Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ¹³C and δ²H_n analyses

Lipids, with fatty acids (FA) as a crucial subset, have become a focal point for diverse medical, physiological, and ecological studies. However, a comprehensive assessment of the various pre-analytical FA extraction methods published in the scientific literature remains lacking. In this study, we examined the efficacy of seven well-established sample preparation methods, specifically focusing on their effectiveness in total lipid and fatty acid extraction and their impact on compound-specific stable hydrogen (δ²H) and carbon (δ¹³C) isotope values. We also considered the repercussions of FA removal efficacy on residual bulk tissue δ²H_n analysis, because lipids typically have low δ²H values. Our findings showed that in most cases chloroform-based extraction methods outperformed those without chloroform. While discrepancies were not as evident for smaller organisms, such as plankton, marked variations were discernible in the extraction efficiencies for muscle and liver samples, which was also manifested in the residual bulk tissue δ²H_n results. Notably, most extraction methods had little effect on specific δ¹³C or δ²H isotope values of FA; instead, an emphasis should be on using an extraction method that achieves optimal baseline peak separation of the chromatograms for C and H isotope measurements.