UV SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF ATENOLOL AND LOSARTAN POTASSIUM BY Q-ANALYSIS

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1 UV SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF ATENOLOL AND LOSARTAN POTASSIUM BY Q-ANALYSIS *K.V.LALITHA 1, R.KIRANJYOTHI 2, B.PADMA 3 ABSTRACT This communication describe simple, sensitive, rapid, accurate, precise and cost effective absorption ratio method(q-analysis)zero crossing method for the simultaneous determination of Atenolol and Losartan Potassium in combined dosage form. The utility of absorption ratio method data processing program is its ability to calculate unknown concentration of components of interest in a mixture containing an interfering component. The wave length used for the estimation of Atenolol is 275nm where Losartan potassium has no absorbance and for Losartan potassium is 282nm where Atenolol has no absorbance respectively..linear correlation was obtained between absorbance and concentrations of Atenolol and Losartan potassiumin the concentration ranges of 5-30 μg/ml, with R 2 value at both the wavelengths respectively. The linearity of the calibration curve was validated by the high values of correlation coefficient of regression. The RSD values of Atenolol and Losartan Potassium were found to be less than 2%.LOD and LOQ values for Atenolol and were found to be 0.74μg/ml and 2.45μg/ml at 275 nm and LOD and LOQ values for Losartan potassium were found to be 0.72 and 1.78 μg/ml at 282 nm, respectively. KEYWORDS Atenolol, Losartan potassium, Absorption ratio method, Zero crossing point AFFILIATIONS Division of Pharmaceutical analysis and Quality Assurance, Center for pharmaceutical Research (CPR), Raghavendra Pharmacy college (RIPER), Anantapur, Andhra Pradesh, India *Correspondence: 54

2 INTRODUCTION Atenolol is chemically2-(4-{2-hydroxy-3-[(propan-2-yl)amino]propoxy}phenyl)acetamide CC(C)NCC(O)COC1=CC=C(CC(N)=O)C=C1.It acts as Antihypertensive Agents, Adrenergic Agents, Adrenergic beta-antagonists, Sympatholytics, Antiarrhythmic Agents, Anti-Arrhythmia Agents.Losartan potassium is chemically[2-butyl-4-chloro-1-({4-[2-(2h-1,2,3,4-tetrazol-5- yl)phenyl]phenyl}methyl)imidazol5yl]methanolccccc1=nc(c1)=c(c=c1)c1=cc=c1c1=n N=N1. It acts as Antihypertensive Agents, Angiotensin II Receptor Antagonists, Antiarrhythmic Agents, Angiotensin II Type 1 Receptor Blockers, Anti-Arrhythmia Agents, susceptible organisims. Literature survey has revealed few methods for their quantification alone (or) in combination by UV spectrophotometry 3-12, HPLC 13, but no UV derivative method was found for simultaneous estimation of both the drugs in dosage forms. Hence, the present work was designed and made attempt to develop Absorption ratio UV spectrophotometric method for Atenolol and Losartan potassium. Structure of Atenolol Structure of Losartan potassium 55

3 MATERIALS AND METHODS Materials A Lab India double beam UV/VIS spectrophotometer with spectral width of 2nm, wavelength accuracy of 0.5nm & a pair of 10mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV Win 5.0 software. Atenolol and Losartan potassium bulk powder were kindly gifted by Hetero Labs ltd. Hyderabad, Andhra Pradesh, India. The commercial combination product procured from the local market. Methanol was used in the study. Preparation of standard solutions 10 mg of standard Atenolol and Losartan potassium were weighed and transferred to 100ml volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 µg/ml each of Atenolol and Losartan potassium. Methodology Atenolol and Losartan Potassium solubility were tested in different organic and aqueous solvents and both drugs are soluble in methanol. The working standard solutions were scanned from 200 to 400 nm to select the wavelengths for estimation. The zero crossing point for Atenolol and Losartan Potassium was 275 nm and 282 nm respectively in methanol. (figure 1.) Fig.1: Overline spectrum of Atenolol and Losartan potassium. 56

