Solid-phase extraction techniques for the determination of glycopyrrolate from equine urine by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.

This research outlines a successful technique for extracting glycopyrrolate, a drug used to enhance breathing, from horse’s urine. By testing the residue after applying solid-phase extraction methods with both liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry, researchers could detect even low concentrations of the drug.

Detail of The Research Process

• The study primarily explores a solid-phase extraction process to obtain a residue from equine urine. This residue could further undergo analysis through liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-mass spectrometry (GC-MS).
• For the LC-MS-MS analysis, researchers extracted a derivative from 5 ml of urine and subjected it to cation-exchange chromatography. They monitored the daughter ion of m/z 318 in positive-ion mode, which turned out to be m/z 116.
• The method ensured a 99.5% recovery rate of glycopyrrolate. The consistency of this technique was verified through a within-run coefficient of variation being less than 5% and a between-run coefficient of less than 6.5% for two different quality control samples.

Detection of Glycopyrrolate

• The concentration of glycopyrrolate in urine is very low, leading the researchers to put significant efforts into its detection. The minimal detectable concentration for the assay was determined to be 250 pg/ml.
• Qualitative detection required XAD-2 extraction from 50 ml of urine, further clean-up using cation-exchange chromatography, and a combined process of hydrolysis-derivatization. The target analyte here was the methyl ester of hydrolyzed glycopyrrolate.
• Even at low concentrations, the post-injection (1mg intravenously) urine samples showed traces of glycopyrrolate for up to 9 hours using both LC-MS-MS and GC-MS analysis methods.

Significance of the Findings

• The main benefit of this method comes from the extreme sensitivity of the LC-MS-MS process, which, when combined with the highly selective glycopyrrolate extraction, allows for a highly sensitive and reliable testing process.
• This research could be instrumental in detecting prohibited drug use in horse racing, as it enables the detection of even low concentrations of glycopyrrolate, a respiratory enhancing drug reportedly used by some to improve a horse’s performance.