Phytohormones, their functions, synthesis and effects, are of great interest. To study them in plant tissues accurate and sensitive methods are required. In the present study we aimed at optimizing experimental conditions to separate and determine not only plant hormones but also their metabolites, by liquid chromatography coupled with a UV-VIS detector. The mixture we analyzed was composed of benzyladenine, kinetin, trans-zeatin, cis-zeatin, dihydrozeatin, meta-topolin, ortho-topolin, alpha-naphthalene acetic acid, indole-3-acetic acid, trans-zeatin-7-glucoside, trans-zeatin-O-glucoside, trans-zeatin-9-riboside, meta-topolin-9-riboside and ortho-topolin-9-riboside. We measured the calibration dependences and estimated limits of detection and quantification under the optimal chromatographic conditions (column: Polaris C(18); mobile phase: gradient starting at 2:98 (methanol:0.001% TFA) and was increasing to 55:45 during twenty minutes, and then decreasing for 10 min to 35:65, flow rate: 200 microL x min(-1), temperature: 50 degrees C, wavelength: 210 nm). The detection limits for the target molecules were estimated as tens of ng per mL. We also studied the effect of flax extracts on the phytohormones' signals. Recovery of aliphatic and aromatic cytokinins, metabolites of cytokinins and auxins were within the range from 87 to 105 %. The experimental conditions were tested on a mass selective detector. In addition we analysed a commercial product used for stimulation of roots formation in cuttings of poorly rooting plants. The determined content of alpha-naphthalene acetic acid was in good agreement with that declared by the manufacturer.

Department of Plant Biology Faculty of Agronomy Mendel University of Agriculture and Forestry Brno Czech Republic

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Swarup K., Benkova E., Swarup R., Casimiro I., Peret B., Yang Y., Parry G., Nielsen E., De Smet I., Vanneste S., Levesque M.P., Carrier D., James N., Calvo V., Ljung K., Kramer E., Roberts R., Graham N., Marillonnet S., Patel K., Jones J.D.G., Taylor C.G., Schachtman D.P., May S., Sandberg G., Benfey P., Friml J., Kerr I., Beeckman T., Laplaze L., Bennett M.J. The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 2008;10:946–954. doi: 10.1038/ncb1754. PubMed DOI

Dhonukshe P., Tanaka H., Goh T., Ebine K., Mahonen A.P., Prasad K., Blilou I., Geldner N., Xu J., Uemura T., Chory J., Ueda T., Nakano A., Scheres B., Friml J. Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions. Nature. 2008;456:962–975. doi: 10.1038/nature07409. PubMed DOI PMC

De Smet I., Vassileva V., De Rybel B., Levesque M.P., Grunewald W., Van Damme D., Van Noorden G., Naudts M., Van Isterdael G., De Clercq R., Wang J.Y., Meuli N., Vanneste S., Friml J., Hilson P., Jurgens G., Ingram G.C., Inze D., Benfey P.N., Beeckman T. Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science. 2008;322:594–597. doi: 10.1126/science.1160158. PubMed DOI

Barendse G.W.M., Peeters T.J.M. Multiple hormonal-control in plants. Acta Bot. Neerl. 1995;44:3–17. doi: 10.1111/j.1438-8677.1995.tb00764.x. DOI

Weyers J.D.B., Paterson N.W. Plant hormones and the control of physiological processes. New Phytol. 2001;152:375–407. doi: 10.1046/j.0028-646X.2001.00281.x. PubMed DOI

Sakakibara H. Cytokinins: Activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 2006;57:431–449. doi: 10.1146/annurev.arplant.57.032905.105231. PubMed DOI

Tarkowski P., Dolezal K., Strnad M. Analytical methods in cytokinin research. Chem. Listy. 2004;98:834–841.

Karadeniz A., Topcuoglu S.F., Inan S. Auxin, gibberellin, cytokinin and abscisic acid production in some bacteria. World J. Microbiol. Biotechnol. 2006;22:1061–1064. doi: 10.1007/s11274-005-4561-1. DOI

Tuomi T., Rosenqvist H. Detection of abscisic, gibberellic and indole-3-acetic-acid from plants and microbes. Plant Physiol. Biochem. 1995;33:725–734.

