Ascorbic Acid Determination with an Automated Enzymatic Procedure (General Clinical Chemistry‪)‬

A wide variety of methods involving different chromophores, detection systems, and enzymes has been developed for the determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in biological samples. (1) The different principles involved have been reviewed and critiqued by Washko et al. [1]. In recent years HPLC has become the preferred method of determination, but this method has the disadvantages of being relatively labor intensive and costly for a clinical laboratory. In fact, very few currently available AA assays are ideally suited to the clinical laboratory. One such procedure, which is based upon measurement of an enzymatic reaction rate and involves a Beckman Synchron CX5 analyzer, has been previously described by Tulley [2]. Given the increasing interest in nutrition testing, and especially in nutrients that serve as antioxidants such as AA, an automated procedure designed for the clinical laboratory is desirable. To meet this need, we have developed an easily adaptable procedure that has significant advantages over chromatographic and spectrophotometric methods. This enzymatic end point procedure was developed by using the Cobas Fara centrifugal analyzer. In principle, AA is selectively and completely oxidized to DHAA by AA oxidase. The product is then coupled with o-phenylenediamine (OPDA) to yield a chromophore, the absorbance of which is measured at 340 nm.