Nestlé Research Center, Lausanne, Switzerland.
J Sep Sci. 2017 Aug;40(16):3289-3300. doi: 10.1002/jssc.201700030. Epub 2017 Jul 31.
As a result of the heterogeneous nature of lipid classes in complex biological matrices such as plasma and erythrocytes, it is imperative to have a robust and validated methodology for fatty acid quantification. The effective method presented here combines available methodology of fast gas chromatography and an improvement of the sample preparation methodology before injection into the gas chromatograph. This methodology ensures complete transesterification and quantification of total and individual fatty acids (and not only in relative amounts) by addition of internal standards. We considered sample preparation key, and we established the use of lysis buffer and ethanol for erythrocytes and plasma sample preparation, respectively. Fatty acid profile was determined by acid methylation and fast gas chromatography equipped with a flame ionization detector. The triacylglycerol 13:0, phosphatidylcholine 23:0, and methyl esters 21:0 were used as internal standards. Within the linearity of the calibration, the ratio of the peak area of each fatty acid over the peak area of the internal standard was constant (coefficient of variation ≤ 2.5). Satisfactory repeatability <15% and intermediate reproducibility < 15% were observed. Finally, this validated method was applied to a pre-clinical trial that investigated the impact of dietary fats on accretion of specific fatty acids in plasma and erythrocytes.
由于血浆和红细胞等复杂生物基质中脂质种类的异质性，因此必须有一种强大且经过验证的脂肪酸定量方法。这里提出的有效方法结合了快速气相色谱法的现有方法，并改进了注入气相色谱仪之前的样品制备方法。该方法通过添加内标物，确保了总脂肪酸和单个脂肪酸（而不仅仅是相对量）的完全酯交换和定量。我们认为样品制备是关键，分别建立了用于红细胞和血浆样品制备的裂解缓冲液和乙醇的使用方法。脂肪酸图谱通过酸甲酯化和配备火焰离子化检测器的快速气相色谱法确定。三酰甘油 13:0、磷脂酰胆碱 23:0 和甲酯 21:0 用作内标物。在校准的线性范围内，每个脂肪酸的峰面积与内标物的峰面积之比保持不变（变异系数≤2.5%）。观察到令人满意的重复性<15%和中间重现性<15%。最后，该经过验证的方法应用于一项临床前试验，该试验研究了饮食脂肪对血浆和红细胞中特定脂肪酸积累的影响。
