Determination of diltiazem and its main metabolites in human plasma by automated solid-phase extraction and high-performance liquid chromatography: a new method overcoming instability of the compounds and interference problems.

An automated sample preparation method, based on solid-phase extraction (SPE) was developed on an ASPEC-Gilson device and combined with HPLC for the determination of diltiazem and three of its metabolites in human plasma (N-desacetylmonodesmethyldiltiazem, N-monodesmethyldiltiazem, O-desacetyldiltiazem). A 1-ml volume of plasma is diluted with 0.5 ml of 0.1 M ammonium dihydrogen phosphate and the sample is automatically loaded onto a SPE silica (C18) column (100 mg); the column is flushed with two different solvents, then eluted with 0.5 ml of a 0.1 M ammonium dihydrogen phosphate-acetonitrile mixture (20:80, v/v) containing 0.06% of triethylamine. The eluate is evaporated to dryness and the residue reconstituted with a suitable solvent and injected onto a C8 silica column connected to a UV detector (lambda = 238 nm). This method overcomes problems caused by the partial instability of diltiazem and metabolites in human plasma during analysis. There is no chromatographic interference from endogenous compounds. The limits of quantitation (LOQ) are 2.5 and 2 ng ml-1 for diltiazem and the metabolites in human plasma, respectively. Linearity between concentrations and detector response for diltiazem and metabolites ranged from 10-200 and 5-100 ng ml-1 in human plasma, respectively. The method has been validated.