Stable Isotope Labeling Combined With Mass Spectrometry: Liposoluble Carboxylic Acids Analysis

Liposoluble carboxylic acids(LCAs)are groups of lipid molecules and play important roles in various physiological processes.Abnormal contents or changes of LCAs are associated with a series of diseases.Therefore,a high sensitive and accurate detection method of LCAs is essential for physiological research and disease diagnosis.LC/MS is the most prominent platforms for the determination and profiling of metabolites because it offers good detection sensitivity and selectivity as well as provides structural information.However,the direct detection of LCAs by MS is arduous in their native form due to their poor ionization.Therefore,the low sensitivity,poor accuracy of quantitation and the limited detection coverage are the major problems in LCAs analysis.Based on the derivatization technology,stable isotope labeling(IL)coupled with MS strategy has been developed used in metabolomics research.To improve the detection sensitivities and accuracy,a pair of isotope labeling reagents with a easily ionized group,a hydrophilic group and a isotope labels,are introduced into the LCA structures.In the typical IL strategy,a light isotope tag is introduced to the analytes in one sample,and another heavy isotope tag is introduced to another sample.Then,the light and heavy isotope-labeled samples are mixed and analysis by LC-MS.In this text,we combined the stable isotope labeling with mass spectrometry techniques to qualitative and quantitative analysis of LCA compounds by using different mass spectral scanning modes.The established method significantly improved the detection sensitivity and accuracy of LCAs,and expanded the sub-metabolomics coverage.The research contents include the following aspects:1.Targeted analysis of fatty acid metabolities:(1)We developed a highly sensitive method to identify and quantify cytochrome P450 metabolites of arachidonic acid in a variety of biological samples based on stable isotope probe labeling coupled with ultra-high performance liquid chromatography/mass spectrometry.A pair of stable isotope probes,2-dimethylaminoethylamine(DMED)and d4-DMED,were utilized to facilely label eicosanoids.The heavy labeled eicosanoid standards were used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis.In addition,the detection sensitivities of DMED labeled eicosanoids improved by 3 to 103 folds in standard solution and 5 to 138 folds in serum matrix compared with unlabeled analytes.Taking advantage of the phenyl column and DMED labeling,a good separation of eicosanoids isomers was achieved.We further quantified cytochrome P450 metabolites of AA in rat liver,heart,brain tissues and human serum using the developed method.The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-,15-,16-,20-HETE,5,6-EET and 14,15-EET in type 2 diabetes mellitus patients and 5-,11-,12-,15-,16-,20-HETE,8,9-EET and 5,6-DHET in myeloid leukemia patients had significant changes,demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.(2)We developed a highly sensitive method for the determination of 16 FAHFAs in biological samples by coupling strong anion exchange solid phase extraction(SAX-SPE)with stable isotope labeling assisted ultra-high performance liquid chromatography/mass spectrometry(SAX-SPE-CL-UHPLC/MS).In the developed method,SAX-SPE was employed to selectively enrich and purify FAHFAs from biological samples.Our results demonstrated that the limits of detections(LODs)of labeled FAHFAs ranged from 0.01 to 0.14 pg and the detection sensitivities of FAHFAs increased approximate two orders of magnitude compared with previous methods.Moreover,a good separation of FAHFAs isomers was achieved on C18 column in a UHPLC system and all FAHFAs could be analyzed in 20 min with sharp peak shape.Using this method,we successfully measured the contents and distribution of FAHFAs in various rat tissues.The results showed that 7 FAHFAs(13-,12-,9-,5-PAHSA,13-,12-and 9-SAHSA)were observed in different tissues of rat and human serum;and among the 7 FAHFAs,13-,9-PAHSA,13-and 12-SAHSA were found remarkably decreased in human breast cancer serum.2.Non-targeted metabolomics analysis of liposoluble carboxylic acids:(1)Neutral loss scan(NLS)and multiple reaction monitoring scan(MRM)are two characteristic scan modes in triple quadrupole mass spectrometry analysis.Here we developed a strategy with stable isotope labeling combined with liquid chromatography-tandem mass spectrometry(IL-LC-MS)analysis for comprehensive profiling and relative quantitation of LCAs in human serum.In this strategy,DMED and d4-DMED were employed to selectively label carboxyl groups of LCAs.The DMED and d4-DMED labeled products can lose four characteristic neutral fragments of 45/49 Da or 63/67 Da in collision-induced dissociation.Therefore,quadruple neutral loss scan(QNLS)mode was established and used for non-targeted profiling of LCAs.The peak pairs of DMED and d4-DMED labeling with the same retention time,intensity and characteristic mass differences were extracted from the two NLS spectra respectively,and assigned as potential LCA candidates.Using this strategy,241 potential LCAs were discovered in the human serum and 21 of these LCAs were successfully identified by standards.Subsequently,a MRM detection mode was developed and used for relative quantification of LCAs in human serum from and healthy controls.As a result,81 LCAs were found to have significant difference between type 2 diabetes mellitus patients and healthy controls.(2)We developed a strategy of stable isotope labeling coupled with liquid chromatography-LTQ-Orbitrap mass spectrometry(IL-LC-full scan-Orbitrap MS)analysis for non-targeted profiling of LCA compounds in mice feces sample.Using this strategy,a total of 853 LCA candidates were discovered in mice feces,which expanded the LCAs detection coverage.Taking advantage of the LTQ trandem Orbitrap mass spectrometry property,we explored the four dependent data acquisition mode for multistage mass spectrometry analysis of LCA candidates to improve the qualitative analysis ability.In potential 853 LCA compounds,447 LCA candidates can be determined by searching METLIN database,16 compounds could be presumed by multistage mass spectrometry analysis and 83 LCAs were successfully identified by standards.