Simultaneous determination of benazepril and hydrochlorothiazide in their binary mixture by spectrophotometric method and stability indicating RP-HPLC of the mixture in presence of benazeprilat in dosage form and its application to human plasma

摘要

Two simple, accurate, and selective methods have been developed and validated for the simultaneous determination of Benazepril (BENZ) and Hydrochlorothiazide (HCTZ) in their binary mixture with or without Benazeprilat (BENZT), the degradation product of Benazepril (BENZ), without prior separation. The first method is extended ratio subtraction spectrophotometric method (EXRSM) coupled with Ratio subtraction method (RSM) where both BENZ and HCTZ were determined simultaneously in bulk powder and dosage form. The second method is stability indicating RP-HPLC method, where BENZ, HCTZ, and the degradation product BENZT, which is the active form of BENZ, were determined simultaneously in bulk powder and dosage form. In the RP-HPLC method, the sample was chromatographed on Inertsil C18 column (250 x 4.6mm, 5μ) and the mobile phase used was consisting of acetonitrile-potassium hydrogen phosphate buffer pH = 7 adjusted by 0.1 N NaOH (40/60 v/v) with a flow rate of 1.0 mL/min and detection at 242 nm. A detailed validation of the methods was performed following the ICH guidelines and the standard curves were found to be linear in the range of 5-30,2-30 μg/mL for BENZ and HCTZ, respectively in the spectrophotometric method, and 0.5-100, 5-60, and 1-100 ug/mL for BENZ, HCTZ, and BENZT in the RP-HPLC method, with mean correlation coefficient of more than 0.9996 for each analyte. The intra-day and inter-day precision and accuracy results were well within the acceptable limits. Also the RP-HPLC method was applied on spiked human plasma.