A new and reliable two-step liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in combination with gas chromatography/mass spectrometry (GC/MS) for the screening and confirmation of adrafinil and its major metabolites, modafinil and modafinil acid, in human urine has been developed and validated. The method involved reversed-phase C18 solid-phase extraction (SPE) cartridge extraction and MS analysis by means of LC/MS/MS and GC/MS. The study illustrated that the ESI capillary temperature played a key role in the formation of the protonated molecule. The limits of detection (LODs) of the developed method for the three compounds were lower than the minimum required performance limit (MRPL) of the World Anti-Doping Agency (WADA). The human urine samples obtained after the oral administration of modafinil and from the Beijing 2008 Olympic Games were analyzed by using the described method, which has also been successfully applied to routine analyses and the WADA Proficiency Test.
