Electrospray ionization mass spectrometric characterization and quantitation of xanthine derivatives using isotopically labelled analogues: an application for equine doping control analysis

Abstract

Isotope‐dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance‐enhancing drugs. Theobromine has to exceed a threshold level of 2 μg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodomethane‐d₃ in the presence of N‐methyl‐N‐trimethylsilyltrifluoroacetamide or potassium carbonate in acetonitrile, respectively. Both compounds were characterized by nuclear magnetic resonance spectroscopy and electrospray ionization tandem mass spectrometry, and a robust and fast assay for the qualitative and quantitative analysis of theobromine in equine urine samples was validated. Urine specimens were extracted by means of solid‐phase extraction cartridges, and concentrated extracts were analyzed by liquid chromatography interfaced to a triple‐quadrupole mass spectrometer. In addition, the dissociation behavior of deuterated analogues to caffeine and theobromine allowed proposals for fragmentation routes of xanthine derivatives after atmospheric pressure ionization and collisionally activated dissociation. Copyright © 2004 John Wiley & Sons, Ltd.