ORGANIC MASS SPECTROMETRY, VOL. 27, 1061-1070 (1992) Application of Gas Chromatography/Mass Spectrometry to Steroid Analysis in Equine Sport Problems with Enzyme Hydrolysist E. Houghton,* L. Grainger, M. C. Dumasia and P. Teale Horseracing Forensic Laboratory Limited, Snailwell Road, Newmarket, Suffolk, CB8 7DT, UK In steroid analysis in biological fluids, cleavage of conjugates is an essential step which can zyx CBA be achieved by either enzymatic or chemical hydrolysis. Where conjugation with both glucuronic acid and sulphate occurs, then the zy IH use of the enzyme preparation from zyxwvut JIHGFED Helix zyxwvut DCBA pomatia, containing both &glucuronidase and aryl sulphatase activities, would appear to be advantageous. However, it has been shown that the sulphatase enzymes of Helix pomatia do not hydrolyse 17&sulphates and that other enzymatic activities also present in the preparation can give rise to a formation with certain steroids. The artefacts produced from incubation of debydroisoandrosterone with the enzyme preparation fro Helix pomatia have been identified by gas chromatography/mass spectrometry (GCWS) as androst-4-ene-3,17-dione, zy BA androsta-4,6diene-3,17dione, androst-4-ene-3,6,17-trione and 6-hydroxyandrost-4-ene-3,17-dione. Incubation of androst-5-ene-3,17-diol produced a similar series of compounds with a 17-hydroxy function. Semi-quantitativ GC/MS analysis has been used to determine the extent of these transformations in the presence of increas amounts of the Helix pomatia preparation. Quantitative conversion in buffer can be obtained but the results from incubation in urine showed a marked modifying effect with minimal artefact formation. The enzyme prepa from Escherichia coli does not yield any artefacts and results are presented for the optimization of its use in th hydrolysis of the glucuronic acid conjugate of %-estrane-3~,17a-diol, the major metabolite of nandrolone in horse. INTRODUCTION Over the past decade, trace analysis by gas chromatography/mass spectrometry (GC/MS) has been extended to numerous scientific disciplines. The prime factor in the extension of the technique has been the development of bench-top systems with fully automated hardware for GC/MS with user-friendly data systems, occupying minimal bench space but yet maintaining the sensitivity of their floor-standing predecessors. The low cost of the instrumentation and simplicity of operation have been major factors in extending the application of the technique away from the specialist, the mass spec- trometrist, into the hands of the chromatographer. GC/MS is finding an increasing role in routine screen- ing applications formerly carried out by GC or other chromatographic techniques. The greater cost of the instrumentation is offset by the enhanced specificity of the data in both qualitative and quantitative modes, the rapidaccess to large mass spectral libraries and the opportunity of establishing in-house target analysis methods. The changing role of GC/MS is clearly evident in the application of the technique to drug detection in sport. In both athletics and equine sports it is mandatory to t To Professor Jim Shannon-a true enthusiast of mass spectrom- etry with whom it was a pleasure and privilege to be associated-EH. identify unequivocally any drug present in the bio- logical fluid under investigation and for many years the sole role of mass spectrometry was one of confirmatory analysis. With the advent of the fully automated bench top systems, in both human and equine sports, the role of GC/MS has now been extended to routine screening operations. The extension to this role was assisted the introduction of the fused-silica capillary column with high resolving power, capillary column GC/MS allowing for the sensitive and specific detection of a large number of prohibited substances in a single analysis. The application of mixed-function solid-phase extrac- tion cartridges to the isolation of both P-agonists a antagonists and their metabolites from equine urine and derivatization of the extracts has resulted in the develop- ment of a GC/MS screening procedure'.' for their detection which has been extended, in this laborato to cover the general class of basic drugs. Other lab atories involved in the detection of prohibited sub- stances in equine sportsare also increasingly using GC/MS screening procedures in both the electron impact (EI) and chemical ionization (CI) However, in sport, it is probably in the field of steroid analysis that GC/MS has found its widest applicatio Routine GC/MS screening procedures have been devel oped for the detection of anabolic steroid abuse in both human5 and equine The technique has also been applied to in vioo metabolism studies," l6 steroid profile studies in biologicalfluids" and 0030-493X/92/lOlO6l-l0 zyxwvuts HGFEDCB $10.00 zyxwvut EDCBA 0 1992 by John Wiley & Sons, Ltd. Received 27 June 1992 Revised manuscript received 16 July 1992 Accepted 24 July I992