Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection
Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection
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DOI：
10.1007/s00216-008-2308-1
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发表时间：
2008-10-01
影响因子：
4.3
通讯作者：
Seeger, Werner
中科院分区：
文献类型：
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作者：
Kiss, Ladislau;Bier, Jens;Seeger, Werner
尽管第一个证据的细胞色素P450介导的顺式-epoxyeicosatrienoic acids（EET）的对映体选择性生物合成和活性，但由于现有的手性方法耗时，劳动密集型，不够灵敏，因此对EET生成和生理学的立体特异性知之甚少。我们提出了一种高灵敏度，直接，同时手性分析所有八个EET对映体的方法，包括（i）固相萃取，（ii）反相高效液相色谱纯化，然后（iii）在一次色谱运行中使用毛细管串联柱手性相液相色谱法连续分离四种未衍生化的EET区域异构体的区域异构体和对映异构体，其中（iv）可靠的双在线光电二极管阵列和温和的电喷雾电离串联质谱鉴定和定量洗脱光学对映体。这种一步、简单、快速和高灵敏度的测量允许同时分析所有八种EET对映体，从而避免物质损失并实现高样品通量。通过使用具有典型的高定量灵敏度的毛细管柱而不是先前方法采用的具有低色谱信号强度的常规柱，实现了低皮克范围内的定量限。对组织匀浆的应用证明了这种方法对于游离内源性E3的常规和可靠的“对映体分析”的适用性，即，未酯化成细胞膜磷脂的雌二醇，通常以极低浓度存在。该技术可以很容易地用于制备性纯化的对映体在微克范围内使用大内径柱。
Despite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable "enantioprofiling" of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns.