Asian Journal of Biochemical and Pharmaceutical Research

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1 ISSN: Research Article Asian Journal of Biochemical and Pharmaceutical Research Analytical Method Validation And Statistical Evaluation of Spectrophotometric Methods For Simultaneous Estimation of Amoxycillin Trihydrate And Ambroxol Hydrochloride Madhura Dhoka*, Shakuntala Sandage and Snehal Dumbre A.I.S.S.M.S.College of Pharmacy, Pune University, Pune-01 Received: 5 May 2011; Revised: 25 May 2011; Accepted: 30 May Abstract: The objective of this study was to evaluate the utility of derivative spectrophotometric and area under curve method for the nonseparative analysis of amoxicillin trihydrate (AMOX) and ambroxol hydrochloride (AMB) in combination. Derivative spectrophotometry allowed specific determination of amoxicillin trihydrate at 282nm with negligible contribution by ambroxol hydrochloride. Similarly, ambroxol hydrochloride was determined at 259nm with negligible interference by amoxicillin trihydrate. The area under curve method is applied because there is considerable difference in λ max of both drugs. The wavelength range selected for amoxicillin trihydrate is nm and for ambroxol hydrochloride is nm.The Beer s law is obeyed for both methods. Parameters such as linearity, accuracy, precision, specificity, LOD and LOQ values were used for validation of the methods. Intra and interday precision %RSD values were <2. The results indicate that proposed method is simple, rapid and specific. The two methods were compared using paired t test, there no significant difference in the results of two methods. Keywords: Amoxycillin, Ambroxol, Derivative spectrophotometric method, Area Under Curve method, Statistical Validation INTRODUCTION: Amoxycillin trihydrate (AMOX) [[2S-[2α, 5α, 6β(S*)]]-6-[[amino(4-hydroxyphenyl) acetyl] amino]-3,3 dimethyl-7-oxo-4-thia-1-azabicyclo[3,2,0]heptane-2-carboxylic acid] is antibacterial agent used in urinary tract infection, respiratory tract infection. Literature survey shows AMOX alone was determined using HPLC method [1-4]. AMOX was determined simultaneously in combined dosage form by HPLC [5-7] and UV spectrophotometry [8-9] method. Ambroxol Hydrochloride (AMB) (2-amino-3, 5-dibromo-N (trans-4-hydroxycyclohexyl)] benzyl amine hydrochloride. AMB alone was determined by HPLC [10-11], UV spectrophotometric method [12-13] and HPTLC [14]. AMB was also determined simultaneously in combined dosage form by UV spectrophotometry [15-18], colorimetry [19]. 585

2 HPLC method was developed on AMOX and AMB combination.but no UV method has been developed on this combination in combined dosage form. In the present investigation, a derivative spectrophotometric method and area under curve method [20-23] were developed for simultaneous estimation of AMOX and AMB. Both methods were validated as per ICH guidelines [22]. Statistical evaluation was done by using t- test [24-25]. MATERIALS AND METHODS: Instrunment: A dual beam Jasco V550 spectrophotometer was used as an instrument. The absorption Spectra of the reference and test solutions were recorded over the range of nm keeping the solutions in 1cm quartz cell. Reagents and chemicals: 0.1M Hydrochloric acid prepared from analytical reagent grade Hydrochloric acid (Merk India, Mumbai) in Distilled water, which is procured locally. Amoxycillin Trihydrate of purity 100% was supplied by Maxim pharmaceuticals, Pune and Ambroxol Hydrochloride of purity 99.7 % was supplied by Centaur pharmaceuticals, Goa. Standard stock and all dilutions of AMOX and AMB were prepared in 0.1 M HCl. Preparation of standard stock solution: 2 mg/ml solution of AMOX and 0.4 mg/ml solution of AMB were prepared by dissolving suitable quantities of each of AMOX and AMB in 0.1 M HCl. Procedure: Method A- Derivative Spectrophotometric Method [20-25] For each drug, appropriate dilutions were done from standard stock solution and were scanned separately in the UV range nm. These spectra were converted to first order derivative spectra. AMOX was determined at 282nm with negligible contribution by AMB and AMB was determined at 259nm with negligible contribution by AMOX. The concentration of AMOX and AMB were calculated by putting the values of absorbance in linearity equations. A) For AMOX Y=0.0014x B) For AMB Y=0.006x Method B- Area Under Curve Method [20-25] The two-wavelength range selected should be such that there is negligible change in absorbance of drug. Within these wavelength ranges peak area of both drugs were measured and used in calculations. So the two wavelength ranges chosen were nm for AMOX and nm for AMB as shown in Figure 2. The area under curve of said concentrations for both the drugs were noted at selected analytical wavelength ranges. These area under curve were then divided by concentration in gm/lit to get X amox and X amb values Following equations were used for calculations. 586

