Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity
Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity
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DOI：
10.1021/ac9806411
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发表时间：
1999-07-01
影响因子：
7.4
通讯作者：
Siuzdak, G
中科院分区：
文献类型：
--
作者：
Chatman, K;Hollenbeck, T;Siuzdak, G
使用前体离子监测对天然存在的类固醇硫酸盐和衍生为其硫酸酯的非共轭类固醇进行纳米电喷雾电离（nanoESI）质谱。最初，基于用于获得最有效的液/液提取方案的组合方法开发了提取方法。新方法允许非共轭甾体及其硫酸化类似物在两步程序中分别分离，第一步使用乙醚/己烷（90：10，v/v）提取非共轭甾体，第二步使用氯仿/2-丁醇（50：50，v/v）提取甾体硫酸盐。用三重四极杆质谱仪进行前体离子扫描，用于定量检测提取的非共轭和硫酸化类固醇，回收率平均分别为70%和87%，此外，一些类固醇可以通过采用串联质谱进行结构解析。仅进样1 μ L样品，生物基质中类固醇硫酸盐的检测限为200阿莫尔/μ L（类似于80 fg/μ L）。内源性水平的非共轭和硫酸化类固醇检测和定量的生理样品，包括尿液和血液。使用内标物--所消耗的少量材料（通常小于进样体积的20%）表明，当与nanoLC方法结合时，nanoESI具有更大的高灵敏度潜力，特别是用于监测生殖和肾上腺类固醇，以及用于分析含量较低的神经类固醇。
Nanoelectrospray ionization (nanoESI) mass spectrometry was performed on naturally occurring steriod sulfates and unconjugated steroids derivatized to their sulfate esters using precursor ion monitoring. Initially, an ex-traction method was developed based on a combinatorial approach employed to obtain the most efficient liquid/liquid extraction protocol. The new method allowed unconjugated steroids and their sulfated analogues to be isolated separately in a two-step procedure using diethyl ether/hexane (90:10, v/v) in the first step to extract the unconjugated steroids and chloroform/2-butanol (50:50, v/v) in the second step to extract steroid sulfates. Precursor ion scanning performed with a triple-quadrupole mass spectrometer was used to examine quantitatively the extracted unconjugated and sulfated steroids, where the recovery efficiency averaged 70 and 87%, respectively, In addition, some steroids could be structurally elucidated by employing tandem mass spectrometry. The limit of detection for steroid sulfates from the biological matrix was 200 amol/mu L (similar to 80 fg/mu L) with only 1 mu L of sample being injected. Endogenous levels of the unconjugated and sulfated steroids were detected and quantified from physiological samples including urine and blood. Internal standards, pregnenolone-d(4) sulfate and dehydroepiandrosterone-d(2) (DHEA), were used for quantitation, Extraction and nanoESI analyses were also performed on cerebrospinal fluid where the neurosteroid DHEA sulfate was detected. The small amount of material consumed (typically less than 20% of the injection volume) suggests that nanoESI has even greater potential for high sensitivity when combined with nanoLC approaches, especially for monitoring reproductive and adrenal steroids, as well as for the analysis of the less abundant neurosteroids.