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 μL plasma samples by a simple liquid–liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm × 3.0 mm, 3.5 μm) 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 → 91.0 (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-amphetamine and 1 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 (r2 ≥ 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.