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Abstract No.: TuP-175
Session: New Instrumentation
Presentation date: Tue, Aug 29, 2006
Presentation time: 09:50 – 11:20

Fast Quantitative Analysis of Endogenous Cytokinins by UPLC-ESI(+)MS/MS Technology

Ondrej Novak1, Petra Amakorova1, Eva Hauserova1, Karel Dolezal1, Miroslav Strnad1

1 Laboratory of Growth Regulators, Palacky University and IEB AS CR, Olomouc, Czech Republic

Correspondence address: Ondrej Novak, UP Olomouc and IEB AS CR, Laboratory of Growth Regulators, Slechtitelu 11, Olomouc, 772 00 Czech Republic.

Keywords: Chromatography, Fast; Mixture Analysis; MS/MS, Liquid Chromatography; Quantitative Analysis.

Novel aspect: New and fast quantitative analysis of endogenous cytokinins in plant materials.


Cytokinins, one group of phytohormones, play a crucial role in controlling the way in which plants grow and develop. The major problem associated with analysis is that the amount of cytokinins is very low, usually in the range of fmol to pmol·g-1 fresh weight. Development of simple purification of real samples by batch immunoextraction1 and application of new analytical methods make possible a new direction in plant hormone research.
New way of purification was tested as routine method of cytokinin analysis. Small (mg) amounts of plant tissues were purified by solid phase extraction and immunoaffinity step. The purification was speed up using batch immunoextraction and completed by fast chromatographic analysis.
A new chromatographic technique, the ultra performance liquid chromatography (Acquity UPLC, Waters), using combination of high pressure and hybrid particles, was coupled to triple quadrupole mass spectrometer (Quatro micro API, Waters) equipped with electrospray interface. Rapid baseline chromatographic separation of 20 cytokinins (trans/cis-zeatin, dihydrozeatin, isopentenyladenine, benzyladenine, meta/ortho-topolin groups) was developed. Using UPLC analysis, the run time was reduced up to seven times in comparison with published cytokinin analysis.2 Retention time stability ranged between 0.1-0.4 % RSD and injection accuracy between 1.5-5.0 % RSD. In multiple reaction monitoring mode, detection limit for most of cytokinins used was below 1 fmol and linear range was at least five orders of magnitude. The accuracy of developed fast separation combined with tandem mass spectrometry was further validated on spiked poplar leaf extracts which also showed good reproducibility.
In conclusion, UPLC-ESI(+)MS/MS technology can be used for fast and sensitive quantitative analysis showing reproducibility in separation of cytokinins in different plant extracts.

1. E. Hauserova, , J. Swaczynova, K. Dolezal, R. Lenobel, I. Popa, M. Hajduch, D.Vydra, , K. Fuksova, M. Strnad, J Chromatogr A 1100, 116 (2005).
2. O. Novak, P. Tarkowski, D.Tarkowska, K. Dolezal, R. Lenobel and M. Strnad, Anal. Chim. Acta. 480, 207 (2003).