Abstract: A sensitive and high throughput method by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for the determination of trihexyphenidyl in human plasma. The method was used to evaluate the bioequivalence of the test preparation and reference preparation, and to investigate the effect of food on the pharmacokinetic behavior of trihexyphenidyl. The trihexyphenidyl and internal standard trihexyphenidyl-d11 were extracted from human plasma by protein precipitation using methanol as the precipitant. Chromatographic separation was achieved on a Waters UPLC BEH C8 column (50 mm×2.1 mm, 1.7 μm) with 0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium acetate and acetonitrile-water (95:5, v/v) as the mobile phases. The analytes were detected by an electrospray ionization source in positive ion and multiple reaction monitoring modes. The linear range of trihexyphenidyl was 0.1-40 ng/mL. This test involved 30 healthy male and female subjects with a single oral administration of a 2-mg trihexyphenidyl hydrochloride tablet each. The 90% confidence intervals under fasting conditions of peak plasma concentration (C_max), area under the plasma concentration-time curve (AUC_0-t) and area under the plasma concentration-time curve from zero to infinity (AUC_0-∞) were 82.2%-99.4%, 82.3%-97.3% and 83.4%-97.9%, and these pharmacokinetics parameters under postprandial conditions were 100.8%-122.8%, 96.8%-112.4% and 96.6%-112.1%, which were all in the range of 80.0%-125.0%, indicating that the test tablets and reference tablets were bioequivalent.

Key words: ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), human pharmacokinetics, bioequivalence, trihexyphenidyl