Determination of L-malate using immobilized malate dehydrogenase and aspartate aminotransferase in a flow system and its application to analyze the L-malate content of beverages

The quantity of L-malate was determined using apparatus comprised of a reactor with immobilized malate dehydrogenase (MDH) and aspartate aminotransferase (AST) in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD(+) in the carrier containing 0.1 M glutamate was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of Tris buffer ranged from 7.0 to 8.5. Various buffer types were also examined as carrier media at pH 8.0 and Tris buffer showed the maximum peak area. When the carrier composed of Tris buffer (0.1 M, pH 8.0) was used, the calibration curve for malate was linear in the range of 0.05-50 mu M (r = 1.000). The detection limit (S/N = 3) was 0.03 mu M. Relative standard deviations of the peak area at I mu M and 10 mu M were 1.5% (n = 7) and 0.36% (n = 7), respectively. Thirty samples of malate (10 mu M) were analyzed for 1 hr. This method was applied to the analysis of malate in several beverages, and malate content determined by this method agreed with that determined by a commercially available test-kit method.