3 Determination of X values : X = AUC of component between selected wavelength ranges Concentration of that component in gm/lit The concentration of drug was then calculated by using following equation. C B = X O AUC M X O AUC M X B X O X B X O C O = X B AUC M X B AUC M X B X O X B X O Where, C B = Concentration of AMB C O = Concentration of AMOX X B = Area under curve of ambroxol at wavelength of nm X O = Area under curve of amoxycillin at wavelength of nm X B = Area under curve of ambroxol at wavelength of nm X O = Area under curve of amoxycillin at wavelength of nm AUC M = Area under curve of mixture at wavelength of nm AUC M = Area under curve of mixture at wavelength of nm 587

4 Application of Proposed Method for the Determination of AMOX and AMB in Tablets: Tablet Composition: Each film coated tablet contains Amoxicillin trihydrate equivalent to Amoxicillin Ambroxol Hydrochloride equivalent to Ambroxol 30 mg. Twenty tablets were weighed and crushed to fine powder. The powder equivalent to 250 mg of AMOX was transferred to 100 ml volumetric flask and volume was made up. The solution was then filtered through a whatmann filter paper and was diluted suitably to get 222 gml -1 of AMOX and 28 gml -1 of AMB. The concentration of both AMOX and AMB were determined by measuring the absorbances of derivative spectra at 282nm and 259nm for derivative spectroscopic method and by determining area under curve in range nm for AMOX and nm for AMB and substituting these values in the above mentioned equations for Area under curve method. Results are shown in Table 1. Method Validation [23] Methods were validated according to ICH guidelines. Accuracy: To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% & 120%). Percent recovery for AMOX and AMB, by both the methods, was found in the range of % to % Linearity: The linearity of measurement was evaluated by analyzing different concentration of the standard solution of AMOX and AMB. For both the methods, the Beer-Lambert s concentration range was found to be µgml -1 for AMOX and µgml -1 for AMB for Derivative spectrophotometric method and µgml -1 for AMOX and µgml -1 for AMB for Area under curve method with correlation coefficient of 0.99 and above. Precision: The reproducibility of the proposed method was determined by performing capsule assay at different time intervals on same day (Intra-day precision) and on three different days (Inter-day precision) and were expressed in % RSD. Percent RSD for Intraday precision was found to be (for AMOX) and (for AMB) in first order derivative method; (for AMOX) and (for AMB) in Area under curve method. Inter-day precision was found to be (for AMOX) and (for AMB) in first order derivative method; (for AMOX) and (for AMB) in Area under curve method. Statistical Evaluation [24-25] The analytical characteristics of the tested methods in area under curve and derivative Spectrophotometric method were validated to ensure the suitability of the analytical requirements and the reliability of the results. Afterwards, the two methods were compared using paired t test. The statistical treatments were performed with the Graphpad Instat statistical software (Table 2). 588

5 RESULTS AND DISCUSSION: The methods were successfully validated with respect to linearity, accuracy, intra and interday precision etc. In Derivative spectroscopic method, linearity for detector response was observed in the concentration range of μg/ml for AMOX and μg/ml for AMB whereas in Area under curve method, it is μg/ml for AMOX and μg/ml for AMB. The %RSD values during both intra and interday precision studies were <2.0% demonstrating repeatability and intermediate precision. Apparently the mean percent recovery values for AMOX and AMB by derivative spectroscopic method were 99.65% and 99.45% respectively. Similarly the mean percent recovery values for AMOX and AMB by Area Under curve method were 99.97% and 99.63% respectively. During assay study, in presence of excipients, results were found within the range, indicating that both the methods were specific. Based on the results obtained, it is found that the proposed methods are accurate, precise, reproducible & economical and can be employed for routine quality control of Amoxicillin trihydrate and Bromhexine hydrochloride in combined oral solid dosage forms. Statistical evaluation of the method showed t = with 4 degrees of freedom for both AMOX and AMB, whereas table t value is As calculated t-value < table t-value. There is no significant difference in the results of two methods. CONCLUSION The proposed spectroscopic methods allow for accurate, precise and reliable measurement of AMOX and AMB simultaneously in combined dosage form. Both the developed methods were found to be simple, rapid, selective, accurate and precise for the concurrent estimation of drugs in respective two-component oral dosage forms of AMOX and AMB. The methods were evaluated for validation parameters, linear relation including coefficient of correlation, accuracy, reproducibility and precision. The RSD for all parameters and assay results obtained by this method are in fair agreement. There is no significant difference in two method as indicated by statistical evaluation. The developed methods can be used for routine quantitative simultaneous estimation of AMOX and BROM in pharmaceutical preparations. ACKNOWLEDGEMENT: The Authors are thankful to Dr. Mrs. A.R. Madgulkar and Dr. K.G. Bothara for their support and for providing required facilities to carry out the work. The authors are grateful to Centaur Pharmaceuticals, Goa and Maxim Pharmaceuticals, Pune for providing gift samples of AMB and AMOX. 589

6 Table 1. Capsule Analysis and Recovery Studies Method Label Claim % Assay % Recovery % RSD AMOX AMB AMOX AMB AMOX AMB AMOX AMB Method A Method B Table 2. Statistical evaluation by t-test Methods Number Mean Standard Standard 95 % of points (n) (%) Deviation Error of Mean confidence From To Amoxycillin AUC Derivative Ambroxol AUC Derivative Fig. 1. Derivative Spectra of Amoxycillin and Ambroxol 590

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