High-Performance Liquid Chromatographic Analysis of Cytokinins Using Isatoic Anhydride as a Fluorescent Derivatizing Reagent

Abstract

A method for quantitative determination of cytokinins containing ribose such as N⁶ -isopentenyladcnosine (i⁶Ado) and zeatin riboside (zR) has been developed. This based on the preparation of fluorescent anthraniloyl derivatives of cytokinins by the reaction of isatoic an hydride with hydroxyl groups of the ribose, and then the resolution of fluorescent derivatives by high-performance liquid chromatography with a spectrofluoro monitor. The fluorescent anthraniloyl derivatives were separated more sharply and symmetrically from each other compared with the separation of the non-derivatized i⁶Ado and zR. The detection limits for anthraniioyl-i⁶Ado and anthraniloyl-zR were 1.1 and 1.2 pmol per 10 μl injection, respectively, and these values indicate that the fluorimetric analyses were approximately 70- to 150-times more sensitive than the U V -monitoring method. The simplicity, speed, sensitivity and selectivity make this method an attractive alternative to established cvtokinin assay systems.