Research Journal of Chemical Sciences Vol. 1(5), 36-39, Aug. (2011) International Science Congress Association Development and V alidation of Cefaclor in Pharmaceutical M.V. Basaveswara Rao Department of Chemistry, Krishna University, Machilipatnam Department of Chemistry, NIMS University, Rajasthan, I (Received 15 Abstract A simple, rapid and precise reverse phase high performance liquid chromatography method has been developed and validated for the determination of Cefaclor in performed by Shimadzu model LC- 20 AT orthophosphoric acid (1%) : 0.01M a analyte was monitored using PDA detector was found to have linearity in the concentration range of 2 Keywords: Cefaclor, accurate, precise, recovery, linearity, IntroductionCefaclor having molecular formulae C15 H molecular weight 367.808 belongs2,3 to the family of second-generation cephalosporin antibiotic the cephalosporins and are used to treat certain infections caused by bacteria such as ear, lung, skin, throat and urinary tract infections. Chemically cefaclor is (6,7R)-7- [[(2 acetyl]-amino] - 3-chloro-8-oxo-5- thia [4.2.0] oct-2-ene- 2- carboxylic acid monohydrate, which is soluble in water and insoluble in methanol, chloroform and benzene. Cefaclor: Cobalt(II) and nickel(II) complexes of the Cefaclor have been synthesized. X Chen sensitive and specific liquid chromatographic spectrometric method for the determination of cefaclor in human plasma. Masaaki Kai etal reported another method for the determination of cefaclor based on the chemical derivatization of the drug with 4-(2 cyanoisoindolyl) phenylisothiocynate (CIPIC) under the reaction conditions with heating at 80°C for 7min in the presence of pyridine. Traje Stafilov, etal presented simple high liquid chromatographic (HPLC) method to measu simultaneously the blood plasma concentration of cefaclor and cephalexine. Research Journal of Chemical Sciences ______ __________________________________ (2011) International Science Congress Association alidation of New RP-HPLC M ethod for the Pharmaceutical Dosage forms and in H uman M.V. Basaveswara Rao *, Prasanthi V., Maiti Sushanta and Raja Department of Chemistry, Krishna University, Machilipatnam -521001, A.P., INDIA Department of Chemistry, NIMS University, Rajasthan, I NDIA Available online at: www.isca.in 15 nd June 2011, revised 23rd June 2011, accepted 7th July 2011) A simple, rapid and precise reverse phase high performance liquid chromatography method has been developed and validated for the determination of Cefaclor in p harmaceutical dosage forms and in serum. Chromatography was 20 AT VP with Kromasil C- 18 column using a mobile phase comprising of a mmonium dihydrogen phosphate (50:45:5 v/v), at a flow rate 1.0ml/min. The PDA detector at 270nm and the Run time was 10min. for Cefaclor. The proposed method was found to have linearity in the concentration range of 2 -10 g/ml. Cefaclor, accurate, precise, recovery, linearity, orthophosphoric acid. H 14ClN and to the family of antibiotic known as and are used to treat 4 such as pneumonia, ear, lung, skin, throat and urinary tract infections. [[(2 )-amino-phenyl thia -1- Azabicyclo carboxylic acid monohydrate, which is soluble in water and insoluble in methanol, chloroform and complexes of the X Chen etal, reported sensitive and specific liquid chromatographic -tandem mass spectrometric method for the determination of cefaclor in reported another method for the determination of cefaclor based on the chemical cyanoisoindolyl) phenylisothiocynate (CIPIC) under the reaction conditions with heating at 80°C for 7min in the presence of pyridine. presented simple high -performance liquid chromatographic (HPLC) method to measu re simultaneously the blood plasma concentration of cefaclor Material and Methods Shimadzu model LC- 20 AT equipped with Empower Kromasil C- 18 column, Particle Size: 5 250 x 4.6mm ID, Injector Type: Injection Volume: 20 l, Detection wavelength: 270nm, Flow rate: 1.0ml/min, Pump Pressure: 25.8 10 min, M obile Phase Used: Acetonitrile: Orthophosphoric acid (1%):0.01M Ammonium dihydrogen phosphate (50:45:5 v/v). Chemicals and Reagents : sample; all the chemicals were procured from E India, Limited. W ater (HPLC grade), orthophosphoric acid and ammonium dihydrogen phosphate ( AR grade) were used. Chromatographic C onditions: consisted of a cetonitrile, 1%Orthophosphoric acid and 0.01M a mmonium dihydrogen phosphate (50:45:5 v/v). Prepared mobile phase was filtered through 0.45 membrane filter and sonicated. Sample solution was prepared by dissolving the drug in mobile phase and sonicated for 30 minutes. The mobile phase was delivered isocratic ally at a flow rate of 1 ml/min. All filtered through a 0.45 m Kromasil C18 column 250 x 4.6 mm ID with 5 size and the column were maintained at ambient temperature. The injection volume was 20 run time was 10min. The detec 270nm. The chemicals were procured from E India, Limited. Preparation of Mobile P hase was prepared by mixing a __________________________________ ISSN 2231-606X Res.J.Chem.Sci. 36 ethod for the Determination of uman Plasma Raja G. INDIA 2011)  A simple, rapid and precise reverse phase high performance liquid chromatography method has been developed and harmaceutical dosage forms and in