Analysis Of Deoxynucleoside Carbonyl-Ammonia Addition Reaction And Oxidative Damage By Mass Spectrometry

Nucleic acid is an important carrier of organism’s genetic information,in which nitrogenous bases are vulnerable to some exogenous or endogenous factors to cause damage or mutation,eventually leading to the wrong expression of genetic information in the body or the occurrence of some diseases.Studies have shown that some active chemical substances such as carbonyl compounds can interact with different bases in various forms,which has attracted the attention of many scholars.In recent years,screening and detection of DNA oxidative damage has become a research hotspot in the field of genomic toxicology and epigenetics.However,existing analytical methods detect only single or limited base-adder derivatives.In view of the current state of the art,a multi-reaction monitoring method for determination of derivatizations of four bases and their deoxynucleosides by two carbonyl compounds was established in this paper,and the detection range of the detection limit was extended by optimizing the derivatization-detection conditions.The specific work is as follows:(1)The addition reaction conditions of acrolein and all four kinds of deoxynucleosides were optimized to obtain a reaction-rate of 91.4%-96.5%under optimal conditions.The intermediate cleavage pathway of the nucleoside adducts in the secondary mass spectrometry dissociation process was discussed.That is,the sugar moity was lost first from the deoxyribose molecule,and the quantitation ion of[M+H]was obtained;then one molecule of water was lost according to the difference of base structure.Based on the fragmentation pathway,an LC-MS/MS method for the simultaneous detection of deoxynucleosides and their acrolein adducts was established.(2)Using BDAPE as a derivatizing agent,the derivatization conditions of all 5 bases and 4 kinds of deoxynucleosides were optimized,and the reaction rate reached 92.47%-98.42%under the optimum derivatization condition.The multi-stage mass spectra of these derivatized products were summarized,i.e.,one molecule of deoxyribose(M=116)was cleaved to obtain the main product ions for quantitation.Then a molecule of water is futher removed to obtain the qualitation ion.In addition,characteristic fragment ions can also be generated depending on the base structure.Based on this fragmentation,a neutral loss scanning mode can be used to screen unknown samples.(3)Using a preparative chromatography to separate and purify the derivative products,a single pure derivatized compound deoxyadenosine(dA),deoxycytidine(dC)and cytosine(C)was obtained to establish a LC-MS/MS method for simultaneous quantitative analysis of real samples.The results showed that the linear range was good(R2>0.9996)with a detection limit of 50.73,41.39 and 392.95 pg/ml,respectively.The recovery rates of whole blood,urine sample,and Chuanbei samples were 99.00-104.95%,95.38-103.21%and 94.26-105.10%,respectively.