AMPEROMETRIC DETERMINATION OF OXALATE IN PLASMA AND URINE BY LIQUID-CHROMATOGRAPHY WITH IMMOBILIZED OXALATE OXIDASE

A detection system for plasma and urinary oxalate involving a postcolumn enzymatic reaction and electrochemical detection is described. Oxalate oxidase was immobilized on AF-Tresyl Toyopearl 650 gel. The immobilized oxalate oxidase was packed into a stainless-steel column (10 x 4 mm I.D.) and used on-line as an immobilized enzyme reactor (IMER). The hydrogen peroxide produced by enzymatic reaction was detected amperometrically at a platinum electrode maintained at +0.5 V vs. Ag/AgCl. The HPLC separation of oxalate was carried out using a Capcell Pak C-8 column and an isocratic mobile phase containing 80 mM KH2PO4 and 5 mM tetra-n-butylammonium phosphate as an ion-pair reagent. The IMER was active and stable in the mobile phase employed. The plot of peak height against concentration of oxalate was linear in the range 0.1-1.6 mu mol/ml with a correlation coefficient of 0.9989. The detection limit was 10 nmol/ml at a signal-to-noise ratio of 3. The within-day and between-day coefficients of variation were 5.3 and 7.9%, respectively.