Endocrine Abstracts

The adrenal and gonadal androgens testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development and fertility as well as in several other processes. We developed a method to assess serum testosterone, androstenedione and DHEA levels in one run using isotope-dilution liquid-chromatography tandem mass spectrometry (ID-LC-MS/MS). Sample preparation consisted of addition of internal standards (¹³C₃-testosterone, ¹³C₃-androstenedione and ²H₆-DHEA) and a liquid–liquid extraction using hexane-ether. The samples were analyzed on an Acquity 2D UPLC system (Waters), equipped with a C4 column (Waters) and a Kinetex Fluorophenyl column (Phenomenex), and a Xevo TQ-S tandem mass spectrometer (Waters). The three analytes were baseline separated in a total run time of 9 min.

The intra-assay CVs were <4, <4.6 and <6.2% for testosterone, androstenedione and DHEA respectively. The inter-assay CVs were <7% for testosterone and androstenedione and <9.3% for DHEA. At the lower concentrations inter-assay CVs were 9, 7 and 9.3%, for testosterone (0.08 nM), androstenedione (0.48 nM) and DHEA (1.18 nM) respectively. Recoveries of spiked analytes were 101–107% and 97–106% for testosterone and androstenedione respectively. Recovery of DHEA is under investigation. Linearity was shown in dilution series (mean R² was >0.999 for all analytes). This method tested negative for interference from several steroids. The method was shown to be suitable for serum as well as EDTA and heparin plasma.

The present testosterone method compared well (y=1.000x+0.035 nmol/l; r=0.9982) to another ID-LC-MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al. 2013). In the near future, the present method will also be compared to another LC-MS/MS method for androstenedione and DHEA.

In conclusion, we developed a sensitive and accurate method to measure serum testosterone, androstenedione and DHEA serum levels in one run.