A simple and rapid method for the routine assay of total ascorbic acid in serum and plasma using ascorbate oxidase and o-phellylenediamine

A simple and rapid analysis of total ascorbic acid (AsA) in serum and plasm a and its automated analysis are described. AsA is oxidized by ascorbate ox idase (AsA oxidase) to dehydroascorbic acid that then reacts with o-phenyle nediamine (OPDA) to form a quinoxaline derivative that absorbs at 340 nm. T he change in absorbance is directly proportional to the total AsA concentra tion. The assay was validated with a linear concentration range of 0.8-80 m g/L, and the within-day and between-day assays precision did not exceed 8.6 % and 12.5%, respectively. On 47 sera, the manual enzymatic procedure gave 0.2 mg/L on average lower values than those of an automated enzymatic proce dure with a correlation coefficient of 0.847. On another 66 sera, results b y automated enzymatic method correlated well with the HPLC method and the r egression equation is Y (enzymatic, automated)=0.97 X (HPLC)+0.1, r=0.980, Sy.x=0.6 mg/L, An experienced analyst can perform about 24 manual assays pe r hour whereas the automated procedure gave a rate of 100 assays per hour.