Quantitative 31P NMR Method for Individual and Concomitant Determination of Phospholipid Classes in Polar Lipid Samples
Quantitative 31P NMR Method for Individual and Concomitant Determination of Phospholipid Classes in Polar Lipid Samples
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DOI：
10.5650/jos.ess18062
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发表时间：
2018-10-01
影响因子：
1.5
通讯作者：
Inagaki, Minoru
中科院分区：
文献类型：
--
作者：
Kato, Tsuyoshi;Nishimiya, Mami;Inagaki, Minoru
在此，为了实现磷脂类别的单独和伴随定量，检查了使用内标物的绝对定量P-31 NMR方法。将磷脂标准品和膳食食品分散在含有EDTA的阴离子表面活性剂（胆酸钠）溶液中制备试验溶液，作为基于报告方法的改进。在pH为6.90 +/-0.04和30.0 +/-0.1 ° C的近中性测试溶液下，每种磷脂类别均显示出可再现的化学位移。使用P-31 NMR测量的合成磷脂酰胆碱的量与使用内标通过H-1 NMR测量的合成磷脂酰胆碱的量一致。由于大豆和蛋黄卵磷脂的主要磷脂类是磷脂酰胆碱，因此优化了P-31 NMR的测量条件（脉冲间隔时间和扫描次数），从而可以同时定量卵磷脂中也存在的次要磷脂（包括溶血磷脂）。使用上述条件分别定量源自猪脑、酵母和大豆的商业极性脂质样品中的磷脂类别。使用磷酸丝氨酸作为内标物质允许磷脂类的绝对摩尔量被精确地测定，并可追溯到SI。然后通过假设每种磷脂类含有两个硬脂酸分子作为组成脂肪酸，将磷脂类的测定摩尔量转化为重量。用P-31 NMR法计算的各磷脂类的总含量与钼蓝比色法测定的结果一致。此外，通过P-31 NMR获得的极性脂质样品中主要磷脂类的定量值在广义上是一致的，然而，对于单个磷脂物质的分离，而不是定量2D薄层色谱法，其信息量更大。
Herein, to achieve individual and concomitant quantifications of phospholipid classes, an absolute quantification P-31 NMR method using an internal standard was examined. Phospholipid standards and dietary foods were dispersed to prepare test solutions in an anionic surfactant (sodium cholate) solution containing EDTA, as a modification based on a reported method. Each phospholipid class showed a reproducible chemical shift at a near-neutral test solution pH of 6.90 +/- 0.04 and temperature of 30.0 +/- 0.1 degrees C The quantity of synthetic phosphatidylcholine measured using P-31 NMR was consistent with that measured by H-1 NMR using an internal standard. As the principal phospholipid class of soybean and egg yolk lecithin is phosphatidylcholine, the measurement conditions of P-31 NMR (pulse interval time and number of scans) were optimized such that minor phospholipids, including lysophospholipids, also present in lecithin could be quantified simultaneously. Phospholipid classes in commercial polar lipid samples derived from porcine brain, yeast, and soybean were individually quantified using the above conditions. Using phosphoserine as the internal standard material allowed the absolute molar quantity of the phospholipid class to be precisely determined with traceability to the SI. The determined molar amounts of phospholipid classes were then translated to the weight amount by assuming that each phospholipid class contained two stearic acid molecules as the constituent fatty acid. The calculated total contents of each phospholipid class by P-31 NMR were in good agreement with those obtained by molybdenum blue colorimetry. Furthermore, the quantitative values of the principal phospholipid classes in the polar lipid samples obtained by P-31 NMR corresponded in a broad view, however, was more informative for the separation of individual phospholipid species rather than the quantitative 2D thin-layer chromatography.