madman
Super Moderator
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
Abstract
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 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 (3βhydroxy-5β-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.
Introduction
Neuroactive steroids rapidly alter neuronal excitability and are synthesized from cholesterol de novo in the brain and in the common steroidogenic organs [1]. These neuroprotective compounds modulate many CNS functions such as memory and cognition in addition to behavioral and emotional effects including anxiety and depression [2–4]. Neuroactive steroids generally mediate their actions at neurotransmitter receptor sites such as γ-aminobutyric acid type A receptor (GABAA), N-methyl-D-aspartate receptor (NMDA), and sigma-1 receptor, instead of at classic steroid hormone receptor sites [5]. Allopregnanolone, the predominant progesterone metabolite, and most studied neuroactive steroid is a potent, positive allosteric GABAA modulator, inhibiting neuronal excitability resulting in anxiolytic, sedative, and anticonvulsant effects on the body [6–9].
*High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides a reliable and accurate multi-analyte technology for quantification of neuroactive steroids in blood for clinical use [21, 35–37]. Initially, most neuroactive steroid analyses were performed using radioimmunoassay (RIA) while gas chromatography-mass spectrometry (GC-MS) has become more common [38]. Both methodologies, while successful, have limitations for clinical use. RIA lacks the specificity of both LC-MS/MS and GC-MS [39]. While GC-MS is highly sensitive, it is labor-intensive and costly for routine clinical use [40]. LC-MS/MS is the preferred assay for clinical applications including many endocrine laboratories [39, 41].
In conclusion, here, we present a straightforward, simple, specific, and sufficiently sensitive multi-analyte LC-MS/MS assay for simultaneous quantification of the 3α-reduced progesterone metabolites, allopregnanolone and pregnanolone, as well as the 3β-reduced metabolite epipregnanolone, their precursor's pregnenolone, and progesterone, as well as cortisol and cortisone in human serum during pregnancy. The simplicity of this validated assay makes its use for clinical research attractive, and the assay has potential use for diagnostic applications in pregnancy.
Abstract
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 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 (3βhydroxy-5β-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.
Introduction
Neuroactive steroids rapidly alter neuronal excitability and are synthesized from cholesterol de novo in the brain and in the common steroidogenic organs [1]. These neuroprotective compounds modulate many CNS functions such as memory and cognition in addition to behavioral and emotional effects including anxiety and depression [2–4]. Neuroactive steroids generally mediate their actions at neurotransmitter receptor sites such as γ-aminobutyric acid type A receptor (GABAA), N-methyl-D-aspartate receptor (NMDA), and sigma-1 receptor, instead of at classic steroid hormone receptor sites [5]. Allopregnanolone, the predominant progesterone metabolite, and most studied neuroactive steroid is a potent, positive allosteric GABAA modulator, inhibiting neuronal excitability resulting in anxiolytic, sedative, and anticonvulsant effects on the body [6–9].
*High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides a reliable and accurate multi-analyte technology for quantification of neuroactive steroids in blood for clinical use [21, 35–37]. Initially, most neuroactive steroid analyses were performed using radioimmunoassay (RIA) while gas chromatography-mass spectrometry (GC-MS) has become more common [38]. Both methodologies, while successful, have limitations for clinical use. RIA lacks the specificity of both LC-MS/MS and GC-MS [39]. While GC-MS is highly sensitive, it is labor-intensive and costly for routine clinical use [40]. LC-MS/MS is the preferred assay for clinical applications including many endocrine laboratories [39, 41].
In conclusion, here, we present a straightforward, simple, specific, and sufficiently sensitive multi-analyte LC-MS/MS assay for simultaneous quantification of the 3α-reduced progesterone metabolites, allopregnanolone and pregnanolone, as well as the 3β-reduced metabolite epipregnanolone, their precursor's pregnenolone, and progesterone, as well as cortisol and cortisone in human serum during pregnancy. The simplicity of this validated assay makes its use for clinical research attractive, and the assay has potential use for diagnostic applications in pregnancy.
