DETERMINATION OF DILTIAZEM AND ITS MAIN METABOLITES IN HUMAN PLASMA BY AUTOMATED SOLID-PHASE EXTRACTION AND HIGHPERFORMANCE LIQUID-CHROMATOGRAPHY - A NEW METHOD OVERCOMING INSTABILITY OF THE COMPOUNDS AND INTERFERENCE PROBLEMS

An automated sample preparation method, based on solid-phase extractio n (SPE) was developed on an ASPEC-Gilson device and combined with HPLC for the determination of diltiazem and three of its metabolites in hu man plasma (N-desacetylmonodesmethyldiitiazem, N-monodesmethyldiltiaze m, 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 automatica lly loaded onto a SPE silica (C-18) column (100 mg); the column is flu shed with two different solvents, then eluted with 0.5 mi of a 0.1 M a mmonium dihydrogen phosphate-acetonitrile mixture (20:80, v/v) contain ing 0.06% of triethylamine. The eluate is evaporated to dryness and th e residue reconstituted with a suitable solvent and injected onto a C- 8 silica column connected to a UV detector (lambda = 238 nm). This met hod overcomes problems caused by the partial instability of diltiazem and metabolites in human plasma during analysis. There is no chromatog raphic interference from endogenous compounds. The limits of quantitat ion (LOQ) are 2.5 and 2 ng ml(-1) for diltiazem and the metabolites in human plasma, respectively. Linearity between concentrations and dete ctor response for diltiazem and metabolites ranged from 10-200 and 5-1 00 ng ml(-1) in human plasma, respectively. The method has been valida ted.