Analytical methodology for the detection of β2-agonists in urine by gas chromatography–mass spectrometry for application in doping control Original Research Article

A comprehensive gas chromatographic–mass spectrometric (GC–MS) procedure for detection in urine of β2-agonists having different alkyl or phenylalkyl chains at the nitrogen atom is described. The method is based on an enzymatic hydrolysis with β-glucuronidase from Helix pomatia, followed by a solid-phase extraction procedure using Bond Elut Certify columns. The influence of urine pH in the extraction recovery has been studied and pH 9.5 was found to give best recovery and cleaner extracts. After pH adjustment, the sample was applied to the pre-conditioned cartridges and after a washing step, the β2-agonists were eluted with a mixture of chloroform and isopropanol (80:20, v/v) containing 2% ammonia. The residues were derivatised with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), and analysed by GC–MS. A validation procedure for qualitative analysis of β2-agonists in urine was performed. Selectivity of the method showed that no interfering peaks were observed for most of the compounds at the retention time of the β2-agonists. Extraction recoveries ranged from 68.1 to 103.7% in urine samples. Detection limits from 0.5 to 5 ng ml−1 were obtained using selected-ion monitoring. Intra-assay precision ranged between 2.3 and 13.8% for all compounds except for fenoterol. The International Olympic Committee (IOC) criteria for compound identification were fulfilled for the compounds studied. The optimised method was successfully applied to analyse urine samples obtained from excretion studies in healthy volunteers.