A GC/C/IRMS-based method for position-specific carbon isotopic analysis of saturated long chain fatty acids

Abstract

Intramolecular carbon isotope profiles (13C/12C) of mid- and long-chain saturated fatty acids represent a potentially rich source of pathway and flux information on fatty lipid biosynthesis. Accessing the PSIC-13C (position-specific carbon isotope composition) of saturated long chain fatty acids, however, proves a challenge as neither 13C NMR, nor online pyrolysis, can measure all carbons successfully. We have developed a wet chemistry procedure (α-bromination, α-hydrolysis and decarboxylation) to stepwise shorten the targeted saturated fatty acid, followed by GC/C/IRMS (gas chromatography/combustion/isotope ratio mass spectrometry) measurement of the 13C/12C ratios of fatty acids, with minimal purification. Isotope mass balance was then applied to calculate the 13C/12C ratios of individual carbons. The triplicated PSIA precision varies from position to position in the range 3.95–5.25 mUr (averaging 4.44 mUr, equivalent to ‰).Applying this method to a commercial C16:0 fatty acid of C3 botanical origin revealed an intramolecular odd-over-even 13C enrichment pattern, consistent with that obtained with 13C NMR. Although PSIA-13C was attempted only on a single fatty acid, the method can potentially be applied to obtain PISC-13C for all fatty acids in a mixture simultaneously, and to long chain saturated alkanes after proper functionalization. The method should prove useful in studies of lipid metabolism and biogeochemistry as both saturated long chain fatty acids and alkanes are the dominant organic archives of climate in petroleum deposits, sediments and soils.