Sensitive quantification of isoprenoid cytokinins in plants by selective immunoaffinity purification and high performance liquid chromatography-quadrupole-time of flight mass spectrometry

In this work, we developed a novel class-specific immunoaffinity column for the natural isoprenoid cytokinins (CTKs) by using trans-zeatin riboside as the hapten to generate a complete antigen. By combination with a mixed-mode solid phase extraction step for pre-cleanup and a high performance liquid chromatography-quadrupole-time of flight mass spectrometry for the quantification, an efficient analytical protocol was established which allowed simultaneous quantification of eight endogenous isoprenoid CTKs in Arabidopsis thaliana leaves with a wide linear range from 25 to 500 pg/g fresh weight and a detection limit of 12.5 pg/g fresh weight. The method will be very useful for comprehensive research on the networks of signaling interactions of the active phytohormones and their regulation of the plant functions. (C) 2011 Elsevier Inc. All rights reserved.