Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system.

Abstract

A liquid chromatographic system consisting of a co-immobilized 3-hydroxybutyrate dehydrogenase-NADH oxidase reactor and an immobilized pyranose oxidase reactor in series and a chemiluminometer was developed for the simultaneous determination of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in plasma. The enzymes were immobilized on toresylated poly(vinyl alcohol) beads. Separation was achieved on a TSK gel SAX column (40 x 4 mm I.D.) with an eluent of 50 mM NaOH containing 30 mM sodium butyrate. The hydrogen peroxide produced was detected by measuring the chemiluminescence emitted on admixing with luminol and potassium hexacyanoferrate(III). The calibration curves were linear from 0.8 to 500 microM (7 ng-4 micrograms) for glucose, from 0.8 to 400 microM (7 ng-3 micrograms) for 1,5-anhydroglucitol and from 1 to 700 microM (5 ng-4 micrograms in a 50-microliter injection) for 3-hydroxybutyrate. The sample throughput was four per hour. The reactors were stable for at least ten days.