Elimination of n-butylated hydroxytoluene methylation during fatty acid analysis by gas chromatography

An improved method for fatty acids analysis with optimum recovery of highly polyunsaturated fatty acids methyl esters in biological systems is presented. The method is based on transesterification of phospholipid and triacylglycerols to fatty acid methyl esters using a commercially available reagent, Methyl-Prep II. Without proper precautions, as much as 50% of n-butylated hydroxytoluene (BHT) added to prevent oxidation of polyunsaturated fatty acids, could be methylated during the transesterification step. Methylated BHT elutes close to 14:0 (myristic acid) and no longer functions as an antioxidant, but the modified conditions virtually eliminate the methylation of BHT. Sample extraction and methylation was completed in 30 min at room temperature. A chelator (diethylenetriamine-pentaacetic acid; DTPA) is also added to prevent peroxidation of metal catalyzed free radical chain reactions. The standard deviations of the major fatty acids from multiple human plasma samples prepared on different days were less than 5%. The recovery of arachidonic acid, 20:4, from plasma was improved using the new method, and the recovery for docosahexaenoic acid, 22:6, spiked to human plasma was found to be 99%.