BACKGROUND: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography-fast scanning tandem mass spectrometry. RESULTS: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. CONCLUSIONS: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.

Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 11 Olomouc CZ 783 71 Czech Republic

Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Umeå SE 901 83 Sweden

Isotope Laboratory Institute of Experimental Botany ASCR v v i Vídeňská 1083 Prague 142 20 Czech Republic

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany AS CR v v i Šlechtitelů 11 Olomouc CZ 783 71 Czech Republic

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