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Routine method for the simultaneous quantification of 17α-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry

Magnisali, P., Dracopoulou, M., Mataragas, M., Dacou-Voutetakis, A., Moutsatsou, P.
Journal of chromatography 2008 v.1206 no.2 pp. 166-177
androstenedione, blood serum, cortisol, detection limit, equations, gas-chromatography-mass spectrometry, humans, immunoassays, iodides, methyltestosterone, neonates, prasterone, pregnenolone, testosterone
Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic-mass spectrometric method for the simultaneous quantification of 17α-hydroxyprogesterone (17αOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Δ4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1ng/mL. The limit of quantification was 0.1ng/mL for all steroids except cortisol which was at 0.25ng/mL. d₃-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10ng/mL (n =10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10ng/mL and ranged from -9.2% to 10.6% (n =10). Linear calibration equations were obtained for all five steroids (0.125-31.25ng/mL) and for cortisol (0.125-200ng/mL). Relative recoveries at concentrations 1.0 and 12.5ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.