Direct determination of the antihypertensive agent Cromakalim and its major metabolites in human urine by high-performance liquid chromatography

Abstract

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of Cromakalim, a novel antihypertensive agent, and its urinary metabolites including diastereomeric glucuronides. The HPLC system employed a strong cation-exchange precolumn (Senshu Pak SCX-2051-N) to allow direct injection of urine samples. The unchanged drug and its three major metabolites were simultaneously separated on a reversed-phase column (Develosil ODS-5) and fluorometrically detected (excitation, 254 nm; emission, 306 nm) by the aid of their native fluorescience. The calibration curves for Cromakalim and a metabolite were linear in the range from 10 to 200 ng ml −1, while those for the diastereomeric glucuronides were linear in the range from 20 to 400 ng ml −1. The detection limits (signal-to-noise ratio = 3) of these compounds were 0.3 ng ml −1 or less in all cases.