4 From the overline spectra of both the drugs wavelengths selected for quantification were 275nm (λmax of Atenolol) for Atenolol & 282nm (Zero crossing point of Losartan potassium) for Losartan potassium. VALIDATION OF THE PROPOSED METHOD The proposed method was validated according to the International Conference on Harmonization (ICH)guidelines Linearity The linearity of calibration curves in pure solution was checked over the concentration ranges of about 5-30 µg/ml for Atenolol and Losartan potassium (Figure 2). The represented data was shown in below Table 1.Appropriate aliquots from the standard stock solutions of Atenolol and Losartan potassium were used to prepare two different sets of dilutions Series A andb stock solutions as follows. Series A consisted of different concentration of Atenolol (5-30 μg/ml). Aliquot from the stock solution ofatenolol5-30 μg/ml) was pipetted out in to a series of 10 ml volumetric flask and diluted with methanol to get final concentration in range of 5-30 μg/ml (5,10, 15, 20, 25 and 30 ml). Series B consisted of varying concentrations of Losartan potassium (5-30 μg/ml). Appropriate volume of the stock solution of Losartan potassium (100 μg/ml) was transferred into a series of 10 ml volumetric flask and the volume was adjusted to the mark with methanol to get final concentration in range of 5-30 μg/ml (5,10,15,20,25,30 ml). The calibration curve were constructed by plotting drug concentration versus the absorbance values of Absorption ratio spectrum 275 nm for Atenolol and 282 nm for Losartan potassium. The concentration of individual drugs present in the mixture was determined from the calibration curves in quantification mode. Fig.2: Linearity Plots of Atenolol and Losartan Potassium. 57

5 Table 1:Regression data of Atenolol and Losartan potassium Parameters Atenolol Losartan potassium Wavelength(nm) Beer s law limit (μg /ml) Regression equation (y = a + bc) Slope (b) Intercept (a) y = x y = x Correlation coefficient (r2) LOD a (μg/ml) LOQ b (μg /ml) Precision (%RSD c, n = 3) Interday Intraday Accuracy (%Recovery, n= 5)

6 a LOD = Limit of detection, b LOQ = Limit of quantification, c RSD = Relative standard deviation. d S.D. =Standard deviation Method Precision (Repeatability) The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions (n= 6) for Atenolol and Losartan potassium (10 μg/ml for both drugs) without changing the parameter of the proposed spectrophotometric method Intermediate Precision (Reproducibility) The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of Atenolol and Losartan potassium (5, 15,.30μg/ml for both Atenolol and Losartan potassium). The intra and inter-day accuracy and precision were calculated and results were presented in the table-2. Precision of the analytical method was found to be reliable based on %RSD (Table 2). Table 2: Precision studies by UV Method S.No. Sample Intra-day (%RSD) Inter-day (% RSD) Atenolol at 275 nm LosartanPotassium at 282 nm Atenolol at 275 nm Losartan Potassium at 282 nm 1 LQC MQC HQC Accuracy The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 80, 100 and 120%. Results of assay and recovery were presented in table-3 59

7 Table 3: Recovery studies on the drugs S.No. Name of the drug Amount of sample (µg/ml) Recovery Level Amount of drug added (µg/ml) Total amount found (µg/ml) ± SD % Recovery % RSD 1. Atenolol 10 80% ± % ± % ± LosartanPotassium 10 80% ± % ± % ± Limit of detection (LOD), Limit of Quantification (LOQ) Limit of detection was found to be 0.74 µg/ml for Atenolol at 275 nm and 0.72 µg/ml for Losartan potassium at 282 nm respectively and Limit of Quantification was found to be 2.45 µg/ml Atenolol at 275nm and 1.78 µg/ml for Losartan Potassium at 282 nm respectively. Analysis of tablet Sample Twenty tablets of REPALOL were weighed and finely powdered and tablet powder equivalent to 100mg of both Atenolol and Losartan potassium is weighed and extracted with methanol in a 100 ml volumetric flask. The flask was sonicated for 15 min and volume was made up to the mark with methanol. 1ml was transferred into a 10ml volumetric flask and the volume was made up to the mark with water, and 1ml of above solution is added to 10ml volumetric flask and made upto the mark with water, finally the solution is filtered by using syringe filter to obtain 10µg/ml of Atenolol and 10 µg/ml of Losartan potassium. The absorbance of the solution was measured under UV spectrophotometer. The assay procedure was made triplicate and weight of sample taken for assay was calculated. The percentage of drug found in formulation, mean and standard deviation in formulation were calculated in the table-4. Table 4: Assay of formulation by Absorption Ratio method Formulation Drug Label claim Amount Found ± SD % Recovery %RSD REPALOL TAB Atenolol 50 mg 195mg± % 0.51 LosartanPotassium 50mg 195mg ± %

