Determination of aliphatic amino acids in serum by HPLC with fluorimetric detection using co-immobilized enzyme reactor

A simple and selective method is presented for the determination of aliphatic amino acids such as L-alanine, L-valine, L-isoleucine and L-leucine in serum using HPLC with detection by co-immobilized alanine dehydrogenase/leucine dehydrogenase post-column reactor and fluorimeter. The enzymes were simultaneously immobilized on chitosan beads. The separation was achieved by means of an ods column with elution with phosphate buffer (pH 7.0). The system gave linear responses over two orders of magnitude and detection limits at 1–2μM levels.