Gomez-Cadenas A., Pozo O.J., Garcia-Augustin P., Sancho J.V. Direct analysis of abscisic acid in crude plant extracts by liquid chromatography-electrospray/tandem mass spectrometry. Phytochem. Anal. 2002;13:228–234. doi: 10.1002/pca.640. PubMed DOI

Prinsen E., Redig P., Vandongen W., Esmans E.L., Vanonckelen H.A. Quantitative-analysis of cytokinins by electrospray tandem mass-spectrometry. Rapid Commun. Mass Spectrom. 1995;9:948–953. doi: 10.1002/rcm.1290091016. DOI

Ge L., Yong J.W.H., Goh N.K., Chia L.S., Tan S.N., Ong E.S. Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography-tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis. J. Chromatogr. B. 2005;829:26–34. doi: 10.1016/j.jchromb.2005.09.026. PubMed DOI

Hahn H. High-performance liquid-chromatography and its use in cytokinin determination in Agrobacterium-tumefaciens B6. Plant Cell Physiol. 1976;17:1053–1058.

Lebuhn M., Hartmann A. Method for the determination of indole-3-acetic-acid and related-compounds of L-tryptophan catabolism in soils. J. Chromatogr. 1993;629:255–266. doi: 10.1016/0021-9673(93)87039-O. DOI

Nordstrom A., Tarkowski P., Tarkowska D., Dolezal K., Astot C., Sandberg G., Moritz T. Derivatization for LC electrospray ionization-MS: A tool for improving reversed-phase separation and ESI responses of bases, ribosides, and intact nucleotides. Anal. Chem. 2004;76:2869–2877. doi: 10.1021/ac0499017. PubMed DOI

Hernandez P., Paton F., Hernandez L. Voltammetric study of kinetin in a carbon fiber microelectrode. Determination by adsorptive stripping in apple extracts. Electroanalysis. 1997;9:1372–1374. doi: 10.1002/elan.1140091714. DOI

Novak O., Tarkowski P., Tarkowska D., Dolezal K., Lenobel R., Strnad M. Quantitative analysis of cytokinins in plants by liquid chromatography-single-quadrupole mass spectrometry. Anal. Chim. Acta. 2003;480:207–218. doi: 10.1016/S0003-2670(03)00025-4. DOI

Chiwocha S., Rouault G., Abrams S., von Aderkas P. Parasitism of seed of Douglas fir (Pseudotsuga menziesii) by the seed chalcid, Megastigmus spermotrophus, and its influence on seed hormone physiology. Sex. Plant Reprod. 2007;20:19–25. doi: 10.1007/s00497-006-0039-z. DOI

Chiwocha S.D.S., Abrams S.R., Ambrose S.J., Cutler A.J., Loewen M., Ross A.R.S., Kermode A.R. A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J. 2003;35:405–417. doi: 10.1046/j.1365-313X.2003.01800.x. PubMed DOI

Chiwocha S.D.S., Cutler A.J., Abrams S.R., Ambrose S.J., Yang J., Ross A.R.S., Kermode A.R. The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination. Plant J. 2005;42:35–48. doi: 10.1111/j.1365-313X.2005.02359.x. PubMed DOI

Ge L.Y., Tan S., Yong J.W.H., Tan S.N. CE for cytokinin analyses: A review. Electrophoresis. 2006;27:4779–4791. doi: 10.1002/elps.200600195. PubMed DOI

Zheng B., Yang H.X., He J.L. Quantitative analysis of plant hormones with capillary electrophoresis. Chin. J. Anal. Chem. 1999;27:704–707.

Liu J., Li S.F.Y. Separation and determination of auxins by capillary electrophoresis. J. Liq. Chromatogr. Relat. Technol. 1996;19:1697–1713. doi: 10.1080/10826079608013998. DOI

Olsson J.C., Andersson P.E., Karlberg B., Nordstrom A.C. Determination of plant indoles by capillary electrophoresis with amperometric detection. J. Chromatogr. A. 1996;755:289–298. doi: 10.1016/S0021-9673(96)00599-7. DOI

Hoyerova K., Gaudinova A., Malbeck J., Dobrev P.I., Kocabek T., Solcova B., Travnickova A., Kaminek M. Efficiency of different methods of extraction and purification of cytokinins. Phytochemistry. 2006;67:1151–1159. doi: 10.1016/j.phytochem.2006.03.010. PubMed DOI

Lopez-Carbonell M., Gabasa M., Jáuregui O. Enhanced determination of abscisic acid (ABA) and abscisic acid glucose ester (ABA-GE) in Cistus albidus plants by liquid chromatography-mass spectrometry in tandem mode. Plant Physiol. Biochem. 2009;47:256–261. doi: 10.1016/j.plaphy.2008.12.016. PubMed DOI

Owen S.J., Abrams S.R. Measurement of plant hormones by liquid chromatography-mass spectrometry. Methods Mol. Biol. 2009;495:39–51. PubMed

