A SENSITIVE, ECONOMICAL BIOANALYTICAL LC-MS/MS METHOD FOR SIMULTANEOUS ANALYSIS OF IRBESARTAN AND HYDROCHLOROTHIAZIDE

Abstract

A novel bioanalytical LC-MS/MS method for irbesartan and hydrochlorothiazide has been developed using deuterated internal standards. Sample pretreatment was carried out using Strata-X Cartridge 33µm. Chromatographic separation was achieved on Chromolith® High Resolution Column, 100X4.6mm, 5µ using a mobile phase of acetonitrile (80 %) and 2mM ammonium acetate (20 %) basified with ammonia solution. A flow rate of 1 mL min-1 was employed with split ratio of approximately 50% outside, with column temperature being 40˚C ±2˚C. The retention time for irbesartan and irbesartanD4 deuterated internal standard was 1.52 min (±0.50min) and for hydrochlorothiazide and hydrochlorothiazide15N213CD2 deuterated internal standard it was 1.43 min (±0.50 min). The developed LC/MS-MS method was validated as per EMA guidelines. The linearity range established for the drugs in human plasma was 50.197 ng mL-1 to 6038.206 ng mL-1 for irbesartan and 1.021 ng mL-1 to 408.480 ng mL-1 for hydrochlorothiazide. The short run time ensured use of less mobile phase and increased sample throughput, making analysis rapid and economical.