Study of cytokinin metabolism using HPLC with radioisotope detection

High performance liquid chromatography (HPLC) with an on-line flow-through radioactivity detector was used to monitor the metabolism of cytokinins ([3H]6-benzylaminopurine and [3H]6-benzylaminopurine riboside) after their incorporation into wheat seedlings. The production and conversion of individual metabolites was assayed within a short time interval (0.5-3 h). Extraction recoveries from plant tissue proved to be 85%. The uptake of both cytokinins was very rapid and differences in their metabolism were already perceptible after 30 min. The individual metabolites were identified as adenine (Ade), adenosine (Ado), benzyladenine-9-glucoside (BA-9G), 6-benzyladenine (BA) and benzyladenosine (BAR). The method is very fast, sensitive and very useful for metabolic studies. The detection limit was 40 pg (220 Bq) for BA at the level of 2 ng ml-1.