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

Abstract

AbstractAn 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%.