A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae

Abstract

A method for the direct preparation of fatty-acid methyl esters (FAME) was simplified for fatty-acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid-catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high-performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty-acid contents displayed a linear relationship. Fatty-acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species.

Keywords: Fatty acid; Gas chromatography; Larval fish; Methyl ester; Methylation; Saponification.