Application Of Normal Pressure Ion Source In Drug - Plasma Protein Affinity Study And Free Blood Concentration Detection

With the development of binding affinity of drug-plasma protein and free drugconcentration, it is very significant to build up the new methods to meet morerequirments for the detection and characterization. Ambient mass spectrometry candirectly provide the real time structural information in situ with high speed, whichwould provide a novel approach for studying these systems. In this work, thetechniques based on ambient mass spectrometry are employed for detection ofbinding affinity of drug-plasma protein and free drug concentration system. Thedetailed study contents are as following:1. We utilized the Desorption Electrospray Ionization Mass Spectrometry toresolve three problems in detection of binding affinity. First, ARID allowed for theprotein with an anionbinding site to act as the reference protein itself withoutliberation of ligand from a specifically bound site. Second, the extracted ion was thequasimolecular ion of the free ligand ([L]) rather than the complex ([PL]). In this way,the gaseous CEID, which acts as a disadvantage on the determination of the complex,plays a positive role in our experiment. Third, the problem of analytical speed wasresolved by our house-made automatic moving platform. We selected AGP as a modelprotein on which all specific ligands bind to one common anion-binding site, andN,Ndimethyloctylamine (DMOA) as a displacer that has been used in earlier research.Our work was divided into three parts. First, the method was verified by using (R),(S)-amlodipine as model ligands, and the enantioselectivity we obtained was inaccordance with earlier research. Next, the high throughput specialty was confirmedby5α-adrenoreceptors and10β-adrenolytics. We obtained qualitativeinformation of15*3samples in2.3min. Finally, atenolol and moxonidine, whichhave been shown to scarcely interact with AGP, were chosen as internal standards tocorrect the volume sprayed into the cone to identify that it is at least asemi-quantitative method. The data we acquired were in great accordance withpreviously reported researc.2. We utilized the Venturi Easy Ambient Sonic-Spray Ionization MassSpectrometry to establish a easy and pretreatment-free detection system for ligands ofproteins with anion-binding sites using a linear ion trap (LTQ) analyzer, and a house-made Venturi Easy Ambient Sonic-Spray Ionization source. And a new anionicregion inhibited dissociation (ARID) mechanism, which describes the inhibiteddissociation of a specific ligand binding to anion-binding site, is verified again basedon our observations. To distinguish these specific ligands from liberated ligandsdissociated from nonspecific interactions, we should first inhibit the dissociation ofspecific binding and evaluate the nonspecific interference. To get precise results ofcontrol tests, two strategies were used to prevent specific bound ligands fromdissociating from the complex. First, we selected AGP, a protein with an anionbinding site, as our model protein. The observation suggested that MS signalsobtained from experiments performed on AGP showed better consistency withbinding affinities than on the proteins without anion-binding site. Second, DESI is asoft ionization technique. Therefore, the complex is impacted with lower energy. It isgood for protecting the specific interactions during the control test.