Gazdik Z., Reznicek V., Adam V., Zitka O., Jurikova T., Krska B., Matuskovic J., Plsek J., Saloun J., Horna A., Kizek R. Use of liquid chromatography with electrochemical detection for the determination of antioxidants in less common fruits. Molecules. 2008;13:2823–2836. doi: 10.3390/molecules131102823. PubMed DOI PMC

Petrlova J., Mikelova R., Stejskal K., Kleckerova A., Zitka O., Petrek J., Havel L., Zehnalek J., Adam V., Trnkova L., Kizek R. Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with Coul-Array detection. J. Sep. Sci. 2005;29:1166–1173. doi: 10.1002/jssc.200500425. PubMed DOI

Potesil D., Petrlova J., Adam V., Vacek J., Klejdus B., Zehnalek J., Trnkova L., Havel L., Kizek R. Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione, and phytochelatin in maize (Zea mays L.) kernels using high-performance liquid chromatography with electrochemical detection. J. Chromatogr. A. 2004;1084:134–144. doi: 10.1016/j.chroma.2005.06.019. PubMed DOI

Klejdus B., Petrlova J., Potesil D., Adam V., Mikelova R., Vacek J., Kizek R., Kuban V. Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection. Anal. Chim. Acta. 2003;520:57–67. doi: 10.1016/j.aca.2004.02.027. DOI

Klejdus B., Vacek J., Adam V., Zehnalek J., Kizek R., Trnkova L., Kuban V. Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection. J. Chromatogr. B. 2004;806:101–111. doi: 10.1016/j.jchromb.2004.03.044. PubMed DOI

Grill E., Winnacker E.L., Zenk M.H. Phytochelatins - the principal heavy-metal complexing peptides of higher-plants. Science. 1985;230:674–676. doi: 10.1126/science.230.4726.674. PubMed DOI

Zenk M.H. Heavy metal detoxification in higher plants - A review. Gene. 1995;179:21–30. doi: 10.1016/S0378-1119(96)00422-2. PubMed DOI

Cobbett C., Goldsbrough P. Phytochelatins and metallothioneins: Roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 2002;53:159–182. doi: 10.1146/annurev.arplant.53.100301.135154. PubMed DOI

Hall J.L. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 2002;53:1–11. doi: 10.1093/jexbot/53.366.1. PubMed DOI

Babula P., Adam V., Opatrilova R., Zehnalek J., Havel L., Kizek R. Uncommon heavy metals, metalloids and their plant toxicity: a review. Environ. Chem. Lett. 2008;6:189–213. doi: 10.1007/s10311-008-0159-9. DOI

Diopan V., Shestivska V., Adam V., Macek T., Mackova M., Havel L., Kizek R. Determination of content of metallothionein and low molecular mass stress peptides in transgenic tobacco plants. Plant Cell Tissue Organ Cult. 2007;94:291–298. doi: 10.1007/s11240-008-9356-2. DOI

Supalkova V., Huska D., Diopan V., Hanustiak P., Zitka O., Stejskal K., Baloun J., Pikula J., Havel L., Zehnalek J., Adam V., Trnkova L., Beklova M., Kizek R. Electroanalysis of plant thiols. Sensors. 2007;7:932–959. doi: 10.3390/s7060932. DOI

Klejdus B., Zehnalek J., Adam V., Petrek J., Kizek R., Vacek J., Trnkova L., Rozik R., Havel L., Kuban V. Sub-picomole high-performance liquid chromatographic/mass spectrometric determination of glutathione in the maize (Zea mays L.) kernels exposed to cadmium. Anal. Chim. Acta. 2003;520:117–124. doi: 10.1016/j.aca.2004.02.060. DOI

Causon R. Validation of chromatographic methods in biomedical analysis - Viewpoint and discussion. J. Chromatogr. B. 1996;689:175–180. doi: 10.1016/S0378-4347(96)00297-6. PubMed DOI

Bugianesi R., Serafini M., Simone F., Wu D.Y., Meydani S., Ferro-Luzzi A., Azzini E., Maiani G. High-performance liquid chromatography with coulometric electrode array detector for the determination of quercetin levels in cells of the immune system. Anal. Biochem. 2000;284:296–300. doi: 10.1006/abio.2000.4697. PubMed DOI

Long G.L., Winefordner J.D. Limit of detection. Anal. Chem. 1983;55:A712–A724.

Annesley T.M. Ion suppression in mass spectrometry. Clin. Chem. 2003;49:1041–1044. doi: 10.1373/49.7.1041. PubMed DOI