Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography-mass spectrometry

This work presents the validation study of the comprehensive two-dimensional gas chromatography (GCxGC)-time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GCxGC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0-999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly "blind" to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new "separation" dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GCxGC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method.