Elucidation of double-bond positions of polyunsaturated alkenes through gas chromatography/mass spectrometry analysis of mono-dimethyl disulfide derivatives.

RATIONALE: Derivatization with dimethyl disulfide (DMDS) followed by gas chromatography/mass spectrometry (GC/MS) analysis is a well-established method for locating double-bond position on the alkyl chain of mono-unsaturated compounds such as alkenes. For alkenes containing more than one double bond, however, the conventional DMDS derivatization approach forms poly- or cyclized DMDS adducts whose mass spectra are difficult to interpret in terms of double-bond positions. In this study, we report an efficient experimental procedure to produce mono-DMDS adducts for polyunsaturated alkenes with two to six double bonds. GC/MS analyses of these mono-DMDS adducts yield highly characteristic mass fragments, allowing unambiguous assignments of double-bond positions on the alkyl chain. We also apply our new approach (i.e., preferential formation of mono-DMDS adducts during derivatization with DMDS) to determine the double-bond positions of unsaturated alkenes produced by laboratory cultured Isochrysis litoralis, a haptophyte algal species. METHODS: Alkenes from different sources were derivatized with DMDS at 25°C for 20 to 160 min. The mass spectra of mono-DMDS adducts were obtained by GC/EI-MS analysis of reaction products which contain chromatographically resolved mono-DMDS adducts. RESULTS: Mass spectra of corresponding mono-DMDS adducts contain prominent diagnostic ions that allow a conclusive elucidation of double-bond positions. In culture samples of Isochrysis litoralis, a series of novel mono- to tri-unsaturated C31 alkenes (9-C31:1 , 6,9-C31:2 , 6,22-C31:2 , 6,25-C31:2 , 9,22-C31:2 , 6,9,25-C31:3 ) were discovered for the first time. CONCLUSIONS: A highly efficient DMDS derivatization approach is developed to yield abundant mono-DMDS adducts of polyunsaturated alkyl alkenes for elucidating double-bond positions using GC/MS.

Group 2i Isochrysidales thrive in marine and lacustrine systems with ice cover.

Global warming is causing rapid changes to the cryosphere. Predicting the future trajectory of the cryosphere requires quantitative reconstruction of its past variations. A recently identified sea-ice-associated haptophyte, known as Group 2i Isochrysidales, has given rise to a new sea-ice proxy with its characteristic alkenone distributions. However, apart from the occurrence of Group 2i Isochrysidales in regions with sea ice, and the empirical relationship between C37:4 alkenone abundance and sea-ice concentration, little is known about the ecology of these haptophyte species. Here, we systematically mapped the spatial and temporal occurrence of known Group 2i Isochrysidales based on environmental DNA in both marine and lacustrine environments. Our results indicate Group 2i is widely distributed in icy marine and lacustrine environments in both Northern and Southern Hemisphere, but is absent in warm environments. Temporally, Group 2i is part of the sea-ice algae bloom during the cold seasons, in contrast to other Isochrysidales that bloom in open waters during warm seasons. Our results indicate that ice is a prerequisite for the occurrence of the psychrophilic Group 2i haptophytes in marine and lacustrine ecosystems and further affirms its value for past ice reconstructions.

Comprehensive analysis of alkenones by reversed-phase HPLC-MS with unprecedented selectivity, linearity and sensitivity.

Alkenones are among the most widely used paleotemperature biomarkers. Traditionally, alkenones are analyzed using gas chromatography-flame ionization detector (GC-FID), or GC-chemical ionization-mass spectrometry (GC-CI-MS). However, these methods encounter considerable challenges for samples that exhibit matrix interference or low concentrations, with GC-FID requiring tedious sample preparations and GC-CI-MS suffering from nonlinear response and a narrow linear dynamic range. Here we demonstrate that reversed-phase high pressure liquid chromatography-mass spectrometry (HPLC-MS) methods provide excellent resolution, selectivity, linearity and sensitivity for alkenones in complex matrices. We systematically compared the advantages and limitations of three mass detectors (quadrupole, Orbitrap, and quadrupole-time of flight) and two ionization modes (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) for alkenone analyses. We demonstrate that ESI performs better than APCI as response factors of various unsaturated alkenones are similar. Among the three mass analyzers tested, orbitrap MS provided the lowest limit of detection (0.4, 3.8 and 8.6 pg injected masses for Orbitrap, qTOF and single quadrupole MS, respectively) and the widest linear dynamic range (600, 20 and 30 folds for Orbitrap, qTOF and single quadrupole MS, respectively). Single quadrupole MS operated in ESI mode provides accurate quantification of proxy measurements over a wide range of injection masses, and with its modest instrument cost, represents an ideal method for routine applications. Analysis of global core-top sediment samples confirmed the efficacy of HPLC-MS methods for the detection and quantification of paleotemperature proxies based on alkenones and their superiority over GC-based methods. The analytical method demonstrated in this study should also allow highly sensitive analyses of diverse aliphatic ketones in complex matrices.

