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

Glycopyrrolate (Robinul) is a quaternary ammonium salt which serves as a respiratory enhancing drug. It is reportedly used in horse racing to improve breathing. Extraction of glycopyrrolate from equine urine employing unique solid-phase extraction techniques gave a residue suitable for liquid chromatography—tandem mass spectrometry (LC—MS—MS) and gas chromatography—mass spectrometry (GC—MS). LC—MS—MS analysis employed an extract derived from 5 ml of urine subjected to cation-exchange chromatography. The daughter ion of m/z 318 monitored in the positive-ion mode was m/z 116. Recovery of glycopyrrolate was 99.5% and the within-run coefficient of variation of two quality control samples (1.0 and 10 ng/ml) was less than 5%. The between-run coefficient of variation for the same two quality control samples was less than 6.5%. The minimal detectable concentration for the assay was 250 pg/ml. Due to the extremely low concentration of glycopyrrolate in urine, qualitative detection via full-scan GC—MS required XAD-2 extraction of 50 ml of urine, cation-exchange chromatography clean-up and a tandem hydrolysis—derivatization procedure. The target analyte for GC—MS qualitative analysis was the methyl ester of hydrolyzed glycopyrrolate. Glycopyrrolate could be detected in post-administration (1 mg intravenously) urine samples for up to 9 h by both LC—MS—MS and GC—MS. The success of the method was due to a combination of the extreme sensitivity of the LC—MS—MS method and the very selective extraction process for quaternary ammonium salts.