微量海洋微藻脂肪酸甾醇及游离氨基酸GC-MS分析研究

 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-CH₃OH甲酯化后再用三氟双(三甲基硅烷基乙酰胺)处理,正己烷定容后用岛津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,七氟丁酸酐异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。

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

OBJECTIVE To develop an sentisitive analytical method for determining the contents of fatty acids, sterols and amino acids in any microalgal species.METHODS For microgal sample of 20 mg, Bligh-Dyer method was applied to separate the chloroform phase and aqueous methanol phase after adding authentic para-amino 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 trimethysilyated 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,0.25 μm (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 min, 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 1 μL 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 carrier 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 min, 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 min. The injection volume was 1 μL with splitless mode. Ion source temperature was set at 200 ℃, and interface temperature of 250 ℃. The solvent cut off time was set at 3.5 min. 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.