ESPE Abstracts (2015) 84 P-2-169

References Values Under Synacthen Test for Six Steroids in Serum by LC-MS/MS

Marie Souillota, Ingrid Plottona,b, Chantal Rigauda, Veronique Tardya, Severine Rueta & Yves Morela

aHospices Civils de Lyon Hormonologie Endocrinologie Moleculaire, Bron, France; bUniversité Claude Bernard Lyon1, U 846, Lyon, France

Background: The response to ACTH test (synacthen®) is a very useful for the screening of steroidogenesis enzymatic deficiency. With the development of steroid quantification by LC-MSMS more specific than most of immunoassays, the determination of reference value is required at basal and under stimulation time.

Objective and hypotheses:: The aim of this study is the determination in the same extraction and chromatography after Synacthen references values for 6 steroids: 21-deoxycortisol (21DF), 11-deoxycortisol (11OH), deoxycorticosterone (DOC), corticosterone, Delta4 androstenedione (Delta4), 17-hydroxyprogesterone (17OHP) in serum by LC-MS/MS method.

Method: Agilent Technology 1290® Infinity was used for HPLC coupled to a mass spectrometer Agilent technology® triple quadrupole 6460. Samples and calibration curve are extracted using SLE after addition of deuterium internal standard. This method was validated according to the Norm (linear response, CV less than 10% for the repetability, less than 15% for the reproducibility). The limit of quantification for 11OH is 0.135 nmol/l, 0.125 nmol/l for 21DF, DOC, Corticosterone (Cortico), Delta4, OHP. Reference values were performed for these six steroids after Synacthen® (T0, T30, T60) in a cohort of patients previously studied in radioimmunoassay for 17OHP and 21DF and genetic status (normal, heterozygous, non- classical form) for mutation of CYP21A2 confirmed by sequencing. In the normal group (normal response to Synacthen determined in radioimmunoassay), steroids were quantified at T0 and T60 min (peak of stimulation for 17OHP and 21DF).

Results: For this study, we analyzed 66 patients. We choose the peak of secretion of steroids at 60 min of this test. We determined normal values for the 6 different steroids (see Table 1). The results for 21 DF exclude a heterozygote form of the the gene CYP21A2 then lower than 0.6 nmol/l and a non-classical form if lower than 9 nmol/l.

Table 1 (for abstract P2.169)
N=66 Normal 21 DF pmol/l11 OH nmol/lDOC nmol/lCortico nmol/lDelta4 nmol/lOHP nmol/l
Mean ±1SD0.606±0.4364.57±5.460.54±0.3758.68±17.294.37±2.94.43±1.99
Range value<0.125–21.1–29.350.130–2.316.24–1050.4–131.41–8.85

Conclusion: An evaluation of normal values for these six steroids may be useful to diagnose affected parents and prevent risk of congenital adrenal hyperplasia for children. The utilisation of LC-MS/MS method showed a sensitive and specific method to detect steroidogenesis enzyme deficiency when multiple precursors were high.

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