Cracking the Code: advancing fatty acid analyses in Antarctic benthic species by Gas Chromatography Mass Spectrometry

Fatty acid (FA) composition is a useful information for investigating trophic ecology of marine organisms and how they interact within food webs. In Antarctica, the diversity and availability of food sources depends on the ice cycle and disturbances. Benthic organisms receive variable quantities of sinking sea ice algae or phytoplankton. Benthic primary producers (e.g. macroalgae, microphytobenthos) may also fuel the food web. The TANGO expedition, held in austral summer 2023, aimed at characterizing the FA profiles of benthic consumers along the West Antarctic Peninsula. Here, the focus was on a frequently encountered benthic mollusk, the limpet Nacella concinna. After transmethylation of lipids and formation of FA methyl ester (FAME), gas chromatography coupled to ionized flame detector (GC-FID) revealed a high diversity of FA. Biomarkers of bacteria (18:1n-7) and macroalgae (20:4n-6) were identified in different proportions depending on the stations. GC coupled to mass spectrometry (GC-MS) highlighted specific aliphatic chains, such as dimethyl acetal (DMA) and non-methylene-interrupted (NMI) FAME, known for their specific association to membrane lipids and their role in stress resistance. Nevertheless, some FAME identification, especially locating double bonds, was not feasible with certainty. Given this, 4,4-dimethyloxazodine (DMOX) derivatives were investigated to clarify molecular structures of these FA. Different protocols were compared in terms of feasibility and efficiency in converting FAME directly into DMOX derivatives. Analysis of various known matrices, such as commercial and lab-made standards with specific FA composition, has enabled protocol development and optimization. Then, analysis of N. concinna DMOX derivatives allowed to assess its FA diversity. Pushing the molecular precision of FA content ultimately results in the creation of libraries covering the variety of compounds present in an organism. Subsequently, a reference base can be established for the identification of future contents and use as trophic markers to delineate complex trophic ecology.