8 RESULTS AND DISCUSSION The selected drugs Atenolol and Losartan Potassium were estimated by using simultaneous estimation by Absorption Ratio method as per ICH guidelines. The method was validated for all validation parameters as per ICH guidelines. From the overlain spectrum shown in figure-1 the wavelength selected for estimation of Atenolol was 275 nm, whereas Losartan Potassium has no absorbance and for Losartan Potassium it was 282 nm, where Atenolol has no absorbance. The linearity range for Atenolol and Losartan Potassium was 5-30 µg/ml with R 2 value of for Atenolol and for Losartan Potassium. Limit of detection was found to be 0.74 µg/ml for Atenolol at 275 nm and 0.72 µg/ml for Losartan potassium at 282 nm respectively. Limit of Quantification was found to be 2.45 µg/ml Atenolol at 275nm and 1.78 µg/ml for Losartan Potassium at 282 nm respectively. The % RSD for intraday and interday precision was <2%. The method has been validated in assay of tablet dosage forms. The accuracy of the method was validated by recovery studies and was found to be significant and it is under specification limits, with % recovery (i.e., within acceptable range %). The assay results were found to be 98% for Atenolol and 97% for Losartan potassium respectively. (i.e., within acceptable range %). CONCLUSION The proposed spectrophotometric method was found to be simple, sensitive, accurate and precise for determination of Atenolol and Losartan potassium in tablet dosage form. The method utilizes easily available and cheap solvent for analysis of Atenolol and Losartan potassium and hence the method was also economic for estimation of Atenolol and Losartan potassium from tablet dosage form. The common excipients and other additives are usually present in the tablet dosage form do not interfere in the analysis of Atenolol and Losartan potassium in method, hence it can be conveniently adopted for routine quality control analysis of the drugs in combined pharmaceutical formulation. ACKNOWLEDGEMENT The authors are thankful to Hetero pharmaceuticals Ltd., Hyderabad, Andhra Pradesh, India for providing gift sample of Atenolol and Losartan potassium for research. The authors are highly thankful to Raghavendra Institute of pharmaceutical Education and Research, Anantapur , Andhrapradesh, India for providing all the facilities to carry out the work. REFERENCES 1. Skoog W: Fundamental of Analytical Chemistry, Saunders College, Publishing, 7 th Edition, 1992, pp Beckett A.H. and Stanlake J.B: Practical Pharmaceutical Chemistry, Part 2, CBS Publishers and Distributors, 4 th Edn, 2002, pp ICH-Guidelines Q2A, Validation of Analytical Procedures: Definition and terminology Geneva, Switzerland

9 4. The United States Pharmacopoeia-32(2009),National Formulary 27,U.S.Pharmacopeial convention Inc., Rockville, MD, p Clarke s analysis of drugs and poisons, 3 rd edition, Pawar1 AKM, Nageswara Rao ABN, Pawar AKM: A validated method for the simultaneous quantification of Hydrochlorothiazide, Olmesartan medoxomil and Amlodipine in bulk and pharmaceutical dosage form.journal of Pharmacy Research 2012; (1): Kardile1DP, Patel HH, Patel MR: Simultaneous Estimation of Amlodipine Besylate And Olmesartan Medoxomil Drug Formulations By HPLC And UV SpectrophotometricMethods.International Journal of Pharmaceutical and Applied Sciences 2011; 2(1). 8. Pournima S. Patil, Harinath N. More, Sachin A. Pishwikar: RP-HPLCmethod for simultaneous estimation of amlodipine besylate andolmesartan medoxomil from tablet. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3(3). 9. Pournima S. Patil, Pramodini D. Kulkarni, Mallappa S. Burkul: Simultaneous estimation of Amlodipine besylate and Olmesartan medoxomil by First Order Derivative Spectroscopy from Tablet. International Journal of PharmTech Research 2011; 3(2): Anandakumar K, Jayamariappan M: Absorption correction method for the simultaneous estimation of Amlodipine besylate, Valsartan and Hydrochlorothiazide in bulk and in combined tablet dosage form. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3(1) 11. Hemendra Kumar Sharma, Nilesh Jain, Surendra Kumar Jain and Sharma: Method development and validation of Olmesartan, Amlodipine and Hydrochlorothiazide in combined tablet dosage form. Pharm Anal Acta 2011;2: Saminathan J, VetrichelvanT: Development of Spectrophotometric Method for Quantitative Estimation of Amlodipine Besylate, Olmesartan Medoxomil and Hydrochlorthiazide in Tablet Dosage Form. International Journal of Pharmaceutical Research & Analysis 2011; 1(1):

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