Abstract: A dual channel FIA manifold with amperometric detection was described for the simultaneous determination of citrate and isocitrate in citrus juices. The channel for citrate determination consisted of a co-immobilized citrate lyase/oxaloacetate decarboxylase reactor followed by a pyruvate oxidase reactor. The generated H2O2 was monitored amperometrically. A carrier stream (0.85 ml/min) of 0.1 M phosphate buffer of pH 7.5 containing 10 mM MgCl2, 80 µM-thiamine pyrophosphate and 10 µM-FAD was used. The channel for isocitrate determination consisted of a NADP-specific isocitrate dehydrogenase reactor and the generated NADPH was monitored amperometrically. A carrier stream (1 ml/min) of 50 mM phosphate buffer of pH 6.5 containing 20 mM MgCl2 was used. The sample and coenzyme (1 mM NADP) were injected using a sandwich method with loops of 80 µL for each solution. The Pt working electrode was set at +0.6V (for H2O2) or +0.75 V (for NADPH) vs. Ag/AgCl. Linear calibration graphs were obtained for 0.25-5 mM citrate and 2.5-50 µM-isocitrate and RSD (n = 10) were 2% for 5 mM citrate and 50 µM-isocitrate. The results were confirmed using F-kits (enzymatic-spectrophotometric methods).

Abstract: Isocitrate dehydrogenase(I) was covalently bound to Eupergit C (Roehm-Pharma, Weiterstadt, W. Germany) and packed into Vertex columns (3 cm x 4 mm). The resulting packed-bed reactor, incorporated into a continuous-flow system, was used in the analysis of orange juice; NADP+ was added to the sample, and the mobile phase (pH 7.4) comprised 64 mM Tris, 2.4 mM MnSO4, 0.3 mM EDTA, 1 mM dithioerythritol and 500 mg L-1 of ethyl 4-hydroxybenzoate in aqueous 2% propan-2-ol. The NADPH produced was detected at 340 nm. The I-containing reactor gave complete substrate conversion (99.9%) over a wide range of flow rates and showed good stability. The method was suitable for automated determination of isocitric acid in orange, grapefruit and raspberry juices. Results agreed well with those by a reference method.