Sequential fluorometric quantification of malic acid enantiomers by a single line flow-injection system using...

A method for the sequential enantiomeric quantification of d-malate and l-malate by a single line flow-injection analysis was developed using immobilized-enzyme reactors and fluorescence detection. An immobilized d-malate dehydrogenase (d-MDH) reactor and an immobilized l-malate dehydrogenase (l-MDH) reactor were introduced into the flow line in series. Sample and coenzyme (NAD(+) or NADP(+)) were injected into the flow line by an open sandwich method. d-Malate was selectively oxidized by d-MDH when NAD(+) was injected with a sample. When NADP(+) was injected with a sample, l-malate was oxidized only by l-MDH. NADH or NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455nm (excitation at 340nm). Linear relationships between the responses and concentrations of d-malate and l-malate were observed in the ranges of 1 x 10(-6)-1 x 10(-4)M and 1 x 10(-6)-2 x 10(-4)M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.1mM level. This analytical method was applied to the sequential quantification of d-malate and l-malate in fruit juices and soft drinks, and the results showed good agreement with those obtained using conventional method (F-kit method).