Endocrine Abstracts

Introduction: Measurement of serum testosterone is necessary for the investigation of androgen disorders in males and females. Measurement of other androgens such as DHEAS and androstenedione (A4) may also be important in the investigation of PCOS and 17-hydroxyprogesterone (17-OHP) is useful to differentiate late onset CAH in patients with raised A4. Although it would be advantageous to measure all four steroids simultaneously this is not always easy because of their differing extraction requirements. We describe a simple method for the simultaneous measurement of testosterone, A4, 17-OHP, and DHEAS using LC–MS/MS after on line sample preparation.

Methods: Initial sample preparation involved the addition of serum (50 μl) to 150 μl ZnSO₄ and 100 μl acetonitrile (inc internal standards) to precipitate proteins. After centrifugation the supernatant was extracted further using on-line solid phase extraction by a Waters Acquity/on-line sample manager (OSM) coupled to a Waters Xevo TQ tandem mass spectrometer.

Results: Separation of all four steroids was achieved within a run time of 5.5 min/sample. The LLOQ for each assay was 0.1 nmol/l for testosterone, 0.25 nmol/l for A4, 0.1 nmol/l for 17-OHP, and 0.1 μmol/l for DHEAS.

The CV for each assay was<8% at concentrations of 0.4 nmol/l (testosterone), 1.7 nmol/l (A4), 0.6 nmol/l (17-OHP), and 0.2 μmol/l (DHEAS).

The comparison with validated assays (n=102) using either liquid/liquid extraction (LLE) or protein precipitation (PPT) gave the following equations: OSM (testosterone)=1.02×LLE (testosterone), r² =1.0; OSM (A4)=1.01×LLE (A4), r² =0.99; and OSM (DHEAS)=1.0×PPT (DHEAS)+0.54, r²=1.0

Discussion: We have developed a rapid assay for the LC–MS/MS measurement of testosterone, A4, 17-OHP, and DHEAS. The assay is suitable for routine clinical use and the small sample volume is good for paediatric work. The assay demonstrated excellent performance compared to existing validated LC–MS/MS methods in our laboratory.