In mammals, mono-N-desethylamiodarone (MDEA) is the only known metabolite of amiodarone. Our previous experiments demonstrated that in vitro MDEA may be hydroxylated, N-dealkylated, and deaminated. In this report, we investigated the concentration of these microsomal metabolites in the plasma of patients receiving amiodarone. The presence of the hydroxy-amiodarone and deiodinated amiodarone was also additionally investigated. A high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS) quantitative assay using morpholine-amiodarone as internal standard was developed for measuring these metabolites in the range of 3-250 ng ml(-1). In the concentration ranges 5-50 and 50-250 ng ml(-1), the coefficients of variation of the measurements were less than 14 and 7%, respectively. The concentrations of investigated compounds in plasma of patients (n=14) receiving amiodarone (0.2 g day(-1), orally for >2 months) varied inter-individually and were 140.0+/-85.2, 39.1+/-20.8, and 26.2+/-15.2 ng ml(-1) for 3'OH-mono-N-desethylamiodarone, di-N-desethylamiodarone, and deaminated amiodarone, respectively. The concentrations of MDEA and amiodarone in these samples were 970+/-347 and 11163+/-435 ng ml(-1), respectively. In contrast, the studied compounds were not detectable in plasma samples from eight patients receiving amiodarone intravenously. Qualitatively, in the plasma of patients receiving amiodarone orally, hydroxylated amiodarone was also positively detected by assaying the [M+H](+) ions at m/z 662, but the deiodo-metabolites of amiodarone were not detected using mass spectrometry. Thus, in humans, amiodarone and MDEA were biotransformed by dealkylation, hydroxylation, and deamination.