Determination of clenbuterol in human urine by GC-MS-MS-MS: confirmation analysis in antidoping control

摘要

This work presents a GC-MS-MS-MS method for the direct determination of clenbuterol in human urine. The method comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC-MS~3 (ion trap) with electron impact ionization. The GC-MS~3 analysis allows isolation and characterization of specific fragments from the original (MS~1) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z = 335-337) characteristic of clenbuterol. The MS~2 product fragment m/z = 300 is in turn used as a further precursor fragment giving rise to a MS~3 spectrum specific for clenbuterol. MS~4 fragmentation spectra were also investigated. However, further fragmentation of MS~3 product ions does not lead to functional MS~4 spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS~3 technique is lower than 0.2 μg/1, with a linear range between 0.5 and 5 μg/1, thus matching the basic requirements for antidoping analysis according to the guidelines of the International Olympic Committee. Due to its overall analytical performance, the method is presently being evaluated as a confirmation protocol to be followed to detect illicit clenbuterol administration to the athletes, and compared with reference GC-MS and GC-MS-MS techniques.