AMPEROMETRIC FLOW-INJECTION DETERMINATION OF CITRIC-ACID IN FOOD USING FREE CITRATE LYASE AND CO-IMMOBILIZED OXALACETATE DECARBOXYLASE AND PYRUVATE OXIDASE

A flow-injection analytical system for the determination of citrate in food has been developed. Citrate lyase, oxalacetate decarboxylase, and pyruvate oxidase were used, and the latter two enzymes were co-immobilized. Hydrogen peroxide, which was produced by three enzyme reactions, was monitored amperometrically with a platinum electrode. The carrier solution used was 0.1 M phosphate buffer (pH 7.0) containing 10 mM MgCl2, 80 mu M thiamine pyrophosphate (TPP), and 10 mu M flavine adenine dinucleotide (FAD). The detection limit was 0.02 mM, and the range of determination was 0.1-1.0 mM. The relative standard deviation was 1.23% for 10 successive determinations at the 0.5 mM level. The determination frequency was about 15 tests per hour. The present system was applied to the determination of citrate in several fruits. The results showed good agreement with those obtained using a conventional method (F-kit method).