DEVELOPMENT AND VALIDATION OF COLUMN-SWITCHING HIGHPERFORMANCE LIQUID-CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF A POTENT AII RECEPTOR ANTAGONIST, TCV-116, AND ITS METABOLITES IN HUMAN SERUM AND URINE

Column-switching HPLC methods have been developed and validated for th e determination of a new antihypertensive prodrug, TCV-116 (I), and it s metabolites, CV-11974 (II) and CV-15959 (III), in human serum and ur ine, Initial sample cleanup was achieved by extracting the analytes in to an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer , the zone of the analyte's retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous pea ks was accomplished by the two-dimensional chromatography. Good precis ion and linearity of the calibration standards, as well as the inter-d ay and intra-day precision and accuracy of quality control samples, we re achieved. The limit of quantitation (LOQ), using 0.5 mi of serum, w as 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes d uring storage, extraction, and chromatography processes was establishe d, The results illustrate the versatile application of column switchin g to method development of multiple analytes in various biological mat rices. The methods have been successfully used for the analyses of I a nd its metabolites in thousands of clinical samples to provide pharmac okinetic data.