ESPE Abstracts (2014) 82 P-D-1-2-8

Normal Value of Steroids in Amniotic Fluid by LC-MS/MS Method

Ingrid Plottona,b, Severine Rueta, Chantal Rigauda, Marianne Tilld, Damien Sanlavillec,d & Yves Morela,c

aHormonology and Endocrinology Molecular, Hospices Civils de Lyon, Bron, France; bU846, University Claude Bernard Lyon, Lyon, France; cUniversity Claude Bernard Lyon, Lyon, France; dCytogenetique, Hospices Civils de Lyon, Bron, France

Background: Determination of steroids in amniotic fluid (AF; Forest et al., J Clin Endocrinol Metab, 1980) has been essentially used in the three past decades for the prenatal diagnosis of 21-OH deficiency. With the recent advances of ultrasound technology (US) and the widespread use of amniocentesis, prenatal diagnosis of DSD appears more common especially if a mismatch between karyotype and external genitalia detected by US occurs. An accurate and specific determination of normal value of steroids in AF appears essential to evaluate DSD during this prenatal period.

Objective: Determination of the pattern of reference value for 17OHP, Δ4-A and testosterone by liquid chromatography–tandem mass spectrometry (LC–MS/MS method) according to sex.

Method: UPLC was performed on an Agilent Technologies 1290 Infinity using a Poroshell C18 4.6×50 2.7 μm coupled to an Agilent Technologies triple quadrupole 6460. In the sample preparation, internal standard deuterium are added in calibration curve and AF before supported liquid extraction (SLE). This method separated the steroids was validated according the Norm (linear response, CV<2% for the repeatability, <15% for the reproducibility and correlation with RIA R2>0.93 but the value was lower than in RIA). The lower limit of quantification is for Δ4A: 0.13, testosterone: 0.05, and 17OHP: 0.11 nmol/l.

Materials: Steroids were quantified in 133 AF with normal karyotype from women having amniocentesis, with informed consent. The amniocentesis was performed at 14.2 and 23 weeks of amenorrhea for increased maternal serum markers. No morphological abnormality (US) has been observed.

Results: The measurement of these steroids allows the determination of normal range: Testostérone is significantly higher in male foetus. The lower values determined by LC–MS/MS vs chromatography–RIA should be due to a more specific determination (absence of cross-reaction with the antibody).

Table 1.
17OHP (mean±S.D.) nmol/lΔ4-A (mean±S.D.) nmol/lTestosterone (mean±S.D.) nmol/l
46,XX (n=74)3.55 (±1.37)0.86 (±0.47)0.08 (±0.31)
46,XY (n=59)3.31 (±1.36)1.46 (±0.84)0.57 (±0.31)

Conclusion: We report a rapid, sensitive and accurate method for simultaneous measurement of three steroids in AF. Moreover, pathological values for DSD (defect of steroid biosynthesis, …) were in progress.

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