摘要/Abstract
摘要： 目的·探究生理病理状态下脂肪酸的种类及含量变化，建立该类代谢物的定量分析技术。方法·基于（-） -氯甲酸薄荷酯与异丁醇作为衍生化试剂的脂肪酸异丁酯化方法，采用气相色谱 - 氢火焰离子化检测器/质谱联用技术（gas chromatography-flame ionization detector/mass spectrometry，GC-FID/MS）对27种代表性脂肪酸进行定性定量分析。通过测量检测限、定量限、日内和日间相对标准偏差检验方法的灵敏度及稳定性。同时，将该方法应用于4类典型生物样本（人类血清、尿液、粪便及大鼠肝脏），以验证其在不同基质中的普适性。结果·27种脂肪酸的异丁酯可在HP-5MS色谱柱上实现分离，在2～3个数量级范围内线性良好（R2>0.99）且具有优异的灵敏度（检测限为柱上0.03～2.96 pmol，定量限为0.09～9.86 pmol）。方法学验证结果显示，该方法在高、中、低3个浓度范围内的日内及日间相对标准偏差均小于10%。在4类典型生物样本中，该方法分别检测并定量出10、7、14、9种脂肪酸。结论·建立了基于异丁酯化并能够对水溶液中的短、中、长链脂肪酸（C1～C24）同步定量的分析方法。该方法的条件温和、灵敏度高且稳定性好，操作简便快捷，可直接用于血清、尿液、粪便及肝脏等多种生物样本的检测，其或将为高通量代谢组分析提供新的思路。
关键词: 脂肪酸, 异丁酯化, 代谢组学, 气相色谱 - 氢火焰离子化检测器/质谱联用技术
Abstract:
Objective · To establish a quantification method for understanding the varieties and concentrational changes of fatty acids under physiological and pathophysiological conditions. Methods · Based on isobutyl esterification using (-)-menthyl chloroformate and isobutanol, 27 typical fatty acids were qualitatively and quantitatively analyzedgas chromatography-flame ionization detector/mass spectrometry (GC-FID/MS). The sensitivity and stability of the method were detectedlimit of detection (LOD), limit of quantification (LOQ), and intra-day and inter-day relative standard deviation (RSD). The method was applied to four typical biological samples (human serum, urine, feces and rat liver) to verify the universality in different substrates. Results · Isobutyl esters of 27 fatty acids were effectively separated with an HP-5MS column. The results showed nice linearity within 2-3 orders of magnitude in terms of concentration with R2>0.99 for all 27 tested fatty acids. The LODs of the method were between 0.03 and 2.96 pmol on column whereas the LOQs were between 0.09 and 9.86 pmol. The results of method validation showed that the intra-day and inter-day RSDs were all under 10% in the range of high, medium and low concentrations. With this method, moreover, 10, 7, 14 and 9 fatty acids were detected in human serum, urine, feces and rat liver, respectively. Conclusion · A quantitative analytical method for fatty acids with short, media and long chains (C1-C24) is established based on isobutyl estrification in aqueous media. This method has the advantages of mild condition, high sensitivity, good stability, and simple and quick operation, and can be directly used in the detection of serum, urine, feces, liver and other biological samples, which may provide a new idea for high-throughput metabolome analysis.
Key words: fatty acid, isobutyl esterification, metabonomics, gas chromatography-flame ionization detector/mass spectrometry (GC-FID/MS)
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