Flow injection assay for the neurotoxin beta-ODAP using an immobilized glutamate oxidase reactor with prereactors to eliminate glutamate interferences.

The neurotoxic amino acid, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP,ODAP) was oxidized by immobilized glutamate oxidase (GlOD) to produce hydrogen peroxide. The peroxide reacts with Trinder reagent in a reactor with immobilized horseradish peroxidase to form a red-colored quinone imine dye, which was detected spectrophotometrically at 512 nm. Determinations were made in a flow injection (FI) setup consisting of four packed-bed enzyme reactors containing GlOD (20 microL), catalase (20 microL), GlOD (250 microL), and peroxidase (50 microL) in series. Glutamate is oxidized quantitatively in the first reactor, but the hydrogen peroxide is destroyed in the second so that interferences from this substrate are removed. This step destroys only a few percent of the ODAP in the sample. Most of the remaining ODAP is oxidized in the third reactor. Injections of 20-microL ODAP standards gave a response curve which was linear within the range 10-650 microM. Phosphate buffer extracts of grass peas (lathyrus sativus) were purified by centrifugation and membrane filtration. Samples were injected into the FI setup to assay the toxin at a rate of 20 samples per hour. The beta-ODAP content of a batch of dry seed corresponded to 0.74% (w/w) with a relative standard deviation of 2.8%. Thermal treatment of ODAP standards at 80-90 degrees C reduced the response to 62% of that before heating. The decrease is due to beta<-->alpha isomerization, and the experiment thus confirms that the method is selective for the toxic beta-isomer.