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

A method for the sequential enantiomeric 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 455 nm (excitation at 340 nm). 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 0.1 mM 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). (C) 2004 Elsevier B.V. All rights reserved.