Analysis Of Coexist Components In The L-phenylalanine Solution By HPLC-MS

With L-aspartate (L-ASP) as the amino donor, L-phenylalanine (L-Phe) can beprepared from phenylpyruvic acid (PPA) via an amination reaction mediated byaminotransferase. And PPA can be prepared by hydrolysis of 5-benzalhydantoin. It isdifficult to reach the required detection limits and many of methods lack specificity.High-performance liquid chromatography-mass spectrometry (HPLC-MS) has become awell established analytical tool in modern laboratories. It can offer superior specificityand often offer lower detection limits than conventional high-performance liquidchromatography (HPLC) detection and thus is ideally suited to the analysis ofcomplicated reactions. In this thesis, a highly sensitive method based on Liquidchromatography-tandem mass spectrometry has been developed for the complicatedreaction. Firstly, the proceed of preparation of PPA by hydrolysis of 5-benzalhydantoin arestudied. A sensitive HPLC method has been developed for the quantitative determinationof phenylpyruvic acid and 5-benzalhydantoin first, then based on the HPLC method, thewhole hydrolysis system has been determined by HPLC-MS. Secondly, a highly sensitive HPLC-MS method has been developed for thequantitative determination of L-Phe, L-ASP and pyruvic acid in the reaction solution ofpreparation of L-Phe via the amination reaction mediated by aminotransferase. Theelectrospray ionization mass spectrometry (ESI-MS) parameters such as DP, FP, CE, EPCEP, CXP, CUR, IS, TEM, GS1 and GS2 have been studied. And on the base of theseparameters, the influential HPLC factors such as the concentration and type of inorganicsalt, polarity of mobile phase, pH of buffer, rate of flow have been studied too. It has beenproved that the HPLC-MS method is accurate, precise and useful in practical solution. Finally, the splitting principle of L-Phe, L-ASP, pyruvic acid, phenylpyruvic acidand 5-benzalhydantoin in ESI-MS has been studied, the possible splitting pathway ofthese materials has been derived from the principle. iThe thesis provides a model to study and determine the composition of mixtures.And the experimental results will play an important role in the produce and qualitycontrol of amino acid.