Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-(p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.