Quantification of fatty acids in erythrocytes and plasma by fast gas chromatography

Abstract

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.

Keywords: biological samples; erythrocytes; fast gas chromatography; fatty acid quantification; plasma.