Study On Rapid Monitoring For Enzyme Reactions Based On Ambient Mass Spectrometry

Mass spectrometry (MS) has become one of the most powerful tool in modern science. There are three generations of mass spectrometry techniques, which are vacuum, atmospheric and ambient (open-air) MS, respectively. The development of mass spectrometry tends to in-situ analysis, direct ionization and soft ionization.Enzymes are biological macromolecules with catalytic function involved a large number of biochemical processes, such as metabolic pathways or pathological dysfunction, played a pivotal role in the biological and biochemical reactions. This paper selected acetylcholinesterase (AChE), angiotensin-converting enzyme (ACE) and lipase to be the subjects of the research.This article pioneered a new type of method named solvent induced phase separation extraction-capillary electrospray ionization mass spectrometry (SIPSE-CESI-MS).It is an effectively supplement of ambient ionization technology, and it can effectively circumvent the disadvantages of ambient mass spectrometry. The main work is as follows:(1) We chose angiotensin converting enzyme (ACE) extracted from lung and Aspergillus niger genus lipase to hydrolyse the substrate of hippuryl-histidine-leucine (HHL) and tributyrin substrate glycerol ester respectively. The solvent induced phase separation extraction (SIPSE) method and CESI-MS method were employed for the extraction and rapid quantitative determination of the products formed during enzymatic reaction, such as hippuric acid and butyric acid. Quinapril and orlistat were used as the model for the verification of the developed method respectly. Then we selected hippuric acid and butyric acid for further verification of methodology quickly, accurately and with less sample consumption. We also provided a theoretical feasibility basis of a mechanical combination of microtiter plate and SIPSE-CESI-MS.(2) Based on the establishment of method of SIPSE-CESI-MS by the previous chapter, to monitor acetylcholinesterase (AChE) under the optimized positive ion mode. Acetylthiocholine iodide was used as substrate. SIPSE method and CESI-MS method were employed for the extraction and rapid quantitative determination of the thiocholine formed during enzymatic reaction under the optimum conditions. Huperzine A was used as the model for the verification of the developed method. To choose the Choline Choline which is the analog of thiocholine for further verification of methodology. In the system of SIPSE, we found a stable peculiar phenomenon of three-phase system, then we initially identified components of the three-phase systems, and made a preliminary estimation. (3) To develop an accurate and fast, low-cost detection method by capillary electrospray ionization mass spectrometry to determine four kinds of dihydropyridine calcium antagonists of nifedipine, felodipine, nicardipine and nitrendipine. In the complex matrix samples, without use of complex sample pre-treatment or traditional chromatographic separation methods can successfully achieve rapid qualitative and semi-quantitative analysis assays for four dihydropyridine calcium antagonists. The method has high specificity and rapid analysis, then it can effectively avoid the disadvantages that matrix is not conducive to mass spectrometric detection.Then it is expected to be mechanizated and commercializated, and thus plays a role in more areas.