AbstractA simple high performance liquid chromatographic method combined with an enzyme sensor has been developed to measure 1,5‐anhydroglucitol in urine. The enzyme sensor consists of a hydrogen peroxide electrode and a chitosan membrane of an immobilized pyranose oxidase. As the system does not resist interfering substances, urine samples are first purified by passing them through a two‐layer column packed with (1) strongly basic anion (OH−form, the upper layer) and (2) strongly acidic (H+form, the lower layer) exchange resins. 1,5‐Anhydroglucitol is efficiently recovered in the flow‐through fraction of the column. In this system, the minimum detectable concentration of 1,5‐anhydroglucitol is 0.1 mg/L, and the measurable range extends from 0.1 to 60 mg/L. The coefficient of variation values of the within‐day and day‐to‐day precisions are 3.0‐6.5% and and 4.4‐6.7% respectively, and there is good agreement between the results measured by our method and those obtained by the gas‐liquid chromatographic/mass spectrometric method (r=0.994). The method we have described here has been successfully used to elucidate a mechanism for the reducing 1,5‐anhydroglucitol level in the ser