Enantiomer resolution of glycerolipid components of fish oils by chromatography

建立了一种快速、灵敏、高效的液相色谱法，以（R）-（+）-1-（1-萘基）乙胺为手性固定相，以3，5-二硝基苯氨基甲酸乙酯为对映体拆分多不饱和甘油二酯（sn-1，2-和sn-2，3-）。用正己烷-1，2-二氯乙烷-乙醇等度洗脱（40：30：1体积比），在15 min内获得了亚油酸、亚麻酸、花生四烯酸、二十碳五烯酸和二十二碳六烯酸的单酸sn-1，2-和sn-2，3-二酰基甘油对映体的完全拆分。3）-三酰基甘油和磷脂酰胆碱的二酰基甘油部分。通过Grignard降解法从金枪鱼眶、太平洋鲳鱼肉、沙丁鱼肉、鲭鱼肉、鲑鱼卵和扇贝肝胰脏的三酰甘油中分离出两组sn-1，2（2，3）-二酰甘油，分别代表sn-1，2-和sn-2，3-二酰甘油对映体。这种清晰的分辨率允许对映体二酰基甘油在手性柱和极性毛细管GLC上对收集的级分进行制备性分离，这将给出详细的对映体组成。从秋刀鱼肉和海胆性腺的三酰基甘油生成的sn-1，2（2，3）-二酰基甘油获得了令人满意的对映体分离度，尽管观察到含有多不饱和酸的sn-1，2-对映体与含有长链单不饱和酸的sn-2，3-对映体之间存在一些峰重叠。秋刀鱼、太平洋银鲳和海胆的磷脂酰胆碱经蛇毒磷脂酶C水解得到的甘油二酯仅为sn-1，2-对映体。该方法还用于猪胰、扇贝肝胰和一些微生物脂肪酶立体专一性的测定。

A rapid ans sensitive high-performance liquid chloromatographic method for the enantiomer resolution of polyunsaturated sn-1,2- and sn-2,3- diacylglycerols as their 3,5-dinitrophenylurethanes was developed with a chiral column (25 cm long) containing (R)-(+)-1-(1-naphthyl)ethylamine. By an isocratic elution with n-hexane-1,2-dichloroethane-ethanol (40 : 30 : 1 by volume), complete enantiomer resolution was obtained within 15 min for monoacid sn-1,2- and sn-2,3-diacylglycerols of linoleic, linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acids.The methos was applied to the enantiomer resolution of the sn-1,2(2,3)-diacylglycerol moieties of triacylglycerols and phosphatidylcholines from some marine organisms. The sn-1,2(2,3)-diacylglycerols generated by Grignard degradation from the triacylglycerols of tuna fish orbital, pacific pomfret flesh, sardine flesh, mackerel flesh, salmon egg and scallop hepatopancreas were clearly resolved into two groups, which represented sn-1,2- and sn-2,3-diacylglycerol enantiomers. This clear resolution permits the preparative separation of the enatimeric diacylglycerols on the chiral column and the polar capillary GLC of the collected fractions, which would give the detailed enantiomer composition. Satisfactory enantiomer resolution was obtained for the sn-1,2(2,3)-diacylglycerols generated from the triacylglycerols of saury flesh and sea urchin gonard, although some peak overlapings between sn-1,2-enantiomers containing polyunsaturated acids and sn-2,3-enantiomers containing long-chain monounsaturated acids were observed. The diacylglycerols derived from phosphatidylcholines of saury, pacific pomfret and sea urchin by venom phospholipase C were only the sn-1,2-enantiomers. The method presented was also applied to the determination of the stereospecificity of lipases from porcine pancreas, scallop hepatopancreas and some microorganisms.