Determination of D-Malate Using Immobilized D-Malate Dehydrogenase in a Flow System and its Application to Analyze the D-Malate Content of Beverages

Abstract

The quantity of D-malate was determined using apparatus comprised of a reactor with immobilized D-malate dehydrogenase (D-MDH) in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD⁺ in the carrier was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) buffer ranged from 7.0 to 8.5. Various buffer types were also examined as carrier media at pH 8.0, and PIPES buffer showed the maximum peak area. When the carrier composed of PIPES buffer (0.1 M, pH 8.0) containing 1 mM NAD⁺ and 5 mM MgCl₂ was used, the calibration curve for D-malate was linear in the range of 0.02-50 μM (r=1.000). The detection limit (S/N=3) was 0.01 μM. Relative standard deviations of the peak area at 1 μM and 10 μM were 1.6% (n=7) and 0.48% (n=7), respectively. This method was applied to the analysis of D-malate in several beverages, and the recovery test of the added D-malate to samples was also carried out to afford good results.