Optimization of a fatty acid methyl ester protocol for quantification of odd- and even-chain fatty acids in yeast

Fatty Acid Methyl Ester (FAME) analysis is widely applied in fields ranging from food and biofuel research to fatty acid profiling in microorganisms. Given the challenges associated with accurately quantifying both even- and odd-chain fatty acids, this study presents a tailored FAME protocol employing a fully deuterated internal standard (dIS) for their simultaneous analysis. Two oleaginous yeast species, Yarrowia lipolytica and Blastobotrys adeninivorans, were chosen for their distinct fatty acid profiles and served as models to evaluate protocol sensitivity and extraction efficiency. Here, we compared two simultaneous extraction and transesterification methods, a microwave-assisted and a shaker-assisted protocol, and found that the microwave-assisted approach not only successfully recovered the dIS but also extracted yeast fatty acids more efficiently than the shaker-assisted method. Incorporating a bead-beating pre-extraction step further improved lipid recovery and reduced variability. Our results suggest that the non-methylated dIS, when added as a free fatty acid, methylates more slowly than yeast-derived fatty acids that are mainly found as triacylglycerols. This can complicate normalization and supports the use of a pre-methylated dIS. While FAME yields varied depending on the extraction method and applied normalization strategies, the FAME profiles remained consistent across protocols, highlighting strong reproducibility in qualitative analyses. The optimized FAME protocol provides a precise and reliable analysis of fatty acids in diverse oleaginous yeast species. Our results underscore the need for species-specific optimization of FAME protocols and warn against relying on a deuterated single fatty acid standard without protocol adjustment.