Silver (I)-dimercaptotriazine functionalized silica: A highly selective liquid chromatography stationary phase targeting unsaturated molecules.

Silver(I)-mercaptopropyl (Ag-MP) functionalized silica gel has demonstrated its effectiveness in separating various unsaturated organic compounds including unsaturated fatty acid ethyl esters (FAEEs), triglycerols (TAGs) and long-chain alkyl ketones (alkenones). While Ag-MP stationary phase displays many advantages over the conventional silver ion-impregnated silica gel (e.g., stability, high recovery, etc.), potential drawbacks of Ag-MP include relatively low retentions for unsaturated molecules, which could limit chromatographic resolutions under certain circumstances. In this study, we evaluate a new silver-thiolate stationary phase: silver(I)-dimercaptotriazine (Ag-DMT) functionalized silica gel targeting the separation of unsaturated compounds. We show Ag-DMT affords substantially higher retention factors, peak resolutions and capacities for TAGs and FAEEs than Ag-MP does. Ag-DMT also yields higher purity eicosapentaenoic acid (EPA) from fish oil FAEE mixtures than Ag-MP. In addition, Ag-DMT resolves double bond positional and cis/trans-isomers of C18:1 fatty acid methyl esters (FAMEs) as well as unsaturated methyl/ethyl alkenones with different number of double bonds. Based on van't Hoff plots, enthalpy changes during the adsorption of unsaturated FAEEs onto Ag-DMT are ~2 times higher than those on Ag-MP. Such difference may be attributed to the stronger electron-withdrawing effect of the thiol group on DMT, which results in more positively charged silver ions hence greater interactions with unsaturated molecules. The stronger interaction between double bonds and Ag-DMT is further corroborated by density-functional theory (DFT) calculations. Ag-DMT shows its high stability for repeated uses in the separation of TAGs over 319 runs, with peak resolutions decreasing by < 3%. Collectively, our data demonstrate the exceptionally high efficiency of Ag-DMT column for separating unsaturated molecules.

Group 2i Isochrysidales produce characteristic alkenones reflecting sea ice distribution.

Alkenones are biomarkers produced solely by algae in the order Isochrysidales that have been used to reconstruct sea surface temperature (SST) since the 1980s. However, alkenone-based SST reconstructions in the northern high latitude oceans show significant bias towards warmer temperatures in core-tops, diverge from other SST proxies in down core records, and are often accompanied by anomalously high relative abundance of the C37 tetra-unsaturated methyl alkenone (%C37:4). Elevated %C37:4 is widely interpreted as an indicator of low sea surface salinity from polar water masses, but its biological source has thus far remained elusive. Here we identify a lineage of Isochrysidales that is responsible for elevated C37:4 methyl alkenone in the northern high latitude oceans through next-generation sequencing and lab-culture experiments. This Isochrysidales lineage co-occurs widely with sea ice in marine environments and is distinct from other known marine alkenone-producers, namely Emiliania huxleyi and Gephyrocapsa oceanica. More importantly, the %C37:4 in seawater filtered particulate organic matter and surface sediments is significantly correlated with annual mean sea ice concentrations. In sediment cores from the Svalbard region, the %C37:4 concentration aligns with the Greenland temperature record and other qualitative regional sea ice records spanning the past 14 kyrs, reflecting sea ice concentrations quantitatively. Our findings imply that %C37:4 is a powerful proxy for reconstructing sea ice conditions in the high latitude oceans on thousand- and, potentially, on million-year timescales.