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%.