Determination of Diphenidol in Mouse Plasma and Application to a Pharmacokinetic Study Using Uplc-Ms/Ms

Objective: Difenidol is widely used in clinical practice due to its good anti-dizziness effect and low side effect rate. This aim was to develop an ultra-high performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) method for the selective and straightforward measurement of diphenidol in mouse plasma. Methods: A total of eighteen mice were divided into three groups: six for intravenous administration at a dose of 0.2 mg/kg, six for oral administration at a dose of 0.4 mg/kg, and another six for oral administration at a dose of 1.6 mg/kg. The analytes were extracted using acetonitrile-mediated protein precipitation following the addition of the internal standard (IS), midazolam. On an Acquity HSS T3 column (50 mm × 2.1 mm, 1.8 μm). The quantification process involved the use of multiple reactions monitoring (MRM) mode, with target fragment ions m/z 310.2→128.9 for diphenidol and m/z 326.2→291.4 for IS. Results: For diphenidol, calibration curves showed a linear distribution between 0.2 and 50 ng/mL. The accuracy of the method was between 94.6% and 110.4%, and the mean recovery of diphenidol in mouse plasma was over 76.5%. The intra-day and inter-day precision RSDs were both limited to 14%. The bioavailability of diphenidol in mice was determined to be 19.9% and 23.56% for the oral dose of 0.4 mg/kg and 1.6 mg/kg, respectively. Conclusion: The UPLC-MS/MS was successfully applied to study the pharmacokinetics of diphenidol in mice, to which it was administered orally and intravenously.