Advances in coupled chromatographic -mass spectrometric analyses of anabolic steroids in human urine

David James Borts, Purdue University

Abstract

Urinary anabolic steroid detection constitutes a substantial portion of the workload in athletic drug testing. The established method for anabolic steroid testing is gas chromatography coupled with quadrupole mass filter mass spectrometry (GC/QMF-MS) operating in the selected ion monitoring (SIM) mode. Detection limits with the QMF are limited by the large number of anabolic steroids and metabolites that must be screened and the sequential nature of SIM. Many anabolic steroids need to be detected at part per trillion levels, so it is desirable to improve limits of detection. In addition, GC/MS steroid analysis requires deconjugation and derivatization of the steroids and metabolites. These steps introduce uncertainties due to losses, interconversions, and decompositions. In this work, gas chromatography coupled with quadrupole ion trap mass spectrometry (GC/QIT-MS) is investigated using selected ion storage (SIS) and tandem mass spectrometry modes of ion trap operation to improve detection limits. Also, liquid phase separations coupled with mass spectrometry are explored for the direct determination of steroid conjugates. ^ Selected ion storage with the quadrupole ion trap results in modest improvements in limits of detection for anabolic steroids in urine. Improvements are limited mainly by chemical matrix interferences at the same unit m/ z value as ions of interest. Tandem mass spectrometry with the ion trap results in substantial improvements in limits of detection for anabolic steroids, typically factors of 50 to 100 for compounds studied. ^ Microbore liquid chromatography tandem mass spectrometry (LC/MS/MS) is used for the direct determination of testosterone and epitestosterone conjugates in urine. Ion suppression effects with electrospray ionization have a detrimental effect on quantitation, but are overcome through the use of a comprehensive set of deuterated internal standards. Analyses of human urine samples indicate that elevated epitestosterone sulfate levels are infrequently the cause of an elevated testosterone/epitestosterone (T/E) ratio. ^ Finally, a microliter flow rate atmospheric pressure chemical ionization (μAPCI) interface for coupling liquid phase separations with mass spectrometry is developed. Improvements in aerosol nebulization enable operation at flow rates a factor of 20 lower and sensitivity a factor of 3 higher than a commercial interface. ^

Degree

Ph.D.

Advisors

Major Professor: Larry D. Bowers, Purdue University.

Subject Area

Chemistry, Analytical

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