Steroidal isomers with uniform mass spectra of their per-TMS derivatives: synthesis of 17-hydroxyandrostan-3-ones, androst-1-, and -4-ene-3,17-diols.

In human sports doping control analysis most of the steroids are analyzed after enzymatic hydrolysis of the glucuronides as per-trimethylsilyl (TMS) derivatives applying gas chromatography-mass spectrometry (GC-MS). According to the recommendations of the World Anti-Doping Agency the identification of analytes should be based on retention time and on mass spectrometric characterization. This study shows that the bis-TMS derivatives of 16 specific C19 steroids, namely the stereoisomers of 5xi-androst-1-ene-3xi,17xi-diol (8 isomers), androst-4-ene-3xi,17xi-diol (4 isomers), and 17xi-hydroxy-5xi-androstan-3-one (4 isomers), reveal very similar mass spectra. As a rule, when taking the retention times, which are provided as Kovac indices for all these isomers, into account, a restriction to two or three possible isomers is possible. Reliable identification should additionally include a comparison of the retention times of the analytes with the reference compounds measured concomitantly. In some cases standard addition may be appropriate. Due to the limited availability, the above mentioned isomers were synthesized by reduction of the corresponding alpha,beta-unsaturated oxo steroids either with K-Selectride or by catalytic hydrogenation (Pd/C as catalyst). The products of the reactions were identified by means of nuclear magnetic resonance (NMR) characterization and by further reduction to the corresponding 5xi-androstane-3xi,17xi-diols and GC-MS comparison with commercially available reference standards.