| The drug metabolites analysis is one focus in the field of life sciences andmedicine, which would contribute to the screening of drug candidates, inspiring thedevelopment of new drugs，improving drug safety and reduce the attrition rate ofdrug candidates. In recent years, the quadrupole time-of-flight mass spectrometry(Q-TOF-MS) with high resolution and excellent qualitative analytical capabilitiesgradually becomes a powerful tool to study drug metabolites and metabolic pathway.Firstly, we established vitro metabolic model for liver microsomes usingtestosterone and midazolam as probe drugs. The probe drugs and metabolites wereanalyzed by high performance liquid chromatography tandem mass spectrometry.The chromatography and mass spectrometry conditions were optimized so that theprobe substrates and their metabolites could be well separated with better MS signalsas well. The validation of the optimized chromatographic conditions was confirmedby our experimental data. The mass spectrum of the drug and its major metaboliteacquired under the optimized MS conditions can provide a theoretical basis for thepreliminary identification of metabolites. Drug metabolic rate can be simplycalculated based on the peak areas, and then, the activity of CYP450isoenzyme canbe evaluated. Properly used specific probe drugs in the determination of CYP450isoenzyme activity not only help in confirming the metabolic characteristics of thedrug, but also have a great significance for the development and clinical applicationof new drugs.GABABreceptor was related to many symptoms of nervous system disorder,such as depression, anxiety, drug addiction, making it one of the most outstandingand promising drug targets. CGP7930is a positive allosteric modifier for GABABreceptor. Studies on CGP7930and its metabolites would contribute to thedevelopment and clinical application of new drugs. In this work, one metabolite ofCGP7930produced by liver microsomes was identified and elucidated usinghigh-performance liquid chromatography quadrupole time-of-flight tandem massspectrometry (HPLC Q-TOF-MS). One metabolite was identified by comparingchromatogram and mass spectra data from the samples and drugs. The metabolitewas characterized by MS/MS spectra and the fragmentation pathways wereconfirmed. The results revealed the main metabolic pathway of CGP7930wasmonohydroxylation reaction and also the hydroxylation sites located in the termination of the tertiary carbon chain. This work contributes to the comprehensiveunderstanding of in-vitro metabolism of CGP7930, and will provide an importantbasis for further in-vivo metabolism study of CGP7930and drug safety evaluation.Last yet not least, we explored the gel permeation chromatography (GPC)method for separating and purifying protein, using the HSA standard protein as amodel. Due to difference in molecular weight among proteins, the retention time forthe proteins through a gel permeation chromatography (GPC) could be distinctive.The outflow time of the target protein from the detector can be accurately calculatedby the flow rate and pipe length, enabling the separation of the pure product of targetprotein other contaminating proteins, meeting the requirements of MS instrument.ESI-MS tests proved the feasibility of this purification method. FAK protein waspurified using the gel permeation chromatography model, but mass spectrometryconditions relatively strict, due to its lower concentrations and other influencingfactors. We cannot find complete FAK proteins after purification, but still, our workprovides theoretical and experimental basis for the follow-up research on the FAKprotein purification and its interaction with the small molecule inhibitors. |
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