A simple and sensitive high-performance liquid chromatography assay of diltiazem and main metabolites in renal transplanted patients

A sensitive and specific reversed-phase high-performance liquid chromatographic method was developed for determination of the benzothiazepine calcium channel blocker diltiazem and three of its main metabolites in human plasma. A solid-phase extraction method (C18) is described for isolating diltiazem and the metabolites N-demethyl-diltiazem (M-A), deacetyldiltiazem (M-1) and N-demethyl-deacetyl-diltiazem (M-2) from human plasma spiked with trans-diltiazem as internal standard. Chromatographic analysis of the eluate was accomplished using a reversed-phase column (C8) and a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate-buffer (pH 2.9), triethylamine and methanol (280:598:2:90 v/v/v/v). The limit of detection was 5 ng/ml for diltiazem and 2.5 ng/ml for the metabolites (UV detection). Recoveries were >75% for all substances. Precision analysis indicated a coefficient of variation below 5% for both diltiazem and metabolites at plasma concentrations ranging from 30 to 120 ng/ml for diltiazem and from 15 to 60 ng/ml for the metabolites. The accuracy was <3% for diltiazem at all concentrations investigated. Metabolites showed an accuracy of <7% at higher concentrations, however at low concentration the accuracy was <16%. A pilot study has been performed in order to study whether the method was applicable to patients and volunteers. Plasma samples from renal transplanted patients treated with cyclosporin A and healthy volunteers did not show any interference by cyclosporin A with the determination of diltiazem and metabolites. (C) 1999 Elsevier Science B.V. All rights reserved.