最後，反應加成物另以LC/MS/MS方法加以分析。由第一次撞擊所得結果顯示，在pH=8.0的緩衝溶液中所形成的反應加成物其主要的斷片質量為463m/z；在pH=10.0緩衝溶液中所形成的反應加成物則得到三種主要的斷片質量，分別為463m/z、449m/z與420m/z；而在pH=12.0時，反應加成物主要的斷片質量為449m/z。綜合一次及二次撞擊所得的主要斷片質量數據，顯示至少有三種不同的加成物在其交互作用之反應中形成，本研究除了解析出其個別化學結構式外，並同時闡明其可能的反應程序機轉。 Previous studies have demonstrated that the interaction between Arecoline and Glutathione may be associated with the etiologic causes of oral cancer, however, the mechanisms have not precisely clarified till present. The aim of this study is to investigate and to clarify the possible mechanisms of the interaction.
Initially, the changes of concentrations of both compounds in buffers with pH range from 2~12 were quantitatively determined by HPLC. The results of the interaction between both compounds in various pH values of buffer solutions found that there were nearly nonreactive in the acidic environment, whereas the significantly rapid interactive reactions were evoked in the buffer solutions with pH>7.0, where we found that the concentrations of both compounds were rapidly decreased within 20 minutes with non-linear patterns. The maximum reactions were occurred at pH 8~9 within 5 minutes. However, when the pH of buffers solution were higher than 10.0, a small portion of Arecoline could be hydrolyzed to Arecaidine, thus the interactive reactions were found to be slightly decreased.
Subsequently, the possible adducts formed in the interacting reaction were then isolated and purified. Their purities were confirmed with HPLC.
Eventually, the adducts were further analyzed with LC/MS/MS. The results of first collision showed that a major fragment mass of adduct formed in pH=8.0 buffer solution was 463 m/z; three major fragments of the adduct formed in pH=10.0 buffer solution were obtained and their masses were 463 m/z, 449 m/z and 420 m/z, respectively; the major fragment of adduct formed at pH=12 had a mass of 449 m/z. Getting togather, from the data including those produced in the second collisions at least of three possible adducts could be formed in the interactive reactions and their chemical structures were determined. The possible reaction schemes were also elucidated.