SIMULTANEOUS ANALYSIS OF DIPHENYLMETHOXYACETIC ACID, A METABOLITE OF DIPHENHYDRAMINE, AND ITS DEUTERIUM-LABELED STABLE-ISOTOPE ANALOG IN OVINE PLASMA AND URINE

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydra mine in monkeys, dogs, and humans. The metabolic fate of diphenhydrami ne (DPHM) in sheep is not yet well understood; however, preliminary st udies have demonstrated the presence of DPMA in the plasma and urine o f sheep following an intravenous bolus of DPHM. Our current studies em ploy the simultaneous intravenous co-administration of DPHM and the st able isotope analog of DPHM to investigate the pharmacokinetics of DPH M in sheep. In these studies, in order to investigate the pharmacokine tics of the DPMA metabolite, measurement of both unlabeled and stable- isotope labeled DPMA is required. Thus, a stable isotope analog of DPM A ([H-2(10)]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chroma tography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [H-2(10)]DPMA used a single step liquid-liquid extraction procedu re using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide. A 1.0-mu l a liquot of the prepared sample was injected into the GC-MS system and q uantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenyla cetic acid, m/z 183 for DPMA, and m/z 177 for [H-2(10)]DPMA. The ion c hromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The i ntra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10 % at all other concentrations). This method was used to measure the co ncentration of DPMA and [H-2(10)]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [H-2(10)]DPHM were admin istered by an intravenous bolus dose via the femoral vein. DPMA appear ed to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [H -2(10)]DPMA in biological samples obtained from sheep (e.g. plasma and urine).