Quantitation of an orally available thrombin inhibitor in rat, monkey and human plasma and in human urine by high-performance liquid chromatography and fluorescent post-column derivatization of arginine

Abstract

An assay for the quantification of plasma and urine levels of CVS 1123, an orally bioavailable thrombin inhibitor, and its desmethyl form, CVS 738, was developed to support clinical and toxicology studies. This assay uses solid-phase extraction, reversed-phase HPLC separation, and post-column fluorescent derivatization with ninhydrin. An internal standard is added to correct for recovery. In aqueous solution, the arginine aldehyde structures of CVS 1123 and CVS 738 exist in multiple forms which can be separated under standard reversed-phase HPLC conditions. HPLC conditions were optimized to give rapid interconversion of the forms on the separation time scale, and consequently a single chromatographic peak. Extraction conditions were modified for quantitative extraction of drug compounds from large volumes of human plasma. The assay was shown to be accurate and precise, with a quantification limit of 17 ng CVS 1123/ml human plasma.