Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC composed Of D-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion- sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination Of D-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 [LL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-CI 8 column (100 mm x 3.0 mm, 3.5 mu m) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0 -> 9 1.0 (D-amphetamine), 256.0 -> 167.0 (diphenhydramine) and 166.1 -> 148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for D-amphetarnine and I ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2) > 0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs. (C) 2007 Elsevier B.V. All rights reserved.