Determination of lactate or oxalate using injected lactate oxidase and peroxidase by capillary electrophoresis with UV detection

Two reactions, catalyzed by lactate oxidase (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2'-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the run buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD(+) which is separated and detected in less than 2 min at 266 nm with a sample throughput of 7 min (including wash steps between samples). Simplex(TM) software is used to optimize the enzyme concentrations and reaction temperature. Consumption of the more expensive LO enzyme is only 1.4 x 10(-3) U per assay assuming 27 nL per injection. Linearity is established within the range from 0.0025 to 1 mm with R-2 of 0.9982. Recoveries of lactate from five spiked serum samples averaged 101%. Application of this method for the determination of oxalate as an inhibitor of LO is demonstrated.