QUANTIFICATION OF DIACYLGLYCEROLS BY CAPILLARY GAS-CHROMATOGRAPHY NEGATIVE-ION CHEMICAL IONIZATION MASS-SPECTROMETRY
QUANTIFICATION OF DIACYLGLYCEROLS BY CAPILLARY GAS-CHROMATOGRAPHY NEGATIVE-ION CHEMICAL IONIZATION MASS-SPECTROMETRY
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DOI：
10.1006/abio.1993.1052
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发表时间：
1993-02-01
影响因子：
2.9
通讯作者：
HUI, R
中科院分区：
文献类型：
--
作者：
FALARDEAU, P;ROBILLARD, M;HUI, R
本文描述了一种毛细管气相色谱-负离子化学电离-质谱法定量二酰基甘油为其1-五氟苯甲酰-2-酰基-3-乙酰基甘油衍生物的方法。该方法的基础是用乙酸酐、胰脂肪酶和五氟苯甲酰氯依次处理二酰基甘油。培养的大鼠肠系膜动脉血管平滑肌细胞（VSMC）在载药剂或加压素（10−7M）存在下孵育20分钟。通过抽吸培养基并加入2 ml含有790pmol内标1-硬脂酰-2-(10,13)-壬二烯酰甘油的甲醇来停止孵育。提取后，用薄层色谱分离二酰基甘油，乙酰化，胰脂肪酶处理。得到的2-酰基-3-乙酰基甘油经薄层色谱纯化，转化为其1-五氟苯甲酰衍生物，并采用毛细管气相色谱-负离子化学电离-质谱法在选定的离子监测模式下进行监测（2-花生四烯基和2-十六烯基分别为m/z 614和604）。静息细胞含花生四烯醇基二酰基甘油脂质磷水平为128±26 pmol/100 nmol，刺激细胞为333±28 pmol/100 nmol （mean±SD, n = 3, P < 0.01）。通过这种方法也可以在VSMC制剂中检测到在第二位置含有油基或亚油基的二酰基甘油。这种新方法可用于定量在甘油分子第二位置具有不同酰基部分的各种二酰基甘油。
We describe a method for quantifying diacylglycerols as their 1-pentafluorobenzoyl-2-acyl-3-acetyl-glycerol derivatives by capillary gas chromatography-negative ion chemical ionization-mass spectrometry. The basis of the method resides in the sequential treatment of diacylglycerols with acetic anhydride, pancreatic lipase, and pentafluorobenzoyl chloride. Cultured rat mesenteric artery vascular smooth muscle cells (VSMC) were incubated for 20 min in the presence of vehicle or vasopressin (10−7M). The incubations were stopped by aspirating the medium and adding 2 ml of methanol containing 790 pmol of internal standard 1-stearoyl-2-(10,13)-nonadecadienoyl-glycerol. After extraction, diacylglycerols were isolated by thin-layer chromatography, acetylated, and treated with pancreatic lipase. The resulting 2-acyl-3-acetylglycerols were then purified by thin-layer chromatography, transformed into their 1-pentafluorobenzoyl-derivatives, and monitored by capillary gas chromatography-negative ion chemical ionization-mass spectrometry on the selected ion-monitoring mode (m/z 614 and 604 for 2-arachidonoyl and 2-nonadecadienoyl species, respectively). The levels of diacylglycerols bearing an arachidonoyl moiety were 128 ± 26 pmol/100 nmol lipid phosphorus in resting cells and 333 ± 28 in stimulated cells (mean ± SD, n = 3, P < 0.01). The presence of diacylglycerol species bearing an oleoyl or a linoleoyl group at the second position could also be detected in VSMC preparations by this approach. This new method can he applied to quantitate various diacylglycerol species bearing distinct acyl moieties at the second position of the glycerol molecule.