Stability studies of testosterone and epitestosterone glucuronides in urine
Article first published online: 8 FEB 2006
Copyright © 2006 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 20, Issue 5, pages 858–864, 15 March 2006
How to Cite
Jiménez, C., Torre, R. d. l., Segura, J. and Ventura, R. (2006), Stability studies of testosterone and epitestosterone glucuronides in urine. Rapid Commun. Mass Spectrom., 20: 858–864. doi: 10.1002/rcm.2387
- Issue published online: 8 FEB 2006
- Article first published online: 8 FEB 2006
- Manuscript Revised: 9 JAN 2006
- Manuscript Accepted: 9 JAN 2006
- Manuscript Received: 7 NOV 2005
- European Commission grant ALADIN 2002: Analytical Laboratories for AntiDoping Control: International Network for External Quality Assessment. Grant Number: G7RT-CT-2000-05022
The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with β-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4°C and −20°C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37°C for 3 and 7 days. The effect of repeated freezing (at −20°C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and −20°C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37°C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes. Copyright © 2006 John Wiley & Sons, Ltd.