Stability studies of testosterone and epitestosterone glucuronides in urine

University Pompeu Fabra, Barcino, Catalonia, Spain
Rapid Communications in Mass Spectrometry (Impact Factor: 2.25). 03/2006; 20(5):858-64. DOI: 10.1002/rcm.2387
Source: PubMed


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 beta-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 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees 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 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees 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.

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    • "Samples were grouped per athlete when the information was available from the sport federations or sport organizations (samples remain nevertheless anonymous). A limit of quantification of 1 ng/ml is used for the concentrations of T and E, in the same range as the limit reported recently by Jimenez et al. [15]. IRMS analysis of androsterone and etiocholanolone (two metabolites of testosterone) was performed on suspicious samples on a routine basis since 2002. "
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