Development and application of novel methods for the detection of anabolic steroids in hair and other matrices using liquid chromatography tandem mass spectrometry

Owing to an increase in the number of doping cases with performance enhancing drugs (PEDs), mainly anabolic androgenic steroids (AASs), and the limitations of urinalysis and self-reports, there is an ever increasing need to develop new methods for detecting doping. We report on the development and application of a series of novel analytical methods for the detection of frequently-used AASs in hair and other matrices using liquid-chromatography tandem mass-spectrometry (LC-MS/MS). Assays capable of detecting 0.5 pg/mg stanozolol and 3.0 pg/mg nandrolone (with 20 mg hair) were developed. The 180 subject samples were analysed by both ELISA and LC-MS/MS, however ELISA showed several false positives for both drugs, showing the inaccuracy of using this technique alone. The analytical findings were successfully employed to verify self-reported drug use data. An assay capable of detecting 0.1 pg/mg testosterone (T) and 0.25 pg/mg epitestosterone (E) (with 50 mg hair) was also developed. Seventy-five hair samples were collected from healthy volunteers. We report for the first time the T/E ratio in hair, 0.5-3.37 in males and 0.56-1.81 in females. Diclofenac was found to significantly reduce the urinary excretion of 3'-hydroxystanozolol in rats, but did not influence the concentration of both compounds in hair. This is the first in vivo study to report this effect. Environmental contamination of stanozolol was also found in Hungarian river and tap water.