Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS Rapid Communications in Mass Spectrometry Pub Date : 2024-07-24 DOI:10.1002/rcm.9875

Tatsuya Higashi, Asuka Tanaka, Shiho Tsubura, Shoichi Nishimoto-Kusunose

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Abstract

Rationale

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.

Conclusion

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.

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通过 LC/ESI-MS/MS 将 5α-雄甾烷-3α,17β-二醇转化为双[(4-二甲基氨基)苯基氨基甲酸酯]衍生物，以灵敏测定其在大鼠大脑中的含量。

理由：5α-雄甾烷-3α,17β-二醇（3α,5α-Adiol）是一种由睾酮衍生的神经类固醇，与由孕酮衍生的神经类固醇异丙孕酮（AP）一样，通过γ-氨基丁酸 A 型受体具有抗焦虑和镇痛作用。虽然人们对精神药物引起的大脑 AP 浓度变化进行了深入研究，但对大脑中 3α,5α-二醇浓度的变化仍然知之甚少。造成这种情况的原因之一是，目前用于定量检测脑内 3α,5α-Adiol、具有足够灵敏度和特异性的有效方法非常有限：为了提高电喷雾离子化-串联质谱法（ESI-MS/MS）对3α,5α-蝶醇的检测能力，采用了4-二甲氨基苯甲酰基叠氮化物进行衍生化。脑样品经固相萃取净化后，对回收的 3α,5α-蝶醇和氘代内标物进行衍生化，然后用液相色谱/ESI-MS/MS 进行选择反应监测：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）提供了强烈的双质子化分子作为前体离子，然后通过碰撞诱导解离得到了含有 3α,5α-Adiol-骨架的特定产物离子。通过衍生化，3α,5α-Adiol 的可检测性最终提高了 1000 倍。衍生化后的 3α,5α-二醇与其立体异构体和干扰脑成分的分离是使用 SunShell 联苯色谱柱和含异丙醇的流动相实现的。验证测试表明，该方法在足够的浓度范围内线性关系良好，精密度和准确度均可接受，基质效应可忽略不计。使用该方法进行的动物（大鼠）研究表明，服用氟西汀（FLX）和氯氮平（CLZ）不会影响大脑中 3α,5α-甲二醇的含量，而 AP 的含量则会显著增加：结论：本研究开发并验证了一种能够利用20毫克组织对大鼠大脑中的3α,5α-二醇进行定量的LC/ESI-MS/MS方法。大鼠大脑中的 3α,5α-二醇含量与服用 FLX 和 CLZ 后的 AP 含量完全不同。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.