Different approaches to the identification of a cortisol isomer compound in horse urine

A threshold concentration for cortisol in equine urine was fixed at 1.0 mu g ml(-1) by the racing authorities in 1994. In some circumstances, interlab oratory discrepancies were observed and structural cortisol modification wa s revealed. In order to elucidate the degradation process and to prevent it , an identification study of the produced compound was carried out. The mod ified substrate was characterised by the same molecular weight as cortisol and a shorter retention time under the conditions used for the cortisol qua ntification (M.A. Popot, PhD Thesis, King's College, London, 1996). To iden tify this isomer, HPLC-APCI-MS and MS-MS methods were applied to the cortis ol post-administration extract diluted in the mobile phase which was either a mixture of methanol-water or labelled methanol-water (CH3OD-D2O). Stereo chemical effects were studied under these conditions. Deuterium-hydrogen ex change was also monitored by HPLC-APCI-MS. Considering MS and MS-MS data, t he hypothesis of isomerisation at C11 giving the 11 alpha-cortisol was reje cted. Isotopic labelling has allowed determination of the number of labile hydrogen atoms of the modified cortisol. Cortisol and modified cortisol hav e the same number of mobile hydrogens. Therefore, the hypothesis of a reduc tion at C20 along with an oxidation at C11 has also been rejected. Deuteriu m-hydrogen exchanges could be a useful tool to elucidate the structure of c ompounds analysed by HPLC-APCI-MS in a complex matrix such as horse urine e xtract.