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 microg/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.

Collision‐induced dissociation studies of caffeine in positive electrospray ionisation mass spectrometry using six deuterated isotopomers and one N1‐ethylated homologue

“…Some comparative studies show that many similarities exist between CID fragments and those which are formed in a collision chamber, particularly when comparing fragmentations of pure protonated molecules . Spectra obtained from preliminary CID fragmentation experiments with caffeine revealed a very close similarity to a published spectrum whose fragments had been generated by means of a collision chamber . Intrigued by this study, it was our aim to gain a deeper insight into the pure CID fragmentation chemistry of xanthine derivatives.…”

“…For the investigation of the fragmentation chemistry of xanthine derivatives in the ESI‐MS mode, in addition to other unlabeled xanthines, a deuterated isotopomer of caffeine has been described and examined . For fragmentation experiments the authors of this work have used a collision cell; however, such a cell is not available in small and simple mass spectrometers.…”

“…Deuteromethylation of 3,7‐dimethylxanthine (theobromine, 1a ), 1,7‐dimethylxanthine (paraxanthine, 1b ), 1,3‐dimethylxanthine (theophylline, 1c ), and xanthine ( 1c ) was performed according to a classical alkylation reaction described using an alkyl halide in the presence of a base …”

“…By performing HPLC/MS experiments using those derivatives 2–7 in combination with deuterated eluents and computer‐based analyses it has been possible to clearly determine the molecular formulae of all caffeine fragments found by low‐resolution CID. One recently published study used a collision fragmentation cell and a deuterated caffeine isotopomer for the clarification of possible fragmentation reactions and was the motivation for the present work …”

Determination of caffeine and its metabolites in urine by capillary electrophoresis‐mass spectrometry

“…The characterization of caffeine and its major metabolites in biological fluids by HPLC-MS and CE has been reported in the literature by various laboratories (see Table 2, [74][75][76][77]); however, the HPLC data differ from the data obtained by CE in several ways: (a) some differences in the migration order or separation of the analytes studied were observed; (b) the LC method was laborious; for example, one laboratory reported that in order to obtain the characterization of all caffeine metabolites using LC-MS/MS, two instrument runs for each sample tested were required, one for the negative ionization mode and another for the positive mode [75]; and (c) a significant difference can be observed in the amount of organic solvents and reagents consumed. Although not tested in this laboratory, CE has the potential for assaying a large number of samples when multicapillary CE instruments are employed [78].…”