摘要：脂肪酸是人体内重要的供能活性物质，越来越多的研究表明，脂肪酸含量和分布与人类疾病具有潜在的关系，它们常常作为某些疾病的生物标志物，为此，脂肪酸的定性定量分析具有重要的临床意义。本实验采用正己烷-异丙醇（4:1 v/v）和经典的氯仿-甲醇（2:1 v/v）溶剂提取生物样本中的脂质并进行比较；采用硫酸-甲醇衍生化法、氢氧化钾-甲醇衍生化法、硅烷化衍生化法3种衍生化方法分别处理总脂肪酸、酯化脂肪酸和游离脂肪酸，最后经气相色谱-质谱联用仪分析。本实验还考察了动物组织中蛋白沉淀剂的选择及其在动物血浆中的用量以及不同样品中硅烷化试剂用量的实验条件。分析总脂肪酸时，正己烷-异丙醇可提取猪肝匀浆中14种脂肪酸，氯仿-甲醇为21种脂肪酸，在兔血浆中测得了9种脂肪酸；分析酯化脂肪酸时，猪肝样品中可得到15种脂肪酸和4种游离脂肪酸；分析游离脂肪酸时，猪肝样品中测到8种脂肪酸，兔血浆中测到5种脂肪酸。通过对实验方法和实验条件的选择以及初步的方法验证，建立了采用三氟乙酸沉淀蛋白的正己烷-异丙醇混合溶剂提取组织和血浆中的脂质，氮气吹干提取液后进行硫酸-甲醇甲酯化和硅烷化分别分析生物样本中的总脂肪酸和游离脂肪酸，最后生成的脂肪酸衍生物用气相色谱-质谱法的选择离子监测（SIM）模式定量，这为组织和血浆样本中的脂肪酸提供了可行简便的分析方法和新的参考。81171

关键词：组织和血浆；脂肪酸；衍生化；气相色谱-质谱联用

Profining of fatty acids in animal tissues and plasma samples

Abstract: Fatty acids are important substances for energy and active small molecules in the human body。 More and more studies have shown that the content and distribution of fatty acids are potentially related to human diseases。 They often pay a role as biomarkers of certain diseases。 To this end, analysis of qualitative and quantitative of fatty acids has important clinical significance。 In this study, the lipids from the biological samples were extracted with n-hexane-isopropanol (4:1 v/v) and the classical chloroform-methanol (2:1 v/v) solvent。 Three kinds of derivatization method for the treatment of total fatty acids, esterified fatty acids and free fatty acids are sulfuric acid- methanol method, silylation, potassium hydroxide - methanol method respectively, and these derivative finally detected by gas chromatography - mass spectrometry。 Besides, there were studied the selection of protein precipitants in animal tissues and the dosage of the selected protein precipitant in animal plasma。The amount of silanizing reagent in different samples were also investigated。 When the total fatty acids were analyzed, n-hexane-isopropanol solvent could extract 14 kinds of fatty acids and chloroform-methanol solvent with 21 fatty acids in the porky liver homogenate while 9 fatty acids were measured in rabbit plasma。 When the esterified fatty acids were analyzed, 15 kinds of fatty acids and 4 kinds of free fatty acids were obtained from the porky liver。 As to free fatty acids, porky liver samples were detected in 8 kinds of fatty acids, rabbit plasma measured 5 kinds of fatty acids。 Through the selection of experimental methods and experimental conditions as well as preliminary method validation, the method of extraction of lipid in tissue and plasma was established by n-hexane - isopropanol mixed solvent with trifluoroacetic acid precipiting protein。 After dried under nitrogen, the fatty acids in lipid derivated by methanol - sulfuric acid and the silane reagent to analyze the total fatty acids and free fatty acids in the biological samples respectively, and these derivatives were finally quantified by selected ion monitoring (SIM) mode of gas chromatography-mass spectrometry, which provided a simple and convenient analytical method and new reference for the detection of fatty acids in tissues and plasma samples。