小硅藻培养过程中游离脂肪酸的GC-NCI-MS分析

目的: 以小硅藻(Nitzschia closterium f.minutissima)为研究对象建立海洋微藻体内及其培养液中游离脂肪酸的高灵敏度分析方法。方法: 小硅藻培养2周，4000 r·min^-1离心收集后冷冻干燥，乙酸乙酯提取藻粉中的游离脂肪酸；培养液经0.45 μm膜过滤后，用三氯甲烷-正己烷(1∶4，v/v)萃取出培养液中的游离脂肪酸。游离脂肪酸样品经五氟苯甲基溴(PFBBr)衍生后进行气相色谱质谱联用分析。色谱分离起始温度150℃，保持3.5 min后以20℃·min^-1升至200℃，200℃中保持5 min后以5℃·min^-1升至280℃，280℃中保持18 min；质谱检测在负化学源SIM模式下进行。结果: 以游离脂肪酸C 14∶0、C 16∶0、C 18∶0、C 20∶4为研究对象，游离脂肪酸PFB衍生物在SPB-1色谱柱上得到良好分离；方法在0~100 μmol·L^-1浓度线性良好；检测限均小于0.05 μmol·L^-1，远优于其他常规的衍生检测方法；方法的平均回收率在藻粉中达到75.3%~87.8%，在藻液中达到66.0%~77.4%，RSD小于5%；小硅藻藻粉中检测出的游离脂肪酸含量在0.39~1.72 μmol·g^-1范围内，而培养液中游离脂肪酸含量在0~399.3 nmol·L^-1范围内。结论: 该方法灵敏高，重现性好，可用于海洋微藻体内及其培养液中游离脂肪酸的定性定量分析。

Objective: To develop a specific,sensitive analysis method for the determination of free fatty acids in microalgae Nitzschia closterium f.minutissima and its culture medium. Methods: Microalgae Nitzschia closterium f.minutissima was collected after culture for 2 weeks by centrifugation at 4000 r· min^-1 and freeze-dried.Free fatty acids in microalgae powder were extracted by ethyl acetate and free fatty acids in the culture medium which was filtrated through 0.45 μm membranes that were extracted by n-hexane-chloroform(4∶1,v/v).After derivatization with pentafluorobenzyl bromide (PFBBr),free fatty acids were analyzed by gas chromatography-negative chemical ion source-mass spectrometry (GC-NCI-MS).The 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 18 min.The mass spectrometer operated in negative chemical ion source on SIM mode. Results: The results showed that efficient separation of free fatty acids (C 14∶0,C 16∶0,C 18∶0 and C 20∶4 as representatives) PFBBr derivative was achieved on the SPB-1 column.The linearity was good in the range of 0 to 100 μmol·L^-1,with lower limit of detection below 0.05 μmol·L^-1,which was superior to other routine derivative methods.The average recovery rates were from 75.3% to 87.8% in microalgae powder and from 66.0% to 77.4% in culture medium,with RSD less than 5%.Using this method,the contents of free fatty acids in microalgae powder were determined to range from 0.39 to 1.72 μmol·g^-1,meanwhile the contents of free fatty acids in culture medium were from 0 to 399.3 nmol·L^-1.Conclusion: This method is sensitive and suitable for the qualitative and quantitative analysis of free fatty acids in marine microalgae.