Postcolumn co-immobilized leucine dehydrogenase-NADH oxidase reactor for the determination of branched-chain amino acids by high-performance liquid chromatography with chemiluminescence detection.

A liquid chromatographic system with a co-immobilized leucine dehydrogenase-NADH oxidase reactor is described for the determination of branched-chain amino acids such as I-leucine, I-isoleucine and I-valine. The enzymes were simultaneously immobilized on tresylate-containing poly(vinyl alcohol) beads. The separation was achieved by means of an ODS column with elution with phosphate buffer (pH 7.5). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The system gave a linear response from 0.3 to 300 mu M for each amino acid and the detection limit was 0.1 mu M.