Oligopeptide Probe-based Mass Spectrometric Characterization Of Reactive Drug Metabolites By Enzymatic Activation

Drug metabolism often generates high polar,low toxicity matabolites,which have excreted from the body easily,but some drugs in the metabolic process will produce a class of chemically reactive intermediates.These materials are easy to covalently bind with biological macro molecules,leading to liver damage,body allergic and other drug side effects.Because of the high reactivity,low stability and low concent of reactive metabolites,we usually detect them in the forms of stable adducts via reaction with capture reagents.At present,it has been reported that capture agents such as glutathione and its derivatives could be used to trap and subsequently detect reactive metabolites,using liquid chromatography and mass spectrometry.But there are still some shortages,such as narrow capture range,low detection sensitivity and inconvenience of detection by LC-MS.Thus exploring a new and efficient capture reagent and MS probe is of great methodological significance for early drug screening,adverse drug reaction mechanism research.Based on the capture characteristics of functional groups(mainly the amine and thiol),the subject is based on amino acids as the structure unit,designing,synthesizing and optimizing a series of oligopeptide probes,and studing about its capturing efficiency and the MS behavior.The main research work of this paper is as follows:1.Based on the amino acid structural configuration,peptide binding sites,amino acid sequence,the terminal residue of the amino acids and other aspects,we designned and synthesized a series of oligopeptide probes for capturing and detecting reactive metabolites.We characterized them using LC-MS analytical method to examine the accuracy of synthesis,and investigate the MS fragmentation mechanism of the oligopeptide probes.2.The paper reported liquid chromatography-tandem mass pectrometry multiple reaction monitoring methods for detecting various adducts of oligopeptide probes and the reactive metabolites of acetaminophen NAPQI,and investigated the trapping and mass spectrometry detection performances of various oligopeptide probes.And finally we screenned out WCK of the L-configuration,?-connection sites as the oligopeptide probe for capturing reactive metabolites.3.We selected six kinds of representative pharmaceutical substrates such as mefenamic acid,furan,estradiol etc,for drug metabolic reaction through in vitro incubation with liver microsomal,by making full use of the neutral loss scan,enhanced product ion scan of MS and multiple reaction monitoring modes to verify the applicability of WCK for capturing reactive metabolites.Furthermore,according to the MS fragmentation mechanism of the adducts,we established a high selectivity,high sensitivity and new general method for characterization drug metabolic activation new method,and successfully applied it to the mass spectrum identification for different structures of reactive metabolites.