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

Area of Science:

• Analytical Chemistry
• Pharmacokinetics
• Equine Science

Background:

• Glycopyrrolate is used in horses, but sensitive detection methods in urine are lacking.
• Understanding glycopyrrolate elimination is vital for anti-doping regulations in equine sports.

Purpose of the Study:

• To develop and validate a sensitive UHPLC-MS/MS method for quantifying glycopyrrolate in horse urine.
• To investigate the pharmacokinetic profile of glycopyrrolate elimination in horses after intravenous and oral administration.

Main Methods:

• Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) was employed.
• Quantification utilized weighted linear regression with a deuterated glycopyrrolate internal standard.
• Method validation included linearity, specificity, precision, accuracy, stability, and matrix effects.

Main Results:

• The validated method demonstrated high sensitivity (LOD 1 pg/mL, LOQ 5 pg/mL) and accuracy (94-104%).
• Glycopyrrolate was detectable in urine up to 168 hours after intravenous and 24 hours after oral administration.
• Urine to plasma concentration ratios were approximately 100:1 from 24 hours onwards.

Conclusions:

• A validated, sensitive, and specific UHPLC-MS/MS method for equine urinary glycopyrrolate detection is established.
• This method is the first capable of regulating glycopyrrolate use in horses more than one day post-administration.
• Findings provide essential data for monitoring glycopyrrolate use and enforcing regulations in Thoroughbred racing.