Recommended methods of fatty acid methylester preparation for conjugated dienes and trienes in food and biological samples

In this study, we compared three acid-catalyzed methods and three base-catalyzed methods for the methylester preparation of conjugated dienoic fatty acids and conjugated trienoic fatty acids in food and biological samples. Among the six methods examined, the sodium methoxide/methanol (NaOCH ₃/MeOH) method and the tetramethylguanidine/ methanol (TMG/MeOH) method of methylester preparation from tung oil were most efficient in preventing the artificial isomerization of α-eleostearic acid (α-ESA; 9c,11t,13t-18:3) to β-eleostearic acid (β-ESA: 9t,11t,13t-18:3) and for avoiding the artificial generation of unknown byproducts. Hydrochloric acid/methanol (HCl/MeOH), sulfuric acid/methanol (H₂SO₄/MeOH) and AOCS (boron trifluoride/methanol (BF ₃/MeOH)) methods of methylester preparation from tung oil resulted in the breakdown of α-ESA due to their long reaction periods and high reaction temperatures. In addition, these three methods did not prevent the generation of β-ESA. For the methylester preparation of tung oil free fatty acids. the BF₃/MeOH method (30 min at room temperature) did not lead to artificial β-ESA formation or byproducts, while the trimethylsilyldiazomethane (TMSN₂CH₃) method did form artifacts. For the methylation of conjugated linoleic acid (CLA, free fatty acid), the BF₃/ MeOH and TMSN₂CH₃ methods completely suppressed artificial isomerization of c,t-CLA and t,c-CLA to t,t-CLA. The results indicated that the BF₃/MeOH method for free fatty acids is the best method for the methylester preparation of both conjugated dienoic and trienoic fatty acids with respect to preventing artificial isomerization and the formation of byproducts. The BF ₃/MeOH method was applicable to both food and biological samples.