Development And Application Of New LC-MS Analysis Methods For Fatty Acids,Isoprenoids And Odd Carbon Chain Lipidomics

According to their function,structure and chemical properties,lipids can be divided into eight categories:（1）fatty acids,（2）glycerolipids,（3）glycerophospholipids,（4）sphingolipids,（5）sterol lipids,（6）prenol lipids,（7）saccharolipids and（8）polyketides.Lipidomics is an independent subject that systematically studies all lipid collections in biological samples.As a methodology for large-scale qualitative and quantitative study of lipid compounds and understanding their functions and changes under different physiological and pathological conditions,it can accurately and comprehensively provide the whole lipid information spectrum of biological samples under different physiological conditions.Sensitive and reliable analysis methods of lipids are thus the key to the study of lipidomics.Based on this,the study intended to develop the analysis methods of fatty acids,mevalonate pathway intermediates（isoprenoids）,and odd carbon chain lipidomics,to decipher the biological roles of certain lipids.The functional regulation of free fatty acids（FFAs）is closely related to the occurrence and development of many diseases.FFAs can be divided into short chain fatty acids（SCFAs）,medium chain fatty acids（MCFAs）and long chain fatty acids（LCFAs）according to their aliphatic chain length.The following three types of fatty acids have different sources and functions.Accurate quantification is thus fundamental to reveal their mechanism of action.However,the accessibility of stable isotope labeled internal standard for absolute quantification,the low response of mass spectrometry of FFAs in negative ionization mode and the suppressed chromatographic resolution due to the wide coverage of FFAs chain length restrict the wide use of stable isotope dilution LC-MS analysis.Here,we developed an isotope-free dual derivatization assisted LC-MS/MS multi reaction monitoring（MRM）strategy to analyze FFAs.Using this strategy,the samples were labeled with"light weight"reagent N,N-dimethyl ethylenediamine（DMED）or dansyl hydrazide（Dns-Hz）,and the standard mixture or control group were labeled with"heavy weight"reagent N,N-diethylethylenediamine（DEEA）or N,N-diethyldansulfonyl hydrazide（Dens-Hz）.The dual derivative internal standard additives（DD-ISs）of the target analyte were synthesized"one-to-one"under mild reaction conditions.The absolute or relative quantification of the target analyte can be achieved in mass spectrometry MRM mode after the two were mixed in identical proportion and effectively separated by chromatography.Taking DMED/DEEA derivatization of SCFAs as an example,the methodological evaluation of the strategy was carried out.The results showed that the detection limit of SCFAs is 0.5～3 n M,the linear R²=0.99374～0.99929.Moreover,there was no significant substrate interference in the derivatization reaction,and no additional quenching steps were required,confirming the feasibility and reliability of the method.Using this method,we successfully analyzed 15SCFAs in fecal samples from patients with human hepatocellular carcinoma（HCC）and healthy controls.Among them,propionate,butyrate,isobutyrate and 2-methylbutyrate in feces of hepatocellular carcinoma group were significantly lower than those of healthy control group.We further expanded the LC-MS/MS MRM analysis of DMED/DEEA or Dns-Hz/Dens-Hz derivatized MCFAs and LCFAs.The results showed that the signals of C8:0～C24:0 derivatives were significantly enhanced.After the chromatographic elution gradient with extended separation time,FFAs with different chain lengths could be detected simultaneously.In addition to fatty acids,the synthesis part of non-sterol squalene in cholesterol synthesis pathway,namely MVA pathway or isoprenoid biosynthesis pathway（IBP）,plays a key role in cell metabolism and is also the target of many clinical drugs.Therefore,monitoring isoprenoid（Iso P）dynamics in cells,plasma and tissues is very essential for evaluating pathological conditions and regulation after therapeutic intervention.At present,different methods of detecting Iso P by LC fluorescence detection and liquid chromatography-mass spectrometry（LC-MS/MS）have been reported previously.However,most analytical methods are developed only for one or two specific compounds due to the large difference in polarity.To solve this problem,we constructed an LC-MS/MS MRM analysis method after chemical derivatization of Iso P with 3-nitrophenylhydrazine（3-NPH）and its isotope analogue ¹³C₆-3-NPH.This method can realize the full coverage of seven metabolic intermediates in IBP pathway including MVA,MVAP,MVAPP,IPP/DMAPP,GPP,FPP and GGPP,and can effectively solve the serious chromatographic peak tailing of Iso P in reverse phase chromatography.Using this method,we detected the potential changes of Iso P intermediates of human prostate cancer cell lines LNCa P and NCI-H660 after treatment with squalene cyclooxygenase inhibitor NB-598 maleate.Based on the above analysis of the building blocks of FFAs and Iso P,we further constructed the analysis method of odd carbon chain lipidomics.At present,there are many studies on the lipidomics with LC-MS methods,but most of them are lipids with even carbon chains（carbon chain length is 2～26）,while the research on odd carbon chain lipidomics is still lacking.Moreover,odd carbon chain lipids are becoming increasingly important in dietary assessment,coronary heart disease（CHD）and type II diabetes mellitus（T2D）risk biomarkers and endogenous metabolic alternative pathways.Using tissue and plasma samples,MRM detection ion pairs were constructed based on the characteristic fragment ions of different types of lipids in ESI ion source.After qualitative analysis by LC-MS/MS,the accuracy of the method was ensured by the regularity of lipid retention time in C18 reverse phase chromatography,and the scheduled MRM method was further established,After adding the internal standard of lipidomics,the lipid group containing odd fatty acyl chain in biological samples can be detected quantitatively.In the analysis of various lipids in tissues,PC contributed the major abundance of OCFA phospholipids,which is largely due to the higher absolute concentration of PC relative to other types of phospholipids.We also noted that C15:0 and C17:0 were preferentially enriched in specific lipid categories.In addition,C13:0,C19:0 and C21:0 were distributed in phospholipids except PG.For monounsaturated odd carbonyl chain lipids,C17:1 and C19:1 were enriched in phospholipids except PG,while C17:2 was only significantly enriched in PS and PI.These results demonstrated that the heterogeneity of OCFA distribution in different tissue lipids may be due to the conversion among lipid categories or the selectivity of fatty acid synthase,which leads to the heterogeneity of OCFA enrichment in different lipid types.This laid a foundation for the functional study of odd carbon chain lipids.We also reviewed the research prospect of LC-MS analysis of free fatty acids and mevalonate pathway intermediates,and the metabolism and function of odd carbon chain lipids.