A chemiluminometric FIA procedure for the enzymatic determination of L-aspartate

An enzymatic FIA procedure was developed for the chemiluminometric determination of L-aspartate in a medium for mammalian cell cultivation. Packed bed flow reactors containing aspartate:aminotransferase and L-glutamate oxidase, respectively, immobilized on sieved porous glass beads were combined with a peroxidase modified fibre optic sensor to detect the produced hydrogen peroxide. Peroxidase from Arthromyces ramosus was immobilized on a preactivated microporous nylone membrane and catalyzes the light generating oxidation of luminol by hydrogen peroxide. To improve the selectivity of the L-aspartate determination a L-glutamate eliminating reactor was prepared by coimmobilization of L-glutamate oxidase and catalase in a packed bed enzyme reactor. Under FIA conditions at least 0.5 mM L-glutamate can be quantitatively eliminated from the preconditioned sample solution. L-Aspartate can be determined in the range between 5 and 1000 muM under FIA conditions. The determination of L-aspartate was optimized with respect to pH, cosubstrate concentration and residence time in the packed bed enzyme reactors. The proposed procedures show high recoveries between 96 and 100% for the L-aspartate determination in a medium for mammalian cell cultivations after the elimination of L-glutamate. (C) 2001 Elsevier Science B.V. All rights reserved.