The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins （free form and their conjugates）. The method includes a protocol of extraction with methanol/water/formic acid （15/4/1, v/v/v） to the micro-scale samples, pre-purification with solid phase extraction （SPE） cartridges of the extracts, separation with a high performance liquid chromatography （HPLC） and detection by an electrospray ionization ion trap mass spectrometry （ESl-lon trap-MS） system in a consecutive ion monitoring （CRM） mode at the three stage fragmentation of mass spectrometry （MS3）. The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg, at the coefficient factors of 0.98-0.99. The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials, such as aerial parts of rice and poplar leaves etc. 12 endogenous cytokinins had been identified and quantified in the plant tissues, with an acceptable relatively higher recovery rate from 40% to 70%.
