Short-term stability of testosterone and epitestosterone conjugates in urine samples: Quantification by liquid chromatography–linear ion trap mass spectrometry

Abstract

A simple method using liquid chromatography–linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1–3 ng/mL), recovery (89–101%), intraday precision (2.0–6.8%), interday precision (3.4–9.6%) and accuracy (101–103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 °C during a time-storage of a week lead to the conclusion that addition of sodium azide (10 mg/mL) is required for preservation of the analytes.