Protocols for sample preparation and compound-specific stable-isotope analyses (δ²H, δ¹³C) of fatty acids in...

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of C-H bonds in acyl chains, hydrogen (δ²H) and carbon (δ¹³C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on δ values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis.•Protocol from obtaining a sample to standardized fatty acid specific δ²H and δ¹³C values.•Separate GC analysis procedures for C and H are recommended for optimal performance.