b)	0.014
Intercept (a)	0.102
Correlation coefficient (γ)	0.998
Standard Deviation (SD)	0.011

DISCUSSION:

The development of a simple, rapid, sensitive and accurate analytical method for the routine quantitative determination of samples will reduce unnecessary tedious sample preparations and the cost of materials and labor. Cephalexin is a UV-absorbing molecule with specific chromophores in the structure that absorb at a particular wavelength and this fact was successfully employed for their quantitative determinations using the UV spectrophotometric method. The λmax of the drug for analysis was determined by taking scans of the drug sample solutions in the entire UV region. It was found to be that only one peak was observed in this method at the wavelength of 261nm in phosphate buffer pH 2.0 and wave length of 257nm in 0.1N HCl.

The spectrum obtained from capsule solution was identical with that of spectrum from standard solution containing an equivalent concentration of cephalexin. Capsule solution showed the same wavelength of maximum absorbance for cephalexin. It was concluded that the excipients did not interfere with quantification of cephalexin in this method and the proposed method could be considered specific. These data showed that there was no spectral interaction in the analysis of cephalexin in pharmaceutical formulations by the proposed method. Therefore, the calibration curve method, which is easier and quicker than the standard addition method, was used in quantitative analysis of cephalexin. These values showed that no significant excipients interference, thus the procedures was able to determination of cephalexin in the presence of excipients. In the proposed method, there was no need for pre-separation and only centrifugation was applied to make the solution clear.

CONCLUSION:

In this study a simple, fast and reliable UV spectrophotometric method was developed and validated for the determination of cephalexin in pharmaceutical formulations. This method was applied directly to the analysis of pharmaceutical dosage forms without the need for separation such as extraction steps prior to the drug analysis. As this proposed method has the lowest LOD value and wider linear range is more sensitive method. From the results obtained, we concluded that the suggested method showed high sensitivity, accuracy, reproducibility and specificity. Moreover, this method is simple and inexpensive and it can be employed for the routine quality control of cephalexin in pharmaceutical formulations.

ACKNOWLEDGEMENTS:

The authors thank to Medrich Ceptech Ltd. Hyderabad, India for providing standard cephalexin. The authors also thank to Management of Annamacharya college of Pharmacy, Rajampet, Kadapa Dist, A.P. India, for providing necessary facilities to carry out this research work.

REFERENCES:

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Received on 09.04.2013 Modified on 19.04.2013

Asian J. Research Chem. 6(5): May 2013; Page 490-494