Studies on neurosteroids XIX -: Development and validation of liquid chromatography-tandem mass spectrometric method for determination of 5α-reduced pregnane-type neurosteroids in rat brain and serum
Studies on neurosteroids XIX -: Development and validation of liquid chromatography-tandem mass spectrometric method for determination of 5α-reduced pregnane-type neurosteroids in rat brain and serum
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DOI：
10.1016/j.jchromb.2006.10.036
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发表时间：
2007-04-01
影响因子：
3
通讯作者：
Shimada, Kazutake
中科院分区：
文献类型：
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作者：
Higashi, Tatsuya;Nagahama, Akihiro;Shimada, Kazutake
建立了同时测定大鼠脑和血清中5种α-还原孕酮类神经类固醇的高效液相色谱-电喷雾电离-串联质谱(LC-ESI-MS-MS)方法。用甲醇-冰醋酸提取脑和血清中的类固醇，用Strata-X小柱纯化，用固定带电试剂2-肼-1-甲基吡啶(HMP)衍生化，然后进行LC阳性的ESI-MS-NIS。衍生化-ESI-MS-MS法对脑组织(0.25 ng/g组织)和血清(0.25 ng/ml)的定量限比未衍生化的常压化学电离-质谱法低60-150倍。以[17α，21，21，21-(2)H(4)]-AP为内标。这种方法可以分别使用20毫克或20亩的L样本重复性和准确地定量大脑或血清中的神经类固醇。该方法的批内和批间变异系数分别小于8.2%和6.0%，精确度为98.5%~103.0%。还介绍了所开发的方法在分析因束缚、应激和乙醇注射而引起的脑和血清神经类固醇水平变化的应用。(C)2006爱思唯尔B.V.保留所有权利。
A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the simultaneous determination of 5 alpha-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5 alpha-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol-acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS-NIS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization-ESI-MS-MS method are 60-150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17 alpha, 21, 21, 21-(2)H(4)]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 mu l sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5-103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented. (c) 2006 Elsevier B.V. All rights reserved.