Derivatization-mass Spectrometry For The Analysis Of Reactive Carbonyl Compounds In Biological Samples

Endogenous reactive carbonyl metabolites are involved in various important biological events related to substance metabolism,oxidative stress and progression of many diseases.However,it is difficult to identify and distinguish them due to the low concentration,chemical instability and complexity of sample matrices.Mass spectrometry is one of the most powerful tools for the determination of metabolites in real biosamples,but it is still limited by the poor ionization efficiency of these carbonyl analytes,especially when using conventional electrospray ionization.At present,the ionization efficiency of the analyte and its analytical behavior can be improved by introducing a chemical group with a derivatization reaction,.which is one of the research hotspots in the field of mass spectrometry.In this paper,in order to screening and quantifying large-scope carbonyl compounds in biological samples,a new method for the analysis of reactive carbonyl metabolites was established by isotope coded derivatization?ICD?combined with liquid chromatography-double neutral loss scanning-mass spectrometry?ICD-LC-DNLS-MS?.The research mainly focused on the following three aspects.1.New quaternized derivatization reagents were designed,synthesized and validated to trap carbonyl compounds.We prepared four functional reagents,and two of them were found as novel derivatization tags.Four carbonyl compounds were used as substrates to evaluate the performance of the derivatization agents by investigating the fragmentation pathways of the derivative products and comparing the chromatographic and mass spectrometry behaviors.The 1-?4-mercapto-4-oxobutyl?pyridine-1-bromide?HPB?derivatization reagent was finally selected for the next experiment.2.A method for the analysis of reactive carbonyl metabolites was established by stable isotope coded derivatization with liquid chromatography-double neutral loss scanning mass spectrometry?ICD-LC-DNLS-MS?for non-targeted screening of reactive carbonyls metabolites in cinnamon extracts,based on a pair of stable isotope labeling reagents HPB and HPB-d₅ as double-derivatizing agents.The extraction and derivatization conditions were optimized,and the established HPLC-MS/MS method was used to investigate 17 potential reactive carbonyl metabolites in the cinnamon extract.3.Based on the ICD-LC-DNLS-MS strategy,we successfully analyzed reactive carbonyl compounds in human urine samples.General MRM parameters were explored for the relative quantitative analysis of the carbonyl compounds.Finally,24 potential active carbonyl metabolites were found in healthy human samples.A relative quantitative analysis was further adopted to detect the urine samples from diabetic patients and healthy objects.We found some reactive carbonyl compounds were significantly different between normal and diabetic samples,which may be used as new potential disease biomarkers.