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Stability studies of testosterone and epitestosterone glucuronides in urine

Authors

  • C. Jiménez,

    1. Unitat de Farmacologia, Institut Municipal d'Investigació Mèdica, Barcelona, Spain
    2. CEXS, Universitat Pompeu Fabra, Barcelona, Spain
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  • R. de la Torre,

    1. Unitat de Farmacologia, Institut Municipal d'Investigació Mèdica, Barcelona, Spain
    2. CEXS, Universitat Pompeu Fabra, Barcelona, Spain
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  • J. Segura,

    1. Unitat de Farmacologia, Institut Municipal d'Investigació Mèdica, Barcelona, Spain
    2. CEXS, Universitat Pompeu Fabra, Barcelona, Spain
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  • R. Ventura

    Corresponding author
    1. Unitat de Farmacologia, Institut Municipal d'Investigació Mèdica, Barcelona, Spain
    2. CEXS, Universitat Pompeu Fabra, Barcelona, Spain
    • Unitat de Recerca en Farmacologia, Institut Municipal d'Investigació Mèdica (IMIM), Doctor Aiguader 80, 08003 Barcelona, Spain.
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Abstract

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.

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