Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry
Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry
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DOI：
10.1194/jlr.d300025-jlr200
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发表时间：
2003-11-01
影响因子：
6.5
通讯作者：
Schmitz, G
中科院分区：
文献类型：
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作者：
Lieser, B;Liebisch, G;Schmitz, G
鞘磷脂（SPH）是鞘脂的主干，被认为是参与细胞生长、分化和凋亡调节的第二信使。在某种程度上，目前可用的SPH定量方法复杂、耗时、不敏感或无选择性。为此，建立了一种快速简便的测定SPH和生物合成中间体鞘氨氨酸（SPA）的方法。该方法是基于高效液相色谱分离耦合电喷雾电离串联质谱（MS/MS）。定量是通过使用恒定浓度的非天然存在的内标，17-碳链SPH (C17-SPH)，以及通过不同浓度的天然存在的鞘碱建立的校准曲线来实现的。SPH和SPA用C17-SPH共洗脱，可对分析物响应进行精确校正。SPH + 2同位素对SPA定量的干扰用实验确定的因子进行了修正。SPH的检出限为9 fmol， SPA的检出限为21 fmol。SPH和SPA的总变异系数分别为8%和13%。jlr开发的高效液相色谱-串联质谱分析方法，分析时间为3.5分钟，样品制备简单，自动化数据分析，允许高通量定量粗脂提取物中的鞘脂碱基，是研究细胞鞘脂代谢和信号传导的宝贵工具。——lieser， B.， G. Liebisch， W. Drobnik， G. SchmitzHPLC电喷雾串联质谱法定量测定粗脂提取物中鞘氨醇和鞘氨氨酸的含量。
Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH + 2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively.jlr The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.-Lieser, B., G. Liebisch, W. Drobnik, and G. Schmitz. Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry.