Rapid screening of anabolic steroids in urine by reactive desorption electrospray ionization

Fast screening for anabolic steroids in whole urine is achieved by combining reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. Seven steroids, including a glycosteroid, were examined. The ion/molecule reaction adduct and the oxime formed via its dehydration were observed using reactive DESI; the protonated and sodiated forms of the ionized steroid were also observed both in reactive DESI and in DESI performed without the added hydroxylamine reagent. Paper, glass, and polytetrafluoroethylene were tested as sample substrates, but the glycosteroid was ionized intact without hydrolysis only from polytetrafluoroethylene. Limits of detection for the pure compounds were less than 1 ng, dynamic ranges were typically 2 orders of magnitude, and analysis times were just a few seconds. Concentration levels of ketosteroids in raw urine relevant to screening for sports doping (approximately 20 ng/mL) can be reached using a simple solid-phase microextraction (SPME) preconcentration step. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect on steroid detection limits. Tandem mass spectrometry provided confirmation of analyte identification in this rapid screening process.