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

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–MS3 (ion trap) with electron impact ionization. The GC–MS3 analysis allows isolation and characterization of specific fragments from the original (MS1) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z=335–337) characteristic of clenbuterol. The MS2 product fragment m/z=300 is in turn used as a further precursor fragment giving rise to a MS3 spectrum specific for clenbuterol. MS4 fragmentation spectra were also investigated. However, further fragmentation of MS3 product ions does not lead to functional MS4 spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS3 technique is lower than 0.2 μg/l, with a linear range between 0.5 and 5 μg/l, 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.