Endocrine Abstracts

Steroid testing has a central role in clinical decision-making and in research studies on diseases such as hypercortisolisms, female hyperandrogenism, male hypogonadism or inborn disorders of steroid synthesis. Recently, liquid chromatography–tandem mass spectrometry (LC–MS/MS) proved its superiority to routine immunoassays (IA) in accurately and sensitively measuring low level steroids. However, the replacement of automated IA with LC–MS/MS platforms is limited by the need for extraction procedures requiring operator handwork, large sample volume and long runtime. Aiming at improving LC–MS/MS practicability, we developed a 2D-LC–MS/MS method for simultaneously determining serum cortisol, testosterone, androstenedione, 17OHprogesterone (OHP), and 17OHpregnenolone (OHp) based on minimal sample preparation. One hundred μl of serum were treated by protein precipitation, diluted with H₂O and injected into the Prominence-LCMS-8050-electrospray ionisation platform (Shimadzu). Sample underwent on-line purification on perfusion column (6 ml/min, 3 min) and separation on Shim-pack XR-ODS-50×3 mm, 2 μm column (Shimadzu) in 5 min H₂O/acetonitrile gradient, before 7 min clean-up and reconditioning program. Total runtime was 15 min. Quantitative and qualitative transitions were monitored for each analyte. Isotopic dilution quantitation was performed by using d4-cortisol, d5-testosterone, d5-androstenedione, d8-OHP, and 13C₃-estrone as internal standard for cortisol, testosterone, androstenedione, OHP, and OHp respectively. Lower limits of quantitation (pg on column) assessed in calibrators diluted in BSA (4%) were 122.1 pg/ml (1.5 pg), 9.77 pg/ml (0.12 pg), 19.5 pg/ml (0.24 pg), 39.1 pg/ml (0.49 pg), and 312.5 pg/ml (3.9 pg), and functional sensitivity assessed in charcoal-stripped serum was 122.1, 19.5, 19.5, 39.1, and 312.5 pg/ml for cortisol, testosterone, androstenedione, OHP, and OHp respectively, and was stable along 150 samples batch. Intra- and inter-assay CV ranged between 3.4–7.2% and 5.8–16.7% respectively. Androstenedione comparison with an established extractive LC–MS/MS assay revealed optimal correlation (r: 0.9933–0.9996) and slope coefficients (0.810–1.060). These preliminary data showed that our 2D-LC–MS/MS method for five steroids based on minimal sample preparation achieves performance and robustness levels that can definitively reconcile routine need for reliability and practicability.

Disclosure: This work was supported by the Emilia Romagna Region-University Program for Young Investigators ‘Alessandro Liberati’ 2010–2012 PRUA 1-2012-004.