微量海洋微藻脂肪酸甾醇及游离氨基酸GC-MS分析研究

Total Microanalysis of Fatty Acids,Sterols and Free Amino Acids in Marine Microalgae by GC-MS

目的从微量海洋微藻样品中测定出脂肪酸、甾醇和游离氨基酸的含量。方法取20mg左右干重海洋微藻，加入对照品对氨基苯甲酸和C19：0脂肪酸，Bligh-Dyer法将氯仿相和水-甲醇相分开。氯仿提取总脂，皂化，调pH小于1，氯仿-正己烷（1：4）提取，提取物用质量分数14％BF3-CH2OH甲酯化后再用三氟双（三甲基硅烷基乙酰胺）处理，正己烷定客后用岛津QP2010GC-MS分析仪分析脂肪酸和甾醇含量；水-甲醇相过Dowex-50阳离子交换树脂，氨水洗脱，七氟丁酸酐并丁醇酰化酯化衍生后用NCI源在SIM模式下进行氨基酸的GC-MS分析。结果脂肪酸和甾醇的方法回收率为93％～107％，氨基酸的方法回收率为61．1％-102．7％之间。方法的重现性也较好。选取巴夫藻（Pavlova sp．）进行分析，可初步确定出26种脂肪酸和11种甾类化合物，含量在0．017，9．288mg·g^-1之间，而19种游离氨基酸的含量则在0．04—2．21mg·g^-1之间。结论使用一份样品，可以在岛津QP2010气相色谱质谱联用分析仪上，实现可靠的脂肪酸、甾醇和游离氨基酸的定性定量分析。

OBJECTIVE To develop an sentisitive analytical method for determining the contents of fatty acids, sterols and amino acids in any mieroalgal species.METHODS For microgal sample of 20 nag, Bligh-Dyer method was applied to separate the chloroform phase and aqueous methanol phase after adding authentic para-araino benzoic acid and 19：0 fatty acid as internal standards. The total lipid in the chloroform phase was saponified, acidified, and re-extracted into chloroform/n-hexane solvent, and methylated using 14%BF3-CH3OH, and subsequently trirnethysilyated with BSTFA, the derivatized fatty acids and sterols were subject to GC-MS analysis. Gas chromatographic analysis was carried out using a SPB-50 fused silica capillary column, 30 m× 0.25 mm,0.25μm （Supelo USA）. The temperature of injector was 250 ℃. High pure helium was used as the carrier gas with the flow rate of 0.81 mL·min^-1 and pre-column pressure of 73.0 kPa. After injection, oven temperature was kept at 150 ℃ for 3.5 rain, and then programmed at a rate of 20 ℃ ·min^-1 to the temperature of 200 ℃ and kept for 5 min, then programmed to a final temperature of 280 ℃ at a rate of 5 ℃·min^- 1, and kept for 30 min. The injection volume was 1μL with the split ratio of 50：1. The mass spectrometer operated in electron compact mode with an electron energy of 70 eV. Ion source temperature was set at 200 ℃, and interface temperature was at 250 ℃. The mass spectrometer scanned from m/z 50 to m/z 600. The solvent cut off time was set at 3.5 min.On the other hand, the aqueous phase was passing through Dowex-50 cationic exchange column, and eluted with aqueous ammonia, and derivatized with HFBI method, and subject to GC-MS analysi under NCI mode with methane as reagent gas. The temperature of injector was 250 ℃. High pure helium was used as the carder gas with the flow rate of 1.00 mL·min^-1 and pre-column pressure of 70.9 kPa. After injection, oven temperature was kept at 100 ℃ for 4.0 rain, and then programmed at a rate of 7 ℃ ·min^-1 to temperature of 200 ℃ and programmed to a final temperature of 280 ℃ at a rate of 15 ℃ ·min^-1 , and kept for 5 rain. The injection volume was 1μL with splifless mode. Ion source temperature was set at 200 ℃, and interface temperature of 250 ℃. The solvent cut off time was set at 3.5 rain. RESULTS The recovery for authentic fatty acids and sterols were between 93 % and 107%, as well as 61.1% to 102.7% for amino acids. The analytical method showed good reproducibility. For case study of Pavlova sp., 26 fatty acids and 11 sterols were identified, with content ranged from 0.017-9.288 mg·g^-1,19 free amino acids were quantified between 0.04- 2.21 mg·g^-1 . CONCLUSION Total microanalysis for fatty acids; sterols and amino acids can be achieved for one biological sample on Shimadzu QP2010 gas chromatography mass spectrometer with satisfactory results.