Application of gas chromatography/mass spectrometry to steroid analysis in equine sports: Problems with enzyme hydrolysis†

In steroid analysis in biological fluids, cleavage of conjugates is an essential step which can be achieved by either enzymatic or chemical hydrolysis. Where conjugation with both glucuronic acid and sulphate occurs, then the use of the enzyme preparation from Helix pomatia, containing both β-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 17β-sulphates and that other enzymatic activities also present in the preparation can give rise to artefact formation with certain steroids. The artefacts produced from incubation of dehydroisoandrosterone with the enzyme preparation from Helix pomatia have been identified by gas chromatography/mass spectrometry (GC/MS) as androst-4-ene-3,17-dione, androsta-4,6-diene-3,17-dioue, 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-quantitative GC/MS analysis has been used to determine the extent of these transformations in the presence of increasing 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 preparation from Escherichia coli does not yield any artefacts and results are presented for the optimization of its use in the hydrolysis of the glucuronic acid conjugate of 5α-estrane-3β,17α-diol, the major metabolite of nandrolone in the horse.