Lionetto Luana, De Andrés Fernando, Capi Matilde, Curto Martina, Sabato Donata, Simmaco Maurizio, Bossù Paola, Sacchinelli Eleonora, Orfei Maria Donata, Piras Federica, Banaj Nerisa, Spalletta Gianfranco
Advanced Molecular Diagnostics Unit, Istituto Dermopatico dell'Immacolata (IDI-IRCCS), Rome 00167, Italy.
CICAB Clinical Research Centre, Extremadura University Hospital & Medical School, Badajoz 06071, Spain.
Bioanalysis. 2017 Mar;9(6):527-539. doi: 10.4155/bio-2016-0262. Epub 2017 Feb 16.
Several neuropsychopharmacological properties have been attributed to the 3α-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate γ-aminobutyric acid (GABA) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC-MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then applied to monitor these neurosteroids in plasma samples from ten volunteers.
For the first time, a straightforward and reliable method for the chromatographic separation of allopregnanolone, epiallopregnanolone and pregnanolone, as well as of dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate was carried out, with optimal accuracy, sensitivity and specificity.
3α-还原孕烷类固醇、别孕烯醇酮和孕烯醇酮以及硫酸脱氢表雄酮具有多种神经心理药理学特性，因为它们能够调节中枢神经系统中的γ-氨基丁酸（GABA）受体。为了更好地了解它们在中枢神经系统多种机制中的作用，提出了一种监测人血浆中这些化合物的新方法。方法与结果：分析物首先用2-肼基吡啶衍生化，然后使用固相萃取从血浆中提取。然后，通过液相色谱-串联质谱法分离和检测这些化合物。水和甲醇中含0.1%甲酸的流动相通过组成梯度和0.3 ml/min的流速实现了分析物的良好分离。在<9分钟内获得了宽浓度范围内的线性响应，定量下限范围为10（硫酸脱氢表雄酮3-硫酸盐）至40 pg/ml（脱氢表雄酮）。所提出的方法已经过验证，然后应用于监测来自十名志愿者的血浆样本中的这些神经甾体。
首次实现了一种直接可靠的方法，用于色谱分离别孕烯醇酮、表别孕烯醇酮和孕烯醇酮，以及脱氢表雄酮和硫酸脱氢表雄酮3-硫酸盐，具有最佳的准确性、灵敏度和特异性。
