Novel Analytical Strategy For Comprehensive Profiling Of Endogenous Lipids Based On High Performance Liquid Chromatography Coupled With Mass Spectrometry

As a fundamental component of organisms,lipids are structural and functional molecules that play important bioliogical roles in a variety of physiological and pathological processes because of the great diversity of their structural,endogenous abundances and physiochemical properties.Lipidomics is the comprehensive study of cellular lipids,which is an important branch of metabolomics.The development of lipidomics has been largely driven by rapid advances in technologies such as chromatography,mass spectrometry(MS)and nuclear magnetic resonance(NMR)in past decade.MS,with its excellent qualitative and quantitative capabilities,has been the most important and widely used technique in lipidomics.It can satisfy the qualitative and quantitative analysis of most endogenous lipids including glycerides,phospholipids,sphingomyelin and ceramides.However,the establishment of MS-based methods for the analysis of some lipid species,such as fatty acid(FA),eicosanoid,fatty aldehyde,acidic glycosphingolipid(AGSL)and fatty acid esters of hydroxyl fatty acid(FAHFA),can be quite challenging due to the following properties of these lipid species:poor MS ionization efficiency,low endogenous abundances,poor chromatography performance and being susceptible to interference from matrix components.The exploring of high performance liquid chromatography coupled with mass spectrometry(HPLC-MS)methods with excellent separation efficiency and high sensitivity was important for the quantification and quantitation of these lipids with poor MS ionization efficiency and low endogenous abundances.In this thesis,comprehensive identification and quantification HPLC-MS methods were established for the profiling of FA,eicosanoid,fatty aldehydes,AGSL and FAHFA,aming to solve the bottleneck problems in their analysis process.FAs play multiple critical roles in living processes:(1)FAs can supply energy through P oxidation process;and(2)FAs are involved in molecular signaling and also modulate biological processes.Studies have shown that FAs are associated with a variety of disease,including tumors,metabolic diseases and heart diseases.Establishment of efficient and rapid detection method for FAs is important for the elucidation of their biological functions,pathogenesis of FA related diseases and discovery of early diagnostic biomarkers.High performance liquid chromatography coupled with tandem mass spectrometry(HPLC-MS/MS)has proven to be a powerful tool for FA analysis.However,it is difficult to satisfy the analysis of endogenous low abundance FAs because of the limited sensitivity resulted from FAs low ionization efficiency and the matrix interfenrence caused by complex bioligical samples.In this study,a new derivatization strategy based on 2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine(T3)and its isotope d20-2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine(D3)were adapted to the comprehensive profiling of FAs by HPLC-MS/MS.Because of the highly nucleophilic of the hydrazine groups of the labelling reagents,the derivatization reaction can be completed within 1 h at 37 ?.Under this mild derivatization conditions,the risk of oxidation was minimized.As a result of this derivatization approach,we achieved an ultra-sensitive FA detection method.The limit of detection(LOD)for FAs is as low as 10 fg and 10 ?l biological body fluid samples can satisfy the FA detection.Multiple reaction monitoring(MEM)mode was employed for FA analysis with high specificity and sensitivity.A general strongest product ion(m/z 254)related to derivatization tags can be achieved for all the FA derivatitives under a similar collision energy in the MRM mode analysis.Thus we developed a general set of conditions for MRM analysis of all the derivatized FAs,which greatly similified the optimization process of MRM conditions.The general MRM conditions remove the dependence on specific MRM condition optimization for FA analysis.Isotope dilution method was employed for the relative quantificaiton of FAs based on the isotopic derivatization reagents.A novel strategy was constructed for the comprehensive discovery and identification of FAs in biological samples according to the retention rules and mass spectrum characteristics of T3 and D3 labled FAs.FAs in plasma from rats of different ages were analyzed using this HPLC-MS/MS method,and several FA species related to the ageing process were identified.Eicosanoids are endogenous compounds oxidized from arachidonic acid(ARA),eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)under the catalysis of cyclooxygenase,lipoxygenase or cytochrome P450.According to the differences in chemical structures,eicosanoids can be divided to several categories,namely,hydroxyl and epoxidated polyunsaturated fatty acids,prostaglandins(PG),thromboxane(TX)and leukotriene(LT).As signalling lipid molecules,eicoanoids can be involved in the inflammatory process and immune system regulation of the organism.Because of the structure similarity and their low ionization efficiency,detection sensitivity,selectivity and chromatography separation resolution were the insurmountable problems for eicosanoids profiling of biological samples.In the thesis,a high sensitive and specific analytical strategy based on T3 derivatization was established to profile and quantify eicosanoids by ultra high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).A milder and more efficient reaction condition were achieved for eicosnaoids derivatization through optimization of the reaction condensation agent,derivative time and derivative temperature refering to established fatty acid derivatization method.Through incubated with T3 under 4 ? for 30 min,carboxy group of eicosanoid was converted to amide with derivatization yield larger than 99%.The risk of eicosanoids alternation was minimized by decreasing reaction temperature and time.The detection sensitivities of derivatized eicosanoids were enhanced by 10-to 5000-folds compared to free eicosanoids.All detected eicoanoids isomers can be separated and quantified in 19 min with good separation efficiency and peak shape.Stabilities of eicosanoids were also highly improved after T3 modification.The newly built UHPLC-MS/MS method was employed to profile and quantitate eicosanoids in rat plasma and heart tissue of control and acute myocardial ischemia(AMI)model rats.The perturbation of the eicosanoids metabolic networks in plasma and heart tissue of rats during AMI was investigated by statistical analysis based on the concentration levels of eicosanoids.Fatty aldehyde is an aldehyde with an aliphatic carbon chain.They are generally catabolized from fatty acids,sphingolipids,plasmalogens,and isoprenoid alcohols via a-or co-oxidation.Numerous studies have shown that fatty aldehydes play important roles in the pathogenesis of neurodegenerative diseases.New method with high sensitivity and efficiency need to be established for the comprehensive profiling of fatty aldehydes in plasma and brain tissue.And this would be of great significance to elucidate the pathogenesis of nervous system diseases.In this thesis,a new HPLC-MS/MS method was developed for comprehensive profiling of fatty aldehydes based on isotopic derivatization reagents T3 and D3.Isotope dilution method was employed for the relative quantification fatty aldehydes,which effectively reduced the interference of matrix.The detection sensitivity of fatty aldehyde was highly improved through derivatization.As low as 0.1 pg/ml fatty aldehyde can be quantified.Approximately 98%fatty aldehydes were converted to their respective phenylhydrazone derivatives through incubated with the derivatization reagent in 15 min at 37 ?.Because of the structure similarity of the derivatized fatty aldehydes,a general strongest product ion(m/z 209)related to derivatization tags can be achieved for all the fatty aldehyde derivatitives under a similar collision energy in the MRM mode analysis.Therefore,the optimization processes of MRM parameters was largely simplified.Fatty aldehydes in biological samples were identified on the basis of isotope-induced retention time shifts(IRS)and collision induced dissociation(CID)products.The new analytical strategy was successfully implemented to globaly profile fatty aldehydes in plasma and brain tissue of the bilateral common carotid artery(2VO)dementia rat model.More than 80 fatty aldehydes were detected in rat plasma and brain tissue,and several fatty aldehydes were found to be closely related to the pathological state of cerebral ischemia.AGSLs belong to the sphingolipid,mainly including gangliosides and sulfatides.Gangliosides are composed of a hdrophobic ceramide moiety and a hydrophilic glycan chain with one or more sialic acids.While sulfatides contain a sulfate group coupled to a galactosyl ceramide,which are also kown as 3-O-sulfogalactosylceramides.A large number of studies have demonstrated that AGSL is closely related to brain tissue development,neurodegenerative disease and cerebral ischemia.However,due to the complexity of AGSL structure and the lack of individual standards,the comprehensive identification and quantitative analysis of AGSL are severely restricted.In this study,a novel analytical strategy was established to comprehensively profile and identify AGSLs in biological samples using ultra high performance performance liquid chromatograph coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS).Ganglioside isomers with different glycan chains such as GDIa/GD1b were completely separated on a C18 column for the first time to our knowledge,facilitated by the addition of formic acid in the mobile phase.High resolution MS and MS/MS data were collected using the positive scanning mode.The glycan chain and ceramide structure of AGSLs can be identified simultaneously based on the high resolution MS and MS/MS data.A mathematical model was established to predict the retention times(RTs)of all theoretically possible AGSLs on the basis of the good logarithmic relationship between the ceramide carbon numbers of the AGSLs in the reference material and their RTs.A data set was created of 598 theoretically possible AGSLs,including the ceramide carbon numbers,RTs,and high-resolution quasi-molecular ions.This new analytical strategy can be used for rapid and accurate identification of AGSL in biological samples with high separation efficicency and wide coverage of AGSLs and 199 AGSLs were identified in rat brain tissue.The number of AGSL compounds identified in biological sample using our methods were larger than those of the previously reported methods.This novel analytical strategy was finally employed to profile AGSLs in brain tissue samples from control rats and model rats with 2-VO cerebral ischemia.The AGSL changes under the pathological state of cerebral ischemia were further interpreted.Fatty acid esters of hydroxy fatty acid(FAHFA)is a recently discovered novel class of endogenous mammalian lipids possessed antidiabetic and anti-inflammatory effects.FAHFA is formed by esterification between the hydroxyl of a hydroxy fatty acid and the carboxyl group of a fatty acid.Concentration levels of FAHFAs in biological system are far lower than triglycerides,phospholipids and other common lipid compounds.They are below 150 pmol/g in adipose tissue(AT),similar to signaling lipids such as eicosanoids and steroids.In this thesis,a novel UHPLC-MS/MS method was developed for comprehensive profiling and quantification of FAHFAs.Through optimization of the chromatographic conditions,FAHFA isomers can be efficiently separated and quantified within 29 min with excellent peak shapes and good robustness.UHPLC-Q-TOF-MS was employed for FAHFAs identification based on the high resolution m/z values and the fragmentation rules.A total of 64 FAHFAs,belonged to 17 different family members,was identified in white adipose tissue(WAT)of golden hamsters.Nine of the 17 FAHFA family members were newly discovered in this paper.And linoleic acid and linolenic acid are newly found building blocks of FAHFAs in WAT.The specific precursor-product ions of FAHFAs identified by UHPLC/Q-TOF-MS were further transmitted to the UHPLC/MS/MS instrument for their quantification under the multiple reaction monitoring(MRM)scan mode.All FAHFAs showed excellent calibration linearity(r>0.99),accuracy and precision within the dynamic range from 1 ng/ml to 400 ng/ml.It has been successfully used for the quantification of FAHFAs in hamster WAT attached to different tissues to explore the disturbance of WAT FAHFAs under the pathological condition of hyperlipidemia.The FAHFA regulation effects of fenofibrate were also investigated with hyperlipidemic hamster.