Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy

Neuroactive steroids are potent neuromodulators that play a critical role in both maternal and fetal health during pregnancy. These stress-responsive compounds are reportedly low in women with perinatal depression and may be associated with poor pregnancy outcomes in animal models. Chronic stress is a risk factor for adverse birth outcomes. Simultaneous quantification of neuroactive steroids, in combination with stress hormones cortisol/cortisone, provides an opportunity to investigate the synergistic relationship of these analytes within the convenience of one assay. A simple, reliable, and sensitive method for quantifying these endogenous compounds is necessary for further research with the potential to advance clinical diagnostic tools during pregnancy. Analytes were extracted from serum with a simple protein precipitation using methanol and then separated and quantified using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). After online extraction, analytes were separated using an Agilent Poroschell 120, 50 × 4.6 mm, 2.7 μm particle size, EC-C18 analytical column. The reliable quantification range was from 0.78 to 1000 ng/mL. QC sample inter- and intraday trueness was between 90 and 110% while inter- and intraday imprecision was less than 10%. Extracted samples were stable up to 7 days at 4 °C and extraction recovery was above 95%. Serum samples from 54 women in pregnancy were analyzed using this method. Here, we provide a validated, fast, and specific assay with sufficient sensitivity that allows for simultaneous quantification of blood serum concentrations of allopregnanolone (3α-hydroxy-5α-pregnan-20-one), pregnanolone (3α-hydroxy-5β-pregnan-20-one), epipregnanolone (3β-hydroxy-5β-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.