serum. Chromatography was 18 column using a mobile phase comprising of acetonitrile: mmonium dihydrogen phosphate (50:45:5 v/v), at a flow rate 1.0ml/min. The and the Run time was 10min. for Cefaclor. The proposed method Material and Methods 20 AT VP using PDA detectorwas Empower software. Column make: 18 column, Particle Size: 5 , Column length: Injector Type: Rheodyne type injector, l, Detection wavelength: 270nm, Flow rate: 1.0ml/min, Pump Pressure: 25.8 Mpa, Run time: obile Phase Used: Acetonitrile: Orthophosphoric Ammonium dihydrogen phosphate : Cefaclor was obtained as a gift all the chemicals were procured from E -Merck, ater (HPLC grade), acetonitrile (HPLC grade), orthophosphoric acid and ammonium dihydrogen AR grade) were used. onditions: The mobile phase cetonitrile, 1%Orthophosphoric acid and mmonium dihydrogen phosphate (50:45:5 v/v). Prepared mobile phase was filtered through 0.45 m membrane filter and sonicated. Sample solution was prepared by dissolving the drug in mobile phase and sonicated for 30 minutes. The mobile phase was delivered isocratic ally at a flow rate of 1 ml/min. All solutions were m membrane filter before use. column 250 x 4.6 mm ID with 5 particle and the column were maintained at ambient temperature. The injection volume was 20 l and the total run time was 10min. The detec tion was carried out at 270nm. The chemicals were procured from E -Merck, hase Solution: The mobile phase a cetonitrile,1% orthophosphoric Research Journal of Chemical Sciences _______________________________________________________ ISSN 2231-606X Vol. 1(5), 36-39, Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 37 acid and 0.01M ammonium dihydrogen phosphate (50:45:5 v/v) by ultra bath sonicated for 30 min. Preparation of Standard: Stock solution of Cefaclor was prepared by dissolving accurately weighed 10mg of drugs in 10ml methanol (final concentration, 1000µg/ml). The prepared stock solutions were stored away from light. From the stock, standard solutions was freshly prepared during the day of analysis. Preparation of Working Standard Solution (a.p.i): From the stock solution 1mmg/ml solution was prepared. Preparation of Working Standards for Linearity: Solutions in the concentration range of 0.1-0.5mg/ml were prepared from the standard working solution. Linearity and Calibration: Linearity was assessed by performing single measurement at several analyte concentration varying quantities of stock standard solution diluted with the mobile phase to give a concentration of 0.1, 0.2, 0.3, 0.4 and 0.5 mg/ml. Injection was made at intervals of 6min. The linearity was tested for the concentration ranging from 0.1-0.5mg/ml. The peak area ratio of the drug was plotted against concentration. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. Precision: Reproducibility was performed by injecting three replicates concentrations of standard and sample solutions which were prepared and analyzed by same analyst on same day. Inter-day variations in the peak area of drug solutions and the amount of drug were calculated in terms of Percentage Relative Standard Deviation. Sample concentration is 1mg/1ml. Accuracy: Recovery assessment was obtained by using standard addition technique which was by adding known quantities of pure standards at three different levels in 80%, 100% and 120% to the pre analyzed sample formulation. Assay: The estimation of drug in pharmaceutical dosage forms. Cefalor tablets of 0.1mg strength were evaluated for the amount of Cefaclor present in the formulation. Each sample was analyzed in triplicate after extracting the drug. The amount of drug present in formulation was calculated by comparing the mean peak area from standard. Intermediate Precision or Ruggedness: Inter-day variations were performed by using six replicate injections of standard and sample solutions of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation. Robustness: Robustness was carried out by varying two parameters from the optimized chromatographic conditionsSpecificity: The method was determined as specific by comparing test results obtained from analyses of sample solution containing excuse ingredients with that of test results those obtained from standard drug. System Suitability Parameter: System suitability tests were carried out on freshly prepared standard stock solutions of Cefaclor and it was calculated by determining the standard deviation of Cefaclor standards by injecting standards in five replicates at 10 minutes interval and the values were recorded. Preparation of Formulation Sample Solution: 20mg Ceflor Dps powder (50mg formulation) was weighed and dissolved in 10ml mobile phase. The resultant sample solution concentration is 2mg/ml then sonicated by ultra bath sonicated for 30 minutes and filtered through 0.45µm membrane filter. The amount of drug present in the 100mg formulation was calculated from linearity graph. Preparation of Serum Sample Solution: 0.5ml of this serum was taken in a test tube and added 100µl of diltizem hydrochloride (1µg/ml) and 0.1ml of 1M NaOH and 5ml of dichloromethane and mixed about 20min in vortex mixer and centrifuged at 3000 rpm for 10min. From this centrifuged solution 4ml of organic layer was separated and evaporated to