Evaluation of equine urine reactivity towards phase II metabolites of 17-hydroxy steroids by liquid chromatography/tandem mass spectrometry
Article first published online: 2 DEC 2008
Copyright © 2008 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 23, Issue 1, pages 65–76, January 2009
How to Cite
Fidani, M., Gamberini, M.C., Pasello, E., Palazzoli, F., De Iuliis, P., Montana, M. and Arioli, F. (2009), Evaluation of equine urine reactivity towards phase II metabolites of 17-hydroxy steroids by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom., 23: 65–76. doi: 10.1002/rcm.3858
- Issue published online: 3 DEC 2008
- Article first published online: 2 DEC 2008
- Manuscript Revised: 31 OCT 2008
- Manuscript Accepted: 31 OCT 2008
- Manuscript Received: 18 JUL 2008
Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (−18°C, 4°C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17β- and 17α-glucuronide steroids were exposed to hydrolysis leading to non-conjugated steroids. Only 17β-hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17α-hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4°C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at −18°C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination. Copyright © 2008 John Wiley & Sons, Ltd.