Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: The identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17β-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dion

For almost two decades we have known that enzymatic hydrolysis of “normal” urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17β-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts.Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of “normal” urine from male horses may not be derived from the 17β-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17β-sulphate conjugate. Thus, if the 19-nortestosterone-17β-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses.These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in “normal” urine from the entire male horse. Purified fractions have then been studied by liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) to identify the precursors.