dryness to get residue. To this residue 100µl of 1M acetic acid and 3ml of n-hexane and mixed for 5 min by vortex mixer and evaporated the organic layer and finally the remaining sample was injected into HPLC and chromatogram was recorded. The amount of drug present in the blood sample was calculated from linearity graph. Results and Discussion The Reverse Phase high performance liquid Chromatography method was developed by a stability indicating assay method. Pure drugs chromatogram was run in different mobile phases containing methanol, acetonitrile, THF, and different buffers like potassium dihydrogen phosphate, sodium dihydrogen phosphate, Ortho phosphoric acid in different volumes ratios. Different columns like C, C18, phenyl, cyano with different dimensions were used. Then retention time and tailing factor were calculated. Finally acetonitrile and 1% orthophosphoric acid and 0.01M ammonium dihydrogen phosphate in the ratio of 50:45:5 v/v (P:3.4) and Kromasil 18 analytical column was selected which gave a sharp and symmetrical peak with 1.82 tailing. Calibration graph was found to be linear at range 0.1mg/ml to 0.5mg/ml. Five different concentrations of Cefaclor in range given above were prepared and 20µl of each concentration injected in HPLC as shown in the table 1, figure 1. The slope (m) and intercept (c) obtained were found to be 170955.95 and -0.03.The correlation of coefficient (r) obtained was found to be 0.9626 as shown in the table 1. It was observed that the concentration range showed a good relationship. The limit of detection for Cefaclor was found to be 40µg/ml and the limit of quantification was found to be 75µg/ml. It Research Journal of Chemical Sciences ______ Vol. 1(5), 36-39, Aug. (2011) International Science Congress Association proves the sensitivity of method. The Percentage assay of Cefaclor in formulation was found to be 67.02%. in the t able 1 and figure 3. The relative standard deviation value obtained was below 1 which indicates the precession of the method. The validation of the proposed method was further verified by recovery studies. The data presented by in the t able 2 and figure 2. The percentage recovery was found to be 104.35% which shows a good index of accuracy of the developed method. The amount of drug present in the human serum sample was calculated from the linearity graph was fo und to be 1.079 mg/ 0.5ml as shown in table 3 and figure 4. Table -1 Optical Chracterisation o f Cefaclor Parameters Linearity range(mg/ml) Correlation coefficient(r) Slope(m) 170955.95 Intercept(c) Limit of detection (LOD; µg/ml) Limit of Quantification (LOQ; µg/ml) Tailing factor Retention time (min) Theoretical plates ( % ) R.S.D ( % )Accuracy ( % )Formulation Serum (mg/0.5ml) Table-2 Recovery Data of Cefaclor Pharmaceutical formulation (brand name) Labeled amount (mg) Percentag e Assay KEFLOR 50 mg 67.02 *Average value of three different levels in triplicate Conclusion The RP- high performance liquid chromatographic method for the analysis of Cefaclor from their formulations was found to be accurate and precise. Thus, the proposed HPLC method can be successfully applied for the routine quality control analysis of Cefaclor formulations. This method could be a simple for the p ractical applications and comparatively better method than the reported ones in the literature. ______ _________________________________ ______________ International Science Congress Association proves the sensitivity of method. The Percentage assay of was found to be 67.02%. as shown able 1 and figure 3. The relative standard deviation value obtained was below 1 which indicates the precession of the method. The validation of the proposed method was further verified by recovery studies. The data was able 2 and figure 2. The percentage recovery was found to be 104.35% which shows a good index of accuracy of the developed method. The amount of drug present in the human serum sample was calculated und to be 1.079 mg/ 0.5ml f Cefaclor Cefaclor 0.1 – 0.5 0.9626 170955.95 -0.03 40 75 1.82 6.053 3927.42 0.148 104.35 67.02 1.079 Cefaclor Percentag Percentage recovery 104.35 *Average value of three different levels in triplicate high performance liquid chromatographic method for the analysis of Cefaclor from their formulations was found to be accurate and precise. Thus, the proposed HPLC method can be successfully applied for the routine quality control analysis of Cefaclor formulations. This ractical applications and comparatively better method than the reported ones in the Figure Chromatogram of Figure Chromatogram of Figure Chromatogram of ( formulation assay) ______________ __ ISSN 2231-606X Res.J.Chem.Sci. 38 Figure -1 Chromatogram of cefaclor (standard) Figure -2 Chromatogram of Cefaclor (Accuracy) Figure -3 Chromatogram of cefaclor formulation assay) Research Journal of Chemical Sciences ______ Vol. 1(5), 36-39, Aug. (2011) International Science Congress Association Figure-4 Chromatogram of cefaclor ( serum) References1.The Merck Index, 13th edition, Merck and company, INC, White House station, NJ, 324 (2001) 2.Hebert A, Sigman E, Levy M., Serum sickness reactions from cefaclor in children Dermatol., 25, 805-8 (1991). 3. 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