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Determination of lactate or oxalate using injected lactate oxidase and peroxidase by capillary electrophoresis with UV detection
Author(s) -
Qi Lining,
Danielson Neil D.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305472
Subject(s) - chemistry , chromatography , oxalate , peroxidase , abts , capillary electrophoresis , substrate (aquarium) , enzyme , biochemistry , organic chemistry , antioxidant , oceanography , dpph , geology
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™ software is used to optimize the enzyme concentrations and reaction temperature. Consumption of the more expensive LO enzyme is only 1.4×10 −3 U per assay assuming 27 nL per injection. Linearity is established within the range from 0.0025 to 1 m M 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.