Simultaneous quantitation of citrate and isocitrate in citrus juice by a flow-injection method based on the use of enzyme reactors

A method for the simultaneous quantitation of citrate and isocitrate in citrus juice by flow-injection analysis (FIA) was developed using two enzyme reactors in parallel and an electrochemical flow-through cell. Citrate was quantitated with a co-immobilized citrate lyase (CL) and oxaloacetate decarboxylase (OD) reactor and an immobilized pyruvate oxidase (PO) reactor as the upstream and downstream reactors, respectively. Hydrogen peroxide produced by the CL·OD and PO reactors was monitored amperometrically with a platinum electrode. Since CL is subjected to end-product inhibition, we tried to increase the tolerated frequency of injections of the CL · OD reactor by lowering the amount of citrate in the reaction solution. The response at the 0.05 mM (2.5 nmol) and 0.10 mM (5.0 nmol) level remained steady for 196 and 116 injections, respectively. Isocitrate was quantitated by monitoring the NADPH formed by NADP-specific isocitrate dehydrogenase (NADP-ICDH) or by measuring the hydrogen peroxide produced in the coupled reactions catalyzed by NAD+-specific isocitrate dehydrogenase (NAD-ICDH) and NADH oxidase (NOD). Since citrate interfered with the signal for isocitrate alone in the case of the NAD-ICDH · NOD reactor, an NADP-ICDH reactor was used for the quantitation of isocitrate in citrus juice. The volume of the sample loop in the citrate line was decreased from 50 μl to 1 μl for the simultaneous quantitation of citrate and isocitrate. Linear relationships between the responses and the concentrations of isocitrate and citrate were observed in the ranges 2.5–50 μM and 0.25–5.00 mM, respectively, i.e. over a range of 0.25–5.00 nmol in the reaction solution. The relative standard deviations for ten successive injections were less than 2% for 5.0 mM citrate and for 50 μM isocitrate. The system was applied to the determination of citrate and isocitrate in citrus juice. The results were in good agreement with those obtained by a conventional method (F-kit method), suggesting that this system might be applicable for the detection of the adulteration of citrus products.