Endocrine Abstracts

Though LC-MS/MS is replacing immunoassays for major glucocorticoids and androgens routine assessment, the measurement of other sex steroids and precursors is still challenging and available only in experienced research laboratories. Our aim was to develop a simple-prep and rapid 2D-LC-MS/MS method for the simultaneous measurement of estrone (E1), estradiol (E2), dihydrotestosterone (DHT) and 17OHpregnenolone.

Serum volumes of 300ul were spiked with internal standards and processed by single-step liquid-liquid-extraction. Extracts were injected onto Prominence HPLC - LCMS-8050 triple-quadrupole platform (Shimadzu). Analytes were purified by perfusion column, separated on a Gemini C6-phenyl (100×2 mm, 5 um) column (Phenomenex), ionized by electrospray and detected by specific multiple reaction monitoring transition in 11min run. The method showed high functional sensitivity (E2 and DHT: 9.8 pg/ml, E1: 4.9 pg/ml, 17OHpregnenolone: 39.1 pg/ml), proper accuracy (86.4–102.1%) and intra- (0.9–4.9%) and inter-assay (2.1–11.6%) imprecision. Sex and age specific reference intervals were estimated. The LC-MS/MS method was compared with routine E1 and E2 immunoassays. The Passing&Bablok analysis revealed high correlation between Roche-Modular III ECLIA and LC-MS/MS for E2 measurement both at all values (n=82; r:0.991) and at E2 levels <100 pg/ml (n=28; r:0.960), with respectively slight or absent proportional (slope (90CI): 1.06 (1.02–1.09) and 096 (0.85–1.11)) and systematic (intercept (90CI): −5.50 (−10.4–−1.68) and −0.78 (−6.94–4.96)) bias. DSL8700 direct RIA for E1 measurement showed poor correlation with LC-MS/MS (n=42; r:0.872), with relevant proportional (slope (90CI): 076 (063–0.94)) and systematic (intercept (90CI): 18.4 (9.4–22.9)) bias.

The LC-MS/MS assay showed optimal performance to sensitively and accurately determine routinely-assayed estrogens, confirming the reliability of new-generation ECLIA for E2, and highlighting direct RIA unreliability in determining E1. The assay further allowed the determination of important though not-routinely assayed DHT and 17OHpregnenolone. The bench- and run-time required by the LC-MS/MS method allow the integration of steroid profiling capability both in research and clinical settings.