Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry

Abstract

The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3'-hydroxystanozolol (II) and 4 beta-hydroxystanozolol (III)] and metandienone (IV) I17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (V) and 18-nor-17,17-dimethyl-5 beta-androsta-1,13-dien-3 alpha-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation.