Neurosteroids in rat brain:: Extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry
Neurosteroids in rat brain:: Extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry
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DOI：
10.1021/ac0346297
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发表时间：
2003-11-01
影响因子：
7.4
通讯作者：
Griffiths, WJ
中科院分区：
文献类型：
--
作者：
Liu, SY;Sjövall, J;Griffiths, WJ
本文建立了一种利用纳米液相色谱-电喷雾（nano-LC-ES）质谱分析大鼠脑内硫酸化神经甾体和非结合酮型神经甾体的方法。采用固相分配、阳离子和阴离子交换色谱法对大鼠脑组织中的神经甾体进行了提取、纯化和分离，将其分为两组：中性非共轭甾体和甾体硫酸盐。通过nano-LC-ES质谱法分析类固醇硫酸盐级分。与预期相反，未检测到双烯醇酮和脱氢表雄酮（DHEA）的硫酸盐。回收了包括硫酸双烯醇酮在内的内标物，该方法的检测限为0.3 ng/g湿脑。检测到硫酸胆固醇的水平为1.2 μ g/g湿脑。用盐酸羟胺将中性非共轭甾体部分衍生化，以将氧代甾体转化为它们的肟。采用阳离子交换色谱法分离肟类化合物，并通过nano-LC-ES串联质谱法进行分析。中性未结合的类固醇馏分的分析证实了在大鼠脑中存在的双烯醇酮，孕烯醇酮异构体，孕酮，睾酮，和DHEA，其特征在于它们的保留时间，质子化分子的质量，和特征碎片离子。通过加入[3，4-C-13（2）]-孕酮作为内标物来估计水平，发现范围为0.04-20纳克/克。
A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 mug/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-C-13(2)]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.