Efficiency of different methods of extraction and purification of cytokinins Kla ´ra Hoyerova ´ a , Alena Gaudinova ´ a , Jir ˇı ´ Malbeck a , Petre I. Dobrev a , Toma ´s ˇ Koca ´bek b , Blanka S ˇ olcova ´ a , Alena Tra ´vnı ´c ˇkova ´ a , Miroslav Kamı ´nek a, * a Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova ´ 135, CZ-165 02 Praha 6, Czech Republic b Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Branis ˇovska ´ 31, CZ-370 05 C ˇ eske ´ Bude ˇjovice, Czech Republic Received 2 December 2005; received in revised form 6 March 2006 Available online 5 May 2006 Abstract The increasing use of advanced methods, such as mass spectrometry, for the determination of cytokinins has raised special require- ments for the extraction and purification of this class of plant hormones. Extraction of Arabidopsis thaliana plants with three different solvents, [80% (v/v) MeOH, Bieleski’s MCF-7, and modified Bieleski’s] provided similar yields of most analyzed cytokinins determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). However, the extraction with a modified Biel- eski’s solvent (MeOH–HCO 2 H–H 2 O [15:1:4, v/v/v]) gave the highest responses of deuterated cytokinins (used as test compounds) in plant extracts as compared to the responses of pure deuterated standards (relative internal standard response, RISR). Purification of cytokinins using Oasis MCX sorbent with reversed-phase and cation-exchange characteristics, in comparison to the DEAE Sephadex RP-C 18 method, provided higher levels of zeatin riboside monophosphate and similar levels of cytokinin bases, ribosides and glucosides. Using this method the content of UV-absorbing contaminates was decreased by about 90% and the RISR values of all tested cytokinin standards but riboside monophosphates were increased about two-fold. The former method provided preparations more suitable for HPLC/MS/MS analysis with respect to simplicity and sample purity. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Arabidopsis thaliana; Cytokinin extraction; Cytokinin purification; Plant hormones; Solid-phase extraction; HPLC/MS/MS 1. Introduction Cytokinins are a class of plant hormones that in coop- eration with auxin play unique role in the control of developmental processes in plants such as cell division and differentiation, formation and growth of roots and shoots, apical dominance and senescence. Natural cytoki- nins are 6-N-substituted purine derivatives. Those which occur in plants as free bases are supposed to be the biologically active compounds. Glycosidic conjugates of cytokinins are transport, storage or inactivated forms of cytokinins; while cytokinin riboside phosphates predomi- nantly represent the primary products of cytokinin biosynthesis. More than 40 natural cytokinins have been identified in plant tissues (Zaz ˇı ´malova ´ et al., 1999). The structures of those cytokinins analyzed in the present report are shown in Fig. 1. Their occurrence in minute amounts in non-transformed plants (<10 8 M), in the presence of structurally related compounds and enzymes catalyzing metabolic conversions and the degradation of cytokinins (Mok and Mok, 2001), complicates both their purification and determination (Horgan and Scott, 1987; Jones et al., 1996). Increasing use of mass spectrometry for the detection and determination of a wide spectrum of cytokinins requires both the operational as well as dependable extraction and purification techniques which prevent metabolic conversions during extraction, and also provide samples of sufficient purity for mass spectrometry. 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.03.010 * Corresponding author. Tel.: +420 2 20390445; fax: +420 2 20390446. E-mail address: kaminek@ueb.cas.cz (M. Kamı ´nek). www.elsevier.com/locate/phytochem Phytochemistