FULLY AUTOMATED METHODS FOR THE DETERMINATION OF HYDROCHLOROTHIAZIDE IN HUMAN PLASMA AND URINE

LC assays utilizing fully automated sample preparation procedures on Z ymark PyTechnology(TM) Robot and BenchMate(TM) Workstation for the qua ntification of hydrochlorothiazide (HCTZ) in human plasma and urine ha ve been developed. After aliquoting plasma and urine samples, and addi ng internal standard (IS) manually, the robot executed buffer and orga nic solvent addition, liquid-liquid extraction, solvent evaporation an d on-line LC injection steps for plasma samples, whereas, BenchMate(TM ) performed buffer and organic solvent addition, liquid-liquid and sol id-phase extractions, and an-line LC injection steps for urine samples . Chromatographic separations were carried out on Beckman Octyl Ultras phere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroac etic acid (urine). The eluent from the column was monitored with UV de tector (271 nm). Peak heights for HCTZ and IS were automatically proce ssed using a PE-Nelson ACCESSCHROM laboratory automation system. The assays have been validated in the concentration range of 2-100 ng ml(- 1) in plasma and 0.1-20 mu g ml(-1) in urine. Both plasma and urine as says have the sensitivity and specificity necessary to determine plasm a and urine concentrations of HCTZ from low dose (6.25/12.5 mg) admini stration of HCTZ to human subjects in the presence or absence of losar tan.