Improvement And Application Of Liquid Chromatography-mass Spectrometry Detection Method For Marine Sterols And Glycerol Dialkylglycerol Tetraether

Biomarkers are organic compounds in the environment and geological bodies that record the molecular structure information of the source organisms.Their content/composition can indicate the contribution of specific biological source to the natural organic matter pool,and can record the transformation of organic matter and the history of environmental evolution as well.In recent years,the application of lipid biomarkers in marine biogeochemical studies has gradually increased,and their roles in indicating and reflecting paleo-oceanic environmental changes,organic matter sources,and biological community structures have attracted widespread attention.Phytosterols and glycerol dialkyl glycerol tetraethers(GDGTs)are two of the widely used lipid biomarkers,but the current analytical methods for these two biomarkers still have shortcomings such as cumbersome experimental procedures and large sample consumption,and further optimization and improvement of the methods are needed.In order to address the above problems,two new methods was developed in this paper for the analysis of the two biomarkers by using liquid chromatography(LC)-mass spectrometry(MS)coupled with accelerated solvent extraction(ASE)and large volume injection(LVI),and applied to the study of the distribution characteristics of phytosterols and GDGTs in the actual marine environment.The main research results obtained are as follows.First,a new method for simultaneous determination of common phytosterols in marine sediments was developed by non-aqueous reversed-phase LC coupled with atmospheric pressure photoionization mass spectrometry(APPI-MS).The APPI-MS characteristics of phytosterols were clarified.Under the positive APPI mode,phytosterols tended to produce stable characteristic ion peaks[M+H-H₂O]⁺,[M+H-H₂]⁺,and[M+H-2H₂]⁺.In the process of APPI-MS~2,abundant fragment ion peaks were produced mainly through i-heterolysis.LC-APPI-MS conditions were optimized,reversed-phase C18 column was used to separate phytosterols with pure methanol as mobile phase,and toluene as APPI ionization dopant,which can realize high sensitivity of detection for phytosterols in sediments.What's more,the method has strong matrix interference resistance ability,as it has a good linear relationship(R~2?0.9990),recovery rate(86.4%?94.9%),and repeatability(relative standard deviations,RSDs?8.3%).The proposed method was applied to detect common phytosterols in the surface sediment of Laizhou Bay in China,and several sterols,such as brassicasterol,fucosterol,and sitosterol,had been detected in every sediment sample.The total concentration of sterols in every gram of dry weight(dw)was in the range of 4.21?g/g dw?12.91?g/g dw,among which the concentration of sitosterol was the highest.Second,in order to reduce the sample consumption of sediment or seawater suspended particulate matter(SPM),a new simple method was developed by using LVI-LC coupled with atmospheric pressure chemical ionization mass spectrometry(APCI-MS)for the analysis of GDGTs in small or semimicro amounts of samples.The parameters of ASE and LVI of target GDGTs were optimized.Under optimal conditions,the LVI-LC-APCI-MS could determine various GDGTs accurately even with 0.02 g?0.10g of lyophilized sediment samples or SPM samples collected from 0.2 L?1.0 L of marginal sea water or 10 L?20 L of open ocean seawater with satisfactory method precision(RSDs?10.2%)and linearity(R~2?0.991).Compared with the classical method,the LVI–LC–APCI–MS required significantly reduced amount of sediment or SPM sample.The proposed method was applied to determine GDGTs in real sediment samples collected from hydrothermal vent sites at the Southwest Indian Ridges.Four groups of GDGTs,namely,isoprenoid GDGTs(iso GDGTs),branched GDGTs(br GDGTs),H-shaped isoprenoidal GDGTs(H-GDGTs)and hydroxylated isoprenoidal GDGTs(OH-GDGTs),and archaeol,glycerol dialkanol diethers and glycerol monoalkanediol diethers were detected with only 0.10 g of lyophilized sediment sample collected from Longqi and Tianzuo hydrothermal fields.The results show that the organic matter in the sediment sample from Tianzuo was significantly affected by hydrothermal activity.In addition,the determination of six common iso GDGTs in the SPM of surface and subsurface seawater in the East Indian Ocean was successfully achieved using the SPM samples collected by filtering only 10 L seawater of open ocean.Compared with the methods in the literature,the LVI method can significantly reduce the workload of collecting seawater SPM,making it possible to conduct extensive studies on GDGTs in deep-sea waters.Finally,the horizontal and vertical distribution characteristics of phytosterols and GDGTs in surface sediments and iso GDGTs in seawater SPM in the Bohai Sea,Yellow Sea and part of East China Sea were investigated.The results showed cholesterol,campesterol,fucosterol,sitosterol,stigmasterol,and dinosterol,were detected in 26sediment samples from the Bohai Sea,32 sediment samples from the Yellow Sea and 6sediment samples from the East China Sea at all stations,and brassicasterol were detected at nearly three-quarters of the stations.The total concentration of various sterols ranged from 0.77?g/g dw to 7.87?g/g dw,and the two sterols with the highest content were cholesterol and fucosterol.The content of marine in situ produced phytosterols and terrestrial input phytosterols are basically comparable in most areas,and marine in situ produced phytosterols are slightly higher than terrestrial input phytosterols.The total concentrations of iso GDGTs in the surface sediments of the Bohai Sea,Yellow Sea and East China Sea ranged from 7.64 ng/g dw to 303.81 ng/g dw,and the two most abundant iso GDGTs were Cren and GDGT-0,respectively.The total concentration of br GDGTs ranged from 0.89 ng/g dw to 52.52 ng/g dw.The content of br GDGTs basically showed the pattern of GDGT-III(II/I)>GDGT-IIIb(IIb/Ib)>GDGT-IIIc(IIc/Ic).The total concentration of OH-GDGTs ranged from 0.35 ng/g dw to 46.95 ng/g dw,and the content of OH-GDGTs showed a pattern of OH-GDGT-0>OH-GDGT-1>OH-GDGT-2.For GDGTs in seawater SPM,the total concentration of iso GDGTs in surface seawater SPM was generally at a low level,ranging from 0.03 ng/L to 6.41 ng/L.The total concentration of iso GDGTs in bottom SPM was significantly higher than that in surface SPM,ranging from 0.88 ng/L to 14.43 ng/L.The total concentration of iso GDGTs in seawater SPM at different depths basically showed a pattern of slightly decreasing first and then increasing with the increase of depth.