The investigation of amine-substituted compounds in peroxyoxalate chemiluminescence reactions for analytical applications

Fluorescent derivatizing agents for carboxylic acid detection by peroxyoxalate chemiluminescence (CL) were investigated. Relative fluorescence and chemiluminescence intensities for several coumarin compounds were compared: 4-(bromomethyl)-7-methoxy-coumarin (Br-Mmc), 7-(diethyl-amino)coumarin-3-carbohydrizide (DCCH), and 7-(diethylamino)-3- (4-((iodoacetyl) amino) phenyl) -4-methylcoumarin (DCIA). These compounds were evaluated as carboxylic acid derivatizing agents to be detected using peroxyoxalate CL with high performance liquid chromatography. No chemiluminescence is observed from Br-Mmc derivatives, but increased intensities can be attributed to the electron donating amino functional group in DCCH and DCIA. The detection limits of aliphatic carboxylic acids derivatized with DCIA and detected with peroxyoxalate CL are reported in the low femtomole range. Fluorescence detectability with DCIA is improved 10-fold relative to Br-Mmc, and an additional 5-fold gain is observed using HPLC/CL detection. The use of DCIA to determine a carboxylic acid analyte in a complex sample matrix was also investigated. HPLC/fluorescence and CL detection of valproic acid in human plasma at clinical levels is demonstrated with no chromatographic interferences from the sample matrix. A linear detector response of the valproate derivative was determined well above and below the therapeutic concentration range.; The complex role and measurement of amine substituted compounds as quenchers and catalysts in buffered and unbuffered peroxyoxalate CL reactions respectively was examined. An important analytical implication is that the quenched CL signal as a function of amine concentration can be used to quantify amines without the need for derivatization, but the linear range is approximately one order of magnitude. Investigation of various quenching mechanisms showed no evidence of amines causing fluorescence quenching nor base hydrolysis of the oxalate which is significant only at high concentrations of amines. At equivalent concentrations of amines and oxalate, the amines can compete with the fluorophor for reaction with the intermediate(s).; A nonlinear response of the catalyzed CL signal as a function of amine concentration is observed and can be attributed to the changing CL intensity-time reaction kinetics and quenching of the CL signal. It was also determined that the sensitivity of the CL response is not a function of the basicity of the catalyst.