Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Quantification of Glycopyrrolate in Horse Plasma

Abstract

A rapid, sensitive, and specific ultra-high-performance liquid chromatography with heated electrospray ionization-tandem mass spectrometry (UHPLC-HESI-MS-MS) method to detect and quantify glycopyrrolate in horse plasma has been developed and validated. We also determined glycopyrrolate in plasma after oral and intravenous administration of clinically relevant doses to Thoroughbred horses. Calibration was accomplished by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). Glycopyrrolate (GLY) and the IS (GLY-d(3)) were isolated from plasma matrices via weak cation exchange using a simple solid-phase extraction technique. Chromatographic analysis was achieved by reversed-phase UHPLC on a C(18) Acquity™ column. Extracts were analyzed in positive electrospray ionization mode and precursor and product ions were detected and quantified by MS-MS using a triple-stage quadrupole (TSQ) instrument. The method was characterized by a linear range of 0.125-25 pg/mL (R(2) > 0.998), a lower limit of quantification of 0.125 pg/mL and a lower limit of detection of 0.025 pg/mL. Recovery of GLY ranged from 78% to 96%, and intra- and interbatch precision were 3.3-14.4%CV and 3.4-14.4%CV, respectively. Glycopyrrolate was stable in plasma for up to 170 days at -80°C, through three freeze/thaw cycles, and for up to 48 h after extraction under 20°C autosampler conditions.