Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography
Enantiomeric analysis of D- and L-pipecolic acid in plasma using a chiral capillary gas chromatography column and mass fragmentography
复制标题
使用手性毛细管气相色谱柱和质量碎片分析对血浆中的 D- 和 L-哌啶酸进行对映体分析
DOI：
--
复制
发表时间：
1999
影响因子：
4.2
通讯作者：
C. Jakobs
中科院分区：
文献类型：
--
作者：
E. Struys;C. Jakobs
哌可酸是过氧化物酶体疾病的重要标志物，仅次于胆汁酸、极长链脂肪酸、植烷酸和降植烷酸。哌啶酸的两种手性形式具有不同的来源：D-哌啶酸来源于肠道细菌和饮食，而L-哌啶酸是L-赖氨酸途径的内源性中间体。本文采用手性毛细管气相色谱柱和质谱法对D和L-哌啶酸进行了对映体分析，其中衍生化和样品制备是基于早期公布的技术（Kok et al 1987 ; Zee et al 1992）。向100 kl血浆中加入1 nmol D/L酸作为内标[2 H11]哌啶，然后加入500 kl磷酸盐缓冲液（1 mol/L，pH 11）和50 kl氯甲酸甲酯，形成N-甲基氨基甲酸酯衍生物。在用乙酸乙酯萃取后，干燥萃取物并形成五氟苄基（PFB）衍生物。从乙醛己烷浸提液中，将1 kl用于气相色谱-电子捕获检测器（GC-ECD）分析，将第二个1 kl用于气相色谱-质谱（GC-MS）分析。对映体分离在CP Chiralsil-Dex CB（25 m] 0.25 mm，μ m厚度0.25 km）分析柱（Chrompack，米德尔堡，荷兰）上进行。烘箱温度从100 ℃（1分钟），以1 ℃的斜坡上升到160 ℃，然后快速上升到240 ℃。以负化学电离（NCI）模式，以氨气为试剂气体进行质谱碎片分析。测量的质量为m/z 186（内源性哌啶酸）和m/z 195（[2 H9]哌啶酸）。单独的对映体纯标准品用于确定D和L-哌啶酸的洗脱顺序。通过测试不同的温度梯度和载气流量来优化分离。采用1.0 ℃/min的升温速率和3 ml/min的载气流速可获得最佳分析性能。在对照和Zellweger血浆样品中，仅观察到哌啶酸的L-异构体，这与其他人发表的结果一致（Armstrong等，1993）。该方法还应用于先前检测到总哌啶酸和植烷酸升高但胆汁酸和极长链脂肪酸正常的患者的血浆样本（Baumgartner et al 1998）。使用我们的程序，我们可以明确地得出结论，只有哌啶酸的L-异构体存在于该血浆样品中。N-甲基氨基甲酸酯-PFB衍生物的稳定性
Pipecolic acid is an important marker for peroxisomal disorders next to bile acids, very long-chain fatty acids, phytanic and pristanic acid. The two chiral forms of pipecolic acid have di†erent origins : D-pipecolic acid derives both from intestinal bacteria and diet, whereas L-pipecolic acid is an endogenous intermediate of the L-lysine pathway. We here present an enantiomeric analysis of Dand L-pipecolic acid using a chiral capillary gas chromatography column and mass fragmentography, wherein derivatization and sample preparation are based on earlier published technology (Kok et al 1987 ; Zee et al 1992). To 100 kl of plasma, 1 nmol D/L acid was added as internal stan[2H11]pipecolic dard followed by addition of 500 kl phosphate bu†er (1 mol/L, pH 11) and 50 kl methylchloroformate to form N-methylcarbamate derivatives. After extraction with ethyl acetate, the extract was dried and pentaÑuorobenzyl (PFB) derivatives were formed. From the Ðnal hexane extract, 1 kl was used for gas chromatography with electron capture detection (GC-ECD) and a second 1kl was used for gas chromatographyÈmass spectrometry (GC-MS) analysis. Enantiomeric separation was performed on a CP Chiralsil-Dex CB (25 m] 0.25 mm, Ðlm thickness 0.25km) analytical column (Chrompack, Middelburg, The Netherlands). The oven temperature was programmed from 100¡C (1 min), with a ramp of 1¡C, to 160¡C, followed by a fast ramp to 240¡C. Mass fragmentography was performed in the negative chemical ionization (NCI) mode with ammonia as reagent gas. The masses measured were m/z 186 (endogenous pipecolic acid) and m/z 195 ([2H9]pipecolic acid). Individual enantiomerically pure standards were used to determine the elution order of Dand L-pipecolic acid. The separation was optimized by testing di†erent temperature ramps and carrier gas Ñows. The best analytical performance was achieved using a temperature ramp of 1.0¡C/min with a carrier gas Ñow of 3 ml/min. In control and Zellweger plasma samples only the L-isomer of pipecolic acid was observed, which is in agreement with results published by others (Armstrong et al 1993). The method was also applied to a plasma sample of a patient with previously detected elevations of total pipecolic acid and phytanic acid but with normal bile acids and very long-chain fatty acids (Baumgartner et al 1998). Using our procedure we could unambiguously conclude that only the L-isomer of pipecolic acid was present in this plasma sample. The stability of the N-methylcarbamate-PFB deriv-