Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photo spray ionization tandem mass spectrometry
Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photo spray ionization tandem mass spectrometry
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DOI：
10.1016/j.cca.2006.03.034
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发表时间：
2006-10-01
影响因子：
5
通讯作者：
Soldin, Steven J.
中科院分区：
文献类型：
--
作者：
Guo, Tiedong;Taylor, Robert L.;Soldin, Steven J.
背景：血清类固醇检测在许多常见内分泌疾病的临床评估中起着重要作用。在各种检测方法中，串联质谱（MS/MS）因其高灵敏度、特异性以及同时进行多分析物定量的能力，在临床实验室中的应用日益增多。我们第一代采用C - 18柱的高效液相色谱 - 串联质谱（HPLC - tandem MS/MS）同位素稀释类固醇谱分析方法，使用760微升血清样本，可在18分钟内测量9种类固醇。我们在此描述第二代类固醇谱分析方法，该方法采用高效液相色谱 - 质谱/质谱（HPLC - MS/MS）和同位素稀释串联质谱，可在11分钟内同时对12种类固醇进行定量，且仅需200微升样本量。
方法：使用API - 5000三重四极杆质谱仪（加拿大康科德市Sciex公司），结合光电喷雾源和岛津高效液相色谱系统（美国马里兰州哥伦比亚市岛津科学仪器公司），对每种分析物采用氘标记内标（IS）进行同位素稀释。向200微升血清中加入300微升含内标的乙腈进行去蛋白处理。离心后，将450微升上清液用900微升水稀释，并取1000微升等分试样注入C - 8柱。3分钟洗涤后，启动阀门以启动梯度洗脱程序，洗脱类固醇。采用多反应监测（MRM）分析进行定量，11种分析物采用正离子模式，醛固酮采用负离子模式。通过与其他MS/MS和免疫分析方法的相关性以及回收率研究，对该方法的日内和日间精密度、可靠性和准确性进行评估。
结果：所有测试分析物的日内变异系数（CVs）均<11.5%，日间变异系数范围为3.5% - 12.2%。比较研究结果得出的相关系数r值在0.908 - 0.999之间，回收率范围为90% - 110%。
结论：该方法可在11分钟内对血清中的12种类固醇进行同时测量，且样本制备过程简单。它可在临床环境中常规使用，因其样本处理简单且通量高而具有吸引力。（c）2006爱思唯尔B.V.版权所有。
Background: Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandern MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 mu l serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 mu l.Methods: An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 mu l of acetonitrile containing internal standards. After centrifugation, 450 mu l of supernatant were diluted with 900 mu l of water and 1000 mu l aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study.Results: Within-day CVs were < 11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%.Conclusions: This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput. (c) 2006 Elsevier B.V. All rights reserved.