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.