ECE2015 Guided Posters Developmental and paediatric endocrinology (10 abstracts)
Endocrinology Unit, Department of Medical and Surgical Sciences, Center for Applied Biomedical Research, S. Orsola‐Malpighi Hospital, University of Bologna, Bologna, Italy.
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 chromatographytandem mass spectrometry (LCMS/MS) proved its superiority to routine immunoassays (IA) in accurately and sensitively measuring low level steroids. However, the replacement of automated IA with LCMS/MS platforms is limited by the need for extraction procedures requiring operator handwork, large sample volume and long runtime. Aiming at improving LCMS/MS practicability, we developed a 2D-LCMS/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 3.47.2% and 5.816.7% respectively. Androstenedione comparison with an established extractive LCMS/MS assay revealed optimal correlation (r: 0.99330.9996) and slope coefficients (0.8101.060). These preliminary data showed that our 2D-LCMS/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 20102012 PRUA 1-2012-004.