Determination of the δ²H values of high molecular weight lipids by high‐temperature gas chromatography coupled to isotope ratio mass spectrometry

Abstract

Rationale: The hydrogen isotopic composition of lipids (δ 2 Hlipid) is widely used in food science and as a proxy for past hydrological conditions. Determining the δ 2 H values of large, well-preserved triacylglycerides and other uniquely microbial lipids, such as glycerol dialkyl glycerol tetraether (GDGT) lipids, is thus of widespread interest but has so far not been This is a non-peer reviewed manuscript submitted to Rapid Communications in Mass Spectrometry. 2 possible due to their size which prohibits analysis by traditional gas chromatography pyrolysis isotope ratio mass spectrometry (GC-P-IRMS). Methods: We determined the δ 2 H values of large, polar molecules and applied high temperature gas chromatography (GC) methods on a modified GC-P-IRMS system. The methods were validated using authentic standards of large, functionalised molecules (triacylglycerides, TAG), purified reference standards of GDGTs, and compared to δ 2 H values determined by elemental analyser pyrolysis isotope ratio mass spectrometry (EA-P-IRMS); and subsequently applied to the analysis of GDGTs in a sample from a methane seep and a Welsh peat. Results: δ 2 H values of TAGs agreed within error between different between GC-P-IRMS and EA-P-IRMS, with GC-P-IRMS showing 3-5 ‰ precision for 10 ng H injected. Archaeal lipid GDGTs with up to three cyclisations could be analysed: δ 2 H values were not significantly different between methods with standard deviations of 5 to 6 ‰. When environmental samples were analysed, δ 2 H values of isoGDGTs were 50 ‰ more negative than those of terrestrial brGDGTs. Conclusions: Our results indicate that the high temperature GC-P-IRMS (HTGC-P-IRMS) method developed here is appropriate to determine the δ 2 H values of TAGs, GDGT lipids with up to two cyclisations, and potentially other high molecular weight compounds. The methodology will widen the current analytical window for biomarker and alimentary light stable isotope analyses. Moreover, our initial measurements suggest that bacterial and archaeal GDGT δ 2 H values can record environmental and ecological conditions.

Revisiting the precursors of the most abundant natural products on Earth: A look back at 30+ years of bacteriohopanepolyol (BHP) research and ahead to new frontiers

“…The next analytical frontiers should include modifying these HTGC-irMS methods (Hemingway et al, 2018;Lengger et al, 2019) to allow δ 2 H analysis of intact BHPs; extending what is thus far only available for hopanols (e.g., Li et al, 2009). The feasibility of analyzing the 2 H isotopic composition of high polarity compounds via HTGC-irMS has been demonstrated by Lengger et al (2021) for GDGTs. Scrutinizing the substantial differences in the hydrogen isotopic composition of precipitation (annual mean δ 2 H varies roughly between −20 to −200‰, depending on latitude) and seawater (δ 2 H=0‰) (Fig.…”

Compound‐specific stable hydrogen isotope (δ2H) analyses of fatty acids: A new method and perspectives for trophic and movement ecology

“…differences in precipitation amounts and ambient temperature, as well as the significant bulk tissue H isotopic differences commonly observed between terrestrial and aquatic primary producers 10,14,15 . The extension of H isotopes towards compound-specific assays of fatty acids has long been seen as encouraging, as it has been shown for amino acids 16,17 . Furthermore, a recent study comparing bulk plasma FA composition with feather bulk  2 H could infer relative inputs to swallows from terrestrial and aquatic insects 18 .…”

“…10,14,15 The extension of H isotopes towards compound-specific assays of fatty acids has long been seen as encouraging, as has been shown for amino acids. 16,17 Furthermore, a recent study comparing bulk plasma fatty acid composition with feather bulk δ 2 H could infer relative inputs to swallows from terrestrial and aquatic insects. 18 Still, the use of 2 H-CSIA for lipids in trophic ecology is largely unexplored and mainly focused on plant lipid and wax extract studies, 17,[19][20][21] or paleobiology and geosciences.…”

“…16,17 Furthermore, a recent study comparing bulk plasma fatty acid composition with feather bulk δ 2 H could infer relative inputs to swallows from terrestrial and aquatic insects. 18 Still, the use of 2 H-CSIA for lipids in trophic ecology is largely unexplored and mainly focused on plant lipid and wax extract studies, 17,[19][20][21] or paleobiology and geosciences. 22,23 Fatty acids are quantifiable tracers used to assess the trophic transfer of specific dietary energy sources, such as bacterial-, algal-, or terrestrial-derived fatty acids.…”

Metabolic imprints in the hydrogen isotopes ofArchaeoglobus fulgidustetraether lipids

“…The δ 2 H of lipids produced by photoautotrophic eukaryotes as well as chemoautotrophic and heterotrophic bacteria have been studied in detail for this reason (e.g., Sessions et al, 1999; Chikaraishi and Naraoka, 2003; Zhang and Sachs, 2007; Zhang et al, 2009; Sachse et al, 2012; Sachs, 2014; Dawson et al, 2015; Osburn et al, 2016; Leavitt et al, 2016; Wijker et al, 2019; McFarlin et al, 2019). The H isotope data on lipids produced by archaea, in contrast, are limited to only a handful of studies (e.g., Kaneko et al, 2011; Dirghangi and Pagani, 2013; Wu et al, 2020; Lengger et al, 2021; Leavitt et al, 2023). As a result, factors controlling the H isotope fractionation between lipid and water (ε L/W ) as well as the extent and limits of proxy applications are relatively well understood for eukaryotic and bacterial lipids, yet remain poorly understood for archaeal lipids.…”