The LC-MS/MS-based Approaches In The Application Of Drug Metabolism And Lipidomics

Liquidchromtography coupled with tandem mass spectrometry(LC-MS/MS)have becomean indispensable analytical technique.Up to now,LC-MS/MS has been widely used in various application fields,such as environmental analysis,food analysis,pharmaceutical and medical research and so on.A variety of methods or strategies were proposed on different research purpose.Among different types of mass spectrometry,triple quadrupole(QQQ)and triple quadruple linear trap(QTRAP)mass spectrometry were mainly applied in quantitative analysis,such aspharmacokinetics,metabolic stability and drug-drug interaction.While high-resolution mass spectrometry(HRMS)was used for qualitative analysis,including drug metabolism,traditional Chinese medicine,metabolomics and proteomics and so on.However,the strategy of using a single LC-MS/MS platform both in quantitative and qualitative analyses or integrating different types of mass spectrometers with complementary advantages is truly needed.Triple quadrupole linear trap(QTRAP)is such a MS platform that can be well suited for both quantitative and qualitative analysis.In addition to the multiple reaction monitoring(MRM)scan mode that was widely used for quantitative analysis in DMPK studied due to its excellent sensitivity and selectivity.QTRAP was also capable of performing information dependent scan method for structural characterization.Nowadays,a variety of strategies based on QTRAP have been proposed for qualitative analysis.However,those strategies have not been widely used due to the lack of selectivity or sensitivity.In this study,through the identification of metabolic soft-spot,and the identification of phase I metabolites and GSH conjugated adducts,and the profiling of lysophospholipids in serum,theuse of QTRAP or integrated use of QTRAP and QTOF was introduced to estabilish more efficient,simple and accurate analytical strategies.1.The application of a QTRAP based method for rapid,accurate and sensitivedetermination of metabolic soft-spotsA simple approach using a single LC-MS/MS platform for fast analysis of metabolic stability and soft-spots was developed.In current study,the half life(t1/2)of each model compound was abtained according to the metabolic stability study.When conducting metabolic soft spot identification,model compounds were incubated at a low substrate concentration of 3.00 ?M and adjustable incubation times based on their respective t1/2 rather than higher substrate concentrations or multiple or fixed incubation time,which was a prerequisite to make the reaction performed under a condition with linear kineticsand controlled generation of secondary metabolites.Since the metabolic pathway is difficult to predict,the multiple ion monitoring(MIM)scan mode with wide scan range and suitable sensitivity and selectivity was used to detect metabolites.Then,the major metabolites were directly determined using an on-line ultraviolet detector,while their MS/MS spectra were acquired and retrieved using different data-mining tools,respectively.Analyses of five model compounds(midazolam,dextromethorphan,verapamil,amodiaquine and clozapine)demonstrated that this liquid chromatography tandem mass spectrometer platform is capable of determining metabolic stability and soft-spots in a manner of high throughput analysis and well suited for lead optimization in drug discovery.This was the first time to improve the accuracy and throughput of metabolic soft spot via controlling bothincubation conditions and analytical method.2.Development of an integrated approach for simultaneous detection and indentification of phase I metabolites and glutathione conjugated adducts using LC-UV-QTRAP-MS/MSIn current study,a new LC-QTRAP-MS/MS based analytical methodology for simultaneous detection,structural characterization and semi-quantitation of in vitro phase I metabolites and glutathione(GSH)trapped reactive metabolites was reported.In the analysis,multiple ion monitoring(MIM)scan mode was used for screening phase I metabolites,while multiple reaction monitoring(MRM)scan mode was used for screening the GSH conjugated adducts,respectively.Additionally,on-line ultraviolet detection was employed to determine relative concentrations of major metabolites.After data acquisition,separation of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches including product ion filter,neutral loss filter and extracted ion chromatography.Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and GSH adducts,but also identified their major oxidative metabolism and bioactivation pathways.From this study,simultaneous detection of phase I metabolites and GSH adducts was demonstrated beneficial to illustrate the biosynthetic pathway of reactive intermediates of drugs.3.Profiling of endogenous lysophospholipidsby two integrated LC-MS/MS platforms and its applicationSince the structural relation and similar core structure of glycerophospholipids results in a common and predictable mass spectrometric fragmentation behavior,six classes of lysoglycerophospholipid(Lyso-PL)were subjected to a new predict-verify approach by LC-QTRAP-MRM complemented with LC-QTOF-MS/MS analyses of biological serum samples in the present study.Firstly,the corresponding specific MRM transitions of individualLyso-PLs were predicted.Then Lyso-PLs were detected with the MRM-IDA-EPI scan mode due to its high sensitivity and good selectivity.If the retrieved product ion spectra confirmed the presence of a Lyso-PLs molecule as well as the type of Lyso-PLs,the formulae were identified subsequently identified by determination of exact masses using LC-QTOF-MS/MS platform.As a result,a total of 96 Lyso-PLs molecular species,consisting of 317 regioisomers were detected in serum.In addition,the qualitative distribution of six classes of Lyso-PLs in serum of four rodent species were calculated and compared with that of human.The results showed that the qualitative distribution of Lyso-PLs in rat and mouse have a relatively higher similarity to that of human.We believe that this improvement for qualitative analysis of endogenous Lyso-PLs should greatly contribute to identification of lipid molecular species in biological systems.Also,the new predict-verify strategy can be applied to qualitative analysis of other targeted endogenous metabolites which occur in a high individual number but sharing either structural similarities or similar functional groups.