Quantification of testosterone and epitestosterone in human urine samples by stir bar sorptive extraction – thermal desorption – gas chromatography/mass spectrometry: Application to HIV‐positive urine samples

Abstract

A simple method is described for the measurement of testosterone (T) and epitestosterone (ET) in human urine samples. The deconjugated steroids were extracted directly from the samples by stir bar sorptive extraction (SBSE) and derivatized in situ on the stir bar by headspace acylation prior to thermal desorption and GC/MS. Extraction and derivatization parameters, namely salt addition, temperature, and time, were optimized to improve the recovery of T and ET by SBSE. The limits of quantification (S/N 10) were 0.9 ng/mL for T and 2.8 ng/mL for ET. Quantification of the steroids in urine samples was performed using standard addition to avoid the influence of matrix effects. The method was applied for the measurement of urinary T and ET in a group of healthy volunteers and HIV+ patients. Decreased levels of T were detected in the HIV+ group, whereas the excretion of ET was comparable for the two groups. Further clinical research is required to elucidate the biomarker significance of the T/ET ratio in HIV infection.