Analysis of Stable Isotope Enrichment of Fatty Acids by Gas Chromatography–Isotope Ratio Mass Spectrometry

In this study, [d₅] or [1-¹³C] enrichment of n-6 or n-3 PUFA, respectively, was measured by GC-thermal conversion or combustion–isotope ratio mass spectrometry as described (46). Briefly, FAMEs were reconstituted in 25 µl of hexane and a 2-µl injection volume in splitless mode onto a Supelcowax 10 capillary column (30 m length × 0.25 mm diameter × 0.25 μm film thickness; Supelco) run with a column flow of 1.5 ml/min on a Thermo Trace 1310 gas chromatograph (ThermoFisher, Loughborough, UK) equipped with a high-temperature (1,000°C/1,400°C) combustion/thermal conversion furnace and a Thermo Delta V IRMS. The ¹³C/¹²C and ²H/¹H ratios for identified fatty acids were measured relative to laboratory reference gas standards calibrated to the international standards (Vienna Standard Mean Ocean Water or Vienna Pee Dee Belemnite, respectively). Stable isotope enrichment was calculated as described (47). The concentration of each labelled fatty acid was calculated from the amount measured by GC-FID normalised to 1 million cells.