Screening Of Fatty Acids And Bile Acids By Fragmentation Patterns In Multistage Mass Spectrometry Based On Stable Isotope Labeling

Liquid chromatography–mass spectrometry（LC-MS）has been one of the most powerful tools for metabolite analysis.It not only exhibits excellent separation performance,good detection sensitivity and selectivity,but also is equipped with plenty of available public databases,which greatly benefit the identification.Nevertheless,for those metabolites without the easily ionizable groups,the detection sensitivity is rather low due to poor ionization,which greatly restricts the detection range.Based on derivatization technology,stable isotope labeling（SIL）has been developed in metabolomics and has resolved the difficulty of poor ionization.To improve the detection sensitivity of the metabolite without easily ionizable group,SIL introduces ionizable group by derivatization reaction.Besides,according to the features of SIL,a multitude of effective analytical methods are established on the LC-MS platform.However,after derivatization reaction,the fragmentation patterns of metabolites have totally changed due to the transformation of the molecular structure,which means that the functions of matching MS spectra in public database has become invalid.Therefore,for identication of metabolites in biological samples,it is significant to elucidate fragmentation patterns of derivatized metabolites.Fatty acids（FAs）and bile acids（BAs）have important physiological and pathological functions in human body.However,as carboxyl compounds,due to their poor ionization efficiency,FAs and BAs are usually labeled with stable isotope derivatization reagents in biological samples to improve the detection sensitivity and establish relevant analysis methods.At this point,their fragmentation patterns change so that the spectrum cannot be matched in database.Therefore,Fatty acids and bile acids were selected as research objects.A complete screening strategy was established by combining SIL with the fragmentation pattern of derivatized FAs and BAs in multistage mass spectrometry.Based on this strategy,FAs and BAs in human serum were comprehensively and accurately screened.The specific work is as follows:With high-resolution mass spectrometry,fragmentation patterns of FAs and BAs before and after derivatization of 2-dimethylaminoethylamine（DMED）in multistage mass spectrometry were compared.The relation was elucidated between the molecular structure and the fragmentation patterns of DMED-derivatized saturated fatty acids（DMED-SFAs）,monounsaturated fatty acids（DMED-MUFAs）,polyunsaturated fatty acids（DMED-PUFAs）and bile acids（DMED-BAs）.According to the relation,a complete set of criteria was established for screening SFAs,MUFAs and PUFAs and BAs with some structure information.For the analysis of FAs and BAs in complex samples,a specific data dependent scan acquisition mode（Full scan-DDMS2-NL3）was created with UPLC-ESI-LTQ-Orbitrap MS as a platform to achieve data acquisition,based on the fragmentation patterns of DMED-and d₄-DMED-FAs and BAs.Revolving around the established screening criteria,a screening strategy with this scanning mode was developed.In this strategy,the full advantage of SIL to identification was taken and the results of identification were ranked according to the qualitative accuracy,all of which made the strategy have high sensitivity,wide coverage and excellent identification capability.The feasibility of this screening strategy was verified in human serum samples,and 12 SFAs,50 UFAs（23 MUFAs and 27 PUFAs）and 14 BAs were successfully screened out,all of which were qualitatively classified into three grades.The results included 16 FAs with a qualitative level of one.According to the strategy,it is reasonably assumed that they were undetected FAs in human serum.