•HPLC–MS protocol for diols, alkenones and iGDGTs in one analysis described.•It enables rapid determination of the SST proxies TEX86, UK′37 and LDI.•As proof of concept, it was applied to a core from the Sea of Marmara.•Reconstructed temperatures differed between the proxies.•Ecology of source organism and change in species composition affect lipid distributions.Reconstructing ocean temperature values is a major target in paleoceanography and climate research. However, most temperature proxies are organism-based and thus suffer from an “ecological bias”. Multiproxy approaches can potentially overcome this bias but typically require more investment in time and resources, while being susceptible to errors induced by sample preparation steps necessary before analysis. Three lipid-based temperature proxies are widely used: UK′37 (based on long chain alkenones from phytoplanktonic haptophytes), TEX86 [based on glycerol dialkyl glycerol tetraethers (GDGTs) from pelagic archaea] and LDI (based on long chain diols from phytoplanktonic eustigmatophytes). So far, separate analytical methods, including gas chromatography (GC) and liquid chromatography (LC), have been used to determine these proxies. Here we present a sensitive method for determining all three in a single normal phase high performance LC–atmospheric pressure chemical ionization mass spectrometry (NP-HPLC–APCI-MS) analysis. Each of the long chain alkenones and long chain diols was separated and unambiguously identified from the accurate masses and characteristic fragmentation during multiple stage MS analysis (MS2). Comparison of conventional GC and HPLC–MS methods showed similar results for UK′37 and LDI, respectively, using diverse environmental samples and an Emiliania huxleyi culture. Including the three sea surface temperature (SST) proxies; the NP-HPLC–APCI-MS method in fact allows simultaneous determination of nine paleoenvironmental proxies. The extent to which the ecology of the source organisms (ecological bias) influences lipid composition and thereby the reconstructed temperature values was demonstrated by applying the new method to a sediment core from the Sea of Marmara, covering the last 21 kyr BP. Reconstructed SST values differed considerably between the proxies for the Last Glacial Maximum (LGM) and the period of Sapropel S1 formation at ca. 10 kyr BP, whereas the trends during the late Holocene were similar. Changes in the composition of alkenone-producing species at the transition from the LGM to the Bølling/Allerød (B/A) were inferred from unreasonably high UK′37-derived SST values (ca. 20 °C) during the LGM. We ascribe discrepancies between the reconstructed temperature records during S1 deposition to habitat change, e.g. a different depth due to changes in nutrient availability.

•A new UHPLC/APCI-MS protocol for ether core lipid analysis is described.•It enables separation of previously co-eluting, unidentified GDGT isomers.•Provides more nuanced exploration of environmental distributions of archaeal and bacterial ether lipids.•Annotated mass spectra of various isomers are presented.In recent decades, microbial membrane lipids have become a focus of geoscientific research because of their proxy potential. The aim of this study was to develop new methods for ultra high performance liquid chromatography (UHPLC) separation of isomers of archaeal and bacterial membrane ether lipids, in particular glycerol dialkyl glycerol tetraethers (GDGTs), because of their tendency to co-elute with related but incompletely characterized derivatives. Our newly developed protocol, involving analysis using two Acquity BEH HILIC amide columns in tandem, enables chromatographic separation of several of these co-eluting compounds, such as the isoprenoid GDGT with four cyclopentyl moieties and other chromatographic shoulders often observed in GDGT analysis. Additionally, resolved peaks were observed for isoprenoid GDGTs, branched GDGTs and isoprenoid glycerol dialkanol diethers (GDDs); these have typically the same molecular mass as the corresponding major compound. Multiple stage mass spectrometry (MS2) indicated that the shoulder peaks represent either regioisomers or other structural isomers with different ring or methyl positions. In some samples, these isomers can be even more abundant than their “regular” counterparts, suggesting that previously hidden clues regarding source organisms and/or community response to environmental forcing factors may be encoded in the distributions.