Measurement of serum testosterone by LC–MS/MS

The calibration standards (6PLUS1 Multilevel Serum Calibrator Set MassChrom Steroid Panel 2) and quality controls (QC) (MassCheck Steroid Panel 2 Level I, II and III) were purchased from ChromSystem (Grafelfing, Germany). They are human-based lyophilized material (0–40 nM for calibrators and three levels for QC). A 100 µg/mL solution of testosterone ¹³C₃ (Cambridge Isotope Laboratories, Tewksbury, MA, USA) was diluted with LC–MS grade methanol (OmniSolv LC–MS, EMD, Mississauga, ON, CA) to prepare the internal standard working solution (6.86 nM).

To perform protein precipitation, 100 µL of the standard, QC or sample were placed into a 1.5 mL polypropylene microcentrifuge tube. Then, 100 µL of 0.1 M zinc sulfate (Thermo Fisher) in water (LC–MS grade, Purelab Ultra, ELGA) were added. This mixture was then vortexed vigorously for 30 s. After vortexing, 250 µL of internal standard was added and the mixture was vortexed for 1 min followed by a 3-min incubation at room temperature. Samples were then centrifuged for 2 min at 15,000 g at room temperature. The supernatant was transferred to an autosampler vial and directly injected into the acquity ultrahigh pressure liquid chromatography and online solid phase extraction system (Waters, Milford, MA, USA) using partial loop mode. Method validation was performed following CLSI guidelines (C62 and C57) (21, 22). This method is evaluated monthly by an external validation program (UK NEQAS), which provides three samples containing low testosterone level to assess the quality and accuracy of the measurements.

Chromatographic separation of testosterone from other components using the guard column and analytical columns maintained at 55°C was performed as described (20). The eluate was injected from the LC directly into a XEVO TQ MS tandem mass spectrometer (Waters) as described (20). Transitions (for details on ions see (20)) were monitored in multiple reaction monitoring mode, with a dwell time of 0.155 s. LLOQ (<0.1 nM) was defined as the lowest amount of analyte that can be detected with a coefficient of variation (CV) of 20% and a signal-to-noise ratio (peak-to-peak method) of ≥10.