Simultaneous determination of diphenhydramine, its N ‐oxide metabolite and their deuterium‐labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry

Our studies on drug disposition in chronically instrumentedpregnant sheep involve simultaneous administration of theantihistamine diphenhydramine (DPHM), its deuteratedanalogue ([ 2 H 10 ]DPHM) andtheir metabolites to the mother or the fetus via various routes. Suchstudies require sensitive and selective mass spectrometric methods forquantitation of these labeled and unlabeled compounds in order toassess comparative maternal and fetal drug metabolism. The objectiveof this study was to develop and validate a liquidchromatographic/tandem mass spectrometric(LC/MS/MS) method for the simultaneous quantitationof DPHM, its N ‐oxide metabolite and theirdeuterium‐labeled analogues in ovine plasma and urine. Samplesspiked with the analytes and the internal standard, orphenadrine, wereprocessed using liquid–liquid extraction. The extract waschromatographed on a propylamino LC column and MS/MS detection wasperformed in the positive ion electrospray mode using multiplereaction monitoring. The linear concentration ranges of thecalibration curves for the N ‐oxides and the parentamines were 0.4–100.0 and 0.2–250.0 ngml ‐1 , respectively. In validation tests, the assayexhibited acceptable variability (⩽15% at analyteconcentrations below 2.0 ng ml ‐1 and <10%at all other concentrations) and bias (<15% at allconcentrations), and the analytes were stable under a variety ofsample handling conditions. Using this method, the labeled andunlabeled N ‐oxide metabolite was identified in fetalplasma after DPHM and [ 2 H 10 ]DPHMadministration. This method will be used further to examine thecomparative metabolism of diphenhydramine to its N ‐oxidemetabolite in the mother and the fetus. © 1998 John Wiley &Sons, Ltd.