背景:5α-雄甾烷-3α,17β-二醇(3α,5α-Adiol)是一种睾丸激素衍生的神经类固醇,与孕酮衍生的神经类固醇一样,通过γ-氨基丁酸A型受体具有抗焦虑和镇痛作用,别孕烯醇酮(AP)。虽然精神药物诱发的脑AP浓度变化已被深入研究,大脑中的3α,5α-Adiol浓度仍然知之甚少。造成这种情况的原因之一是量化大脑3α的有效方法的可用性有限,5α-Adiol具有足够的灵敏度和特异性,这是在这项研究中描述的。
方法:为了增强3α的可检测性,5α-Adiol电喷雾串联质谱(ESI-MS/MS),采用4-二甲基氨基苯甲酰基叠氮化物的衍生化。通过固相萃取纯化脑样品,回收的3α,5α-Adiol和氘代内标被衍生化,然后通过液相色谱(LC)/ESI-MS/MS进行测量,并选择反应监测。
结果:衍生化的3α,5α-Adiol,即,双[(4-二甲基氨基)苯基氨基甲酸酯]衍生物,提供强烈的双质子化分子作为前体离子,然后是含有3α的特定产物离子,5α-Adiol骨架通过碰撞诱导解离。3α的可检测性,5α-Adiol最终通过衍生化增加了1000倍。衍生化3α的分离,使用具有含异丙醇流动相的SunShell联苯色谱柱,从其立体异构体和干扰脑成分中获得5α-Adiol。在足够的浓度范围内具有良好的线性,可接受的精度和准确度,验证试验证明了基质效应可以忽略不计。使用这种方法的动物(大鼠)研究表明,大脑3α,5α-Adiol水平不受氟西汀(FLX)和氯氮平(CLZ)的影响,与AP水平的显着增加相反。
结论:能够定量3α的LC/ESI-MS/MS方法,使用20mg组织在大鼠脑中开发并验证了5α-Adiol。3α的大脑水平,由于FLX和CLZ的施用,5α-Adiol具有与AP完全不同的行为。
BACKGROUND: 5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study.
METHODS: To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring.
RESULTS: The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels.
CONCLUSIONS: An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.