Abstract

For the first time the author bring forward newly developed RP-HPLC method for the simultaneous separation and determination of Atenolol (ATN), Metoprolol succinate (MET), Hydrochlorothiazide (HZT) and Amlodipine besylate (AML) individually and in combination with ATN with AML in tablet formulations which is quite distinct from that of other methods in vogue till today. The most important advantage of developed method was that the 4 separate drugs and combinations can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase composition by RP-HPLC. The chromatographic separation of the selected drugs was carried out on Welchrom C18 column consisting of 250 mm X 4.6 mm, 5 μm particle size utilizing mixture of 10 mM phosphate buffer (pH 3.0): Acetonitrile in the ratio of 50:50, v/v as mobile phase at the flow rate of 1mL/min with detection wave length at 235 nm by using UV spectrophotometric detector with total run time of 6 minutes. The retention times for ATN, MET, HZT, and AML were found to be 2.240, 2.813, 3.543, 3.753 and Combination of ATN and AML were arrived at 2.240 and 3.753 minutes respectively. All calibration curves for four drugs showed linearity over a concentration range of 2 - 10 μg/mL and for Amlokind - AT tablet 5 - 25 μg/mL for ATN and 1 - 5 μg/mL for AML. The corresponding correlation coefficient were also calculated from the linear regression analysis and found to be above 0.9995 in all cases. For intra and inter-day precision the % RSD values for each drug were evaluated and found RSD percentages of all above specified drugs were found less than 2 %. The results relating to limit of detection (LOD) and limit of quantitation (LOQ) for ATN, MET, HZT, AML and (ATN and AML combination) were found to be 0.3177 μg/mL and 0.9627 μg/mL; 0.1840 μg/mL and 0.5578 μg/mL; 0.1266 μg/mL and 0.3836 μg/mL; 0.2251 μg/mL and 0.6823 μg/mL; and for combination of drugs are 0.4941 μg/mL and 1.4973 μg/mL; 0.1181 μg/mL and 0.3580 μg/mL, respectively. The method was validated for its recovery, intra- and interday precision, selectivity and specificity. Therefore it is concluded that these methods are successfully feasible for the application of routine analysis within the said four antihypertensive drugs and binary combination of ATN and AML.