Tetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC-ESI-mass spectrometry.

Interconversion of hormonally active cortisol (F) into the corresponding inactive 11-keto form, cortisone (E), is catalyzed by 11beta-hydroxysteroid dehydrogenases (11ß-HSDs). With a view to estimating in vivo activities of some 11ß-HSD isoforms, the measurement of urinary F and E and their tetrahydro metabolites (tetrahydrocortisol, THF, allotetrahydrocortisol, ATHF, tetrahydrocortisone, THE) has been suggested. The basic knowledge of THF, ATHF and THE levels in farm cattle is limited. Therefore the aim of this study was first to optimize a simple and quick method to determine F and E tetrahydro-metabolites in bovine urine by HPLC-mass spectrometry with electrospray ionization (HPLC-ESI-MS) and then to apply the method to real urine of bovines treated with prednisolone. The samples underwent filtration, deconjugation, solid-phase extraction (SPE) and the relevant analytes were measured by HPLC-ESI-MS. The method described in this paper is simple and efficient, featuring good linearity (up to 0.996) and reproducibility (6.8-12.5%, CV). Especially, good LODs were obtained, from 1.63 to 2.67 ppb, depending on the analyte. The chromatographic conditions were optimized in order to obtain a resolution which would allow to simultaneously measure two diastereoisomers, i.e. THF and ATHF. In our study, ATHF turns out to be below the detection limit, while for 18 samples tested the contents of examinated metabolites were as followed: THF (12.5±4.8 ppb), THE (10.9±5.5 ppb), F (11.6±3.3 ppb) and E (5.0±2.2 ppb). When the method was applied to the subject treated with prednisolone a major increase in the concentration of tetrahydro metabolites was observed before the slaughter, mainly due to stress conditions; prednisolone treatment, most presumably, influenced the 11ß-HSD activity, as indicated by the decrease in the F/E ratio. This work may provide a useful methodological contribution to the future definition of F, E, THF, ATHF and THE urinary baseline values in order to obtain indirect evaluations of HSDs activity in farm cattle and possible applications in screenings for suspected abuse of synthetic corticosteroids in bovines.