A combined TLC–GC approach for the regiospecific analysis of triacylglycerols in Yarrowia lipolytica

This study developed a robust, cost-effective thin-layer chromatography (TLC) combined with gas chromatography (GC) protocol for the regiospecific analysis of triacylglycerols (TAGs), with application to microbial oils from engineered Yarrowia lipolytica . Key steps, such as sn -1,3-specific lipase hydrolysis, TLC separation, staining, and fatty acid methyl ester (FAME) derivatization, were systematically optimized. Porcine pancreatic lipase was identified as the most efficient and economical enzyme for hydrolysis. A petroleum ether/diethyl ether/acetic acid (32:8:0.8, v/v/v) TLC solvent system provided optimal resolution, with iodine vapor as the preferred non-destructive staining method. BF 3 -methanol methylation prevented artifact formation observed with methanol-H 2 SO 4 . Applied to oils from strains producing nervonic acid (NA) and γ-linolenic acid (GLA), the method revealed distinct regiospecific patterns: NA was almost exclusively esterified at the sn -1,3 positions, whereas approximately 64.3% of GLA resided at the sn -2 position. This TLC-GC approach offers an accessible alternative for rapid screening of fatty acid positional distribution in TAGs.