An Improved Method for Determining Medium‐ and Long‐Chain FAMEs Using Gas Chromatography

Abstract

The existing protocols for analyzing fatty acid methyl esters (FAMEs) using a one-step acetyl chloride (AC) catalyzed transesterification and extraction procedure cannot accurately determine the medium- and long-chain fatty acids simultaneously in clinical (enteral, parenteral) formulations. For example: (1) addition of AC at room temperature generates an exothermic reaction that often results in loss of sample and possible injury to the analyst; (2) certain polyunsaturated fatty acids (PUFAs) are less stable at elevated temperatures during the transesterification and contribute to the over-estimation of the C16:0 and C18:1 fatty acids; and (3) the flame-ionization detector (FID) response varies depending on the carbon chain length of the fatty acids, that consequently impacts the underestimation of medium-chain fatty acid (C6-C10) recoveries. To overcome these deficiencies and accurately determine FAMEs, we have developed an improved one-step transesterification method that employs the addition of AC in tubes kept on a dry ice bath, the transesterification at room temperature, and the data analysis using relative response factors. Using this modified protocol, we determined the fatty acid composition of lipid emulsions (Omegaven and Lipidem) on a Shimadzu GC2010 gas chromatography (GC) system using a capillary GC column (Zebron ZB-WAX plus, 30 m, 0.25 mm ID, 0.25 microm). Our data suggest that the improved method can be easily used to accurately determine fatty acids (C6-C24) in functional foods and lipid emulsions.

Single‐step transesterification with simultaneous concentration and stable isotope analysis of fatty acid methyl esters by gas chromatography‐combustion‐isotope ratio mass spectrometry

“…Acetyl chloride (AcCl) was then added to the cooled methanol and shaken to ensure even dissolution. Previous authors have noted the exothermic reaction between AcCl and methanol and recommended keeping the reaction mixture cold over dry ice while slowly adding AcCl . However, we found that pre‐cooling the methanol was a more convenient approach that rendered the reaction much less violent.…”

Attenuation of niacin-induced prostaglandin D2 generation by omega-3 fatty acids in THP-1 macrophages and Langerhans dendritic cells

“…An internal standard (C23:0) was added to 500 μl of cell lysate and a portion of the remaining 100 μl was used to establish a protein concentration in order to normalize fatty acid content to the amount of protein present. Lipids were extracted with chloroform: methanol (2:1) using the Folch method50 and fatty acids were converted into methyl esters at room temperature for 24 hours as described previously 51. The fatty acids were separated on a gas chromatography system equipped with an auto sampler, flame ionizing detector (GC2010; Shimadzu Corporation, Kyoto, Japan), and a Zebron ZB-WAX plus column (100 m, 0.25 mm ID, 0.25 m; Phenomenex, Torrance CA).…”

Microplate assay for quantitation of neutral lipids in extracts from microalgae

“…A subset of the lipid extracts was transesterified and analyzed by GC-MS using a modified version of the procedure by Xu et al [30]. 250 µl of extract were transferred to an 8 ml glass tube with a Teflon-lined lid.…”