Regulatory control of glycopyrrolate in performance horses using validated UHPLC/MS–MS methods

We describe a validated, rapid, sensitive, and specific UHPLC–MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5–2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The intra and inter-batch imprecisions were <10% RSD and accuracy of the method ranged between 94 and 104%. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24 h after oral administration. Analytical method validation requirements for linearity, specificity, precision, accuracy, stability, dilution integrity, matrix effect, and ruggedness have been fulfilled. The urine method described in this report is simple and efficient and is the first reported method with sufficient sensitivity, accuracy, and precision to regulate the use of glycopyrrolate in urine samples collected more than one day after dosing of horses. Urine to plasma glycopyrrolate concentration ratios were calculated and were approximately 100:1 in samples collected from 24 h through the end of sample collection.

► We develop and validate an LC–MS/MS method for glycopyrrolate in horse urine.
► We construct tolerance intervals for glycopyrrolate in urine and plasma for threshold limits.
► Urine to plasma concentration ratios of glycopyrrolate can enhance the regulatory control of glycopyrrolate.
► Renal clearance of glycopyrrolate in the horse can be estimated without volumetric urine collections.