Quantification of neurosteroids during pregnancy using selective ion monitoring mass spectrometry

•A simplified GC–MS method was developed to simultaneously measure multiple neurosteroids in plasma.•Versatility of method was demonstrated by inclusion of additional steroids and 25-hydroxy-vitamin D3.•The GC–MS method was able to detect marked changes in neurosteroid profiles over the course of pregnancy and postpartum.

Analytical techniques used to quantify neurosteroids in biological samples are often compromised by non-specificity and limited dynamic range which can result in erroneous results. A relatively rapid and inexpensive gas chromatography–mass spectrometry (GC–MS) was developed to simultaneously measure nine neurosteroids, including allopregnanolone, estradiol, and progesterone, as well as 25-hydroxy-vitamin D3 in plasma samples collected from adult women subjects during and after pregnancy. Sample preparation involved solid-phase extraction and derivatization, followed by automated injection on a GC equipped with a mass selective detector (MSD) operated in single ion monitoring (SIM) mode to yield a run time of less than 11 min. Method detection limits for all neurosteroids ranged from 30 to 200 pg/mL (parts per trillion), with coefficients of variation that ranged from 3% to 5% based on intra-assay comparisons run in triplicate. Although concentrations of estradiol measured by chemiluminescent immunoassay (CIA) were consistent with values determined by GC–MS values, CIA yielded considerable higher values of progesterone, suggesting antibody cross reactions resulting from low specificity. Mean neurosteroid levels and representative time-course data demonstrate the ability of the method to quantify changes in multiple neurosteroids during pregnancy, including rapid declines in neurosteroid levels associated with delivery. This simplified GC–MS method holds particular promise for research and clinical laboratories that require simultaneous quantification of multiple neurosteroids, but lack the resources and expertise to support advanced liquid chromatography–tandem mass spectrometry facilities.

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