HIGHLY SENSITIVE DETERMINATION OF L-LACTATE AND PYRUVATE BY LIQUID-CHROMATOGRAPHY AND AMPEROMETRIC DETECTION WITH LACTATE OXIDASE LACTATE-DEHYDROGENASE COIMMOBILIZED REACTOR INVOLVING AMPLIFICATION BY SUBSTRATE RECYCLING

An amperometric flow injection system with a lactate oxidase-lactate dehydrogenase coimmobilized reactor involving amplification by substrate recycling is used as a specific postcolumn detector system in liquid chromatography, to detect L-lactate and pyruvate with high sensitivity. Both components are separated at a reversed phase column and are recycled enzymatically during the passage through the enzyme reactor in the presence of reduced nicotinamide adenine dinucleotide (NADH) and oxygen in the carrier stream. As a result of this recycling reaction, a large amount of hydrogen peroxidase is generated in the enzyme reactor which is detected amperometrically at a flow-through peroxidase electrode in the presence of hexacyanoferrate(II) as a mediator. Linear determination range is 0.2-200 pmol for both L-lactate and pyruvate. The detection limit is 0.02 pmol. The relative standard deviation obtained for this system at 2 pmol L-lactate and pyruvate (n = 5) is about 3.8%.