Stability studies of testosterone and epitestosterone glucuronides in urine

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 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.

Factors affecting urinary excretion of testosterone metabolites conjugated with cysteine

“…The results observed for the T/E ratio (Figure c) in this study were similar to those found in a previous study, where no significant variations (less than 30%) in the T/E values were observed …”

Preclinical challenges in steroid analysis of human samples

“…Before urine samples are measured by immunoassay they have to be extracted for the separation of unwanted interfering substances and to concentrate for fitting the lower urine levels compared to serum to the optimal measuring range of the assay. Urine steroids were also relatively stabile in their storage, for LC-MS/MS the stability of testosterone and epitestosterone was shown for at least 22 months [81].…”

Evaluation of equine urine reactivity towards phase II metabolites of 17‐hydroxy steroids by liquid chromatography/tandem mass spectrometry

“…Some authors have tried to identify the microorganisms involved in altering the profile of urinary endogenous steroids 17, 18. Some studies on conjugate or non‐conjugate compounds have been carried out on human urine,19–23 but few studies of equine urine stability have been reported.…”