Profiling neuroactive steroids in pregnancy. A non-derivatised liquid chromatography tandem mass spectrometry method for the quantitation of allopregnanolone and four related isomers in maternal serum.

Abstract

Neuroactive steroids are metabolites of progesterone, synthesised during pregnancy by the placenta. Here, we describe development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantitation of allopregnanolone, pregnanolone, isopregnanolone, epipregnanolone and allopregnan-20α-ol-3-one in maternal serum. Following addition of deuterated internal standards, 200 μL of serum was subjected to solid phase extraction. Chromatography was performed using a pentafluorophenyl column, and LC-MS/MS on a Sciex 6500+. Sample injection volume was 20 μL, and injection-to-injection time 10.0 min. The assay was validated according to published guidelines; assay linearity and lower limit of quantification were suitable for analysis of each steroid in maternal serum, for all analytes mean recoveries were 100 % ± 15 %, intra- and inter-assay imprecision <15 %, and matrix effects negligible, and specificity experiments confirmed nil interference from a wide range of endogenous metabolites of progesterone. The method was applied to human serum samples obtained from a large cohort of third trimester pregnancies which were subsequently characterised by normal fetal and maternal outcomes, and relationships between maternal neuroactive steroid concentrations and fetal gestational age assessed. Positive correlations between maternal serum concentration and fetal gestational age were observed for isopregnanolone, allopregnanolone and allopregnan-20α-ol-3-one. The LC-MS/MS method offers significant advantages over previously published approaches for quantitation of neuroactive steroids in human maternal serum, notably obviating the need for derivatisation, whilst achieving exceptional specificity. Characterisation of normal maternal neuroactive steroid concentrations will aid future research as dysregulated placental progesterone metabolism is observed in pregnancies with poor outcomes.