The Derivatization of Proquinoidal Analytes with 1,2-Diphenylethane-1,2-diamine (DPE) and Benzylamine (BA): An Investigation of Products, Yields, Kinetics and Reagent Selectivity
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文摘
The fluorogenic derivatization of 5-hydroxyindoles, catecholamines and catechols with benzylamine (BA) and diphenylethylenediamine (DPE), mediated by the presence of an oxidant such as potassium hexacyanoferrate, followed by liquid chromatography with fluorescence detection has become an established approach for the bioanalysis of this group of biologically important substances. The derivatization products have been established except for the product arising from reaction of DPE with catechols. Previous reports have described the selective derivatization of the 5-hydroxyindoles with BA and likewise a selective reaction of catecholamines and catechols with DPE, which has been the basis of numerous prior protocols. In the present investigation, the products for each analyte class resulting from reaction of either reagent are either re-examined or established, via synthesis of authentic product standards, as well the determination of the absolute yield and reaction kinetics for each analyte class via the use of a representative model substance (5-hydroxyindoles, 5-hydroxyindole-2-carboxylic acid ethyl ester; catecholamines, epinephrine; catechols, 4-methylcatechol). The data provided herein establish that either reagent undergoes reaction with 5-hydroxyindole and epinephrine, forming the same product for each respective analyte in equivalent yields albeit with differing reaction rates. However, the two reagents result in the formation of related but different products formed at different rates and yields in the derivatization of 4-methylcatechol, especially when glycine was included as a regent component. As prior reports advocate use of glycine as an accelerant in the DPE derivatization of catecholamines and catechols, this aspect has been investigated with the present finding that glycine does not serve in such a role for epinephrine; however, in the case of 4-methylcatechol derivatization with DPE in the presence of gly, both substances compete with the first formed intermediate, thus glycine appears to serve as an accelerant, but in fact provides for a competing alternative reaction pathway which ultimately results in a significantly reduced yield of the desired product. Integration of the results obtained from the kinetic and yield investigations for each analyte class leads to the suggestion of plausible reaction schemes. These implied chemical pathways are of high analytical importance as they provide a basis for the rationale in development of an optimal derivatization protocol for these analytes. In a further aspect, based on the proposed mechanism of catechol derivatization, the examination of the effect of exposure of the first oxidized analyte species to ammonia was explored to determine the feasibility of creating a DPE-like derivative using the more water-soluble reagent BA, through exposure of the catechol to ammonia in the presence of an oxidant and while successful an overall lower yield was realized.

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