Determination of doxepin and desmethyldoxepin in human plasma using liquidchromatography-tandem mass spectrometry

A sensitive method for the simultaneous determination of doxepin and its ac tive metabolite desmethyldoxepin in plasma was established, using high-perf ormance liquid chromatographic separation with tandem mass spectrometric de tection. The samples were extracted with hexane-isoamyl alcohol, separated on a Phenomenex Luna C-18 5 mu m, 150x2,1 mm column with a mobile phase con sisting of methanol-water-formic acid (600:400:0.5, v/v) at a flow-rate of 0.25 ml/min. Detection was achieved by a Perkin-Elmer API 2000 mass spectro meter at unit resolution in multiple reaction monitoring mode monitoring th e transition of the protonated molecular ions m/z 280.2, 266.2 and 250.1 to the product ions mit 107.1, 107.1 and 191.0 for analyte, metabolite and in ternal standard (benzoctamine-HCl), respectively. TurboIonSpray ionisation was used for ion production. The mean recovery for doxepin and desmethyldox epin was 90% and 75%, respectively, with a lower limit of quantification at 0.320 ng/ml and 0.178 ng/ml for the analyte and its metabolite, respective ly, using 0.5 mi plasma for extraction. This is the first assay method desc ribed for the simultaneous determination of doxepin and desmethyldoxepin in plasma using LC-MS-MS. The method is sensitive enough to be used in drug b ioavailablity studies with doxepin. (C) 2000 Elsevier Science B.V. All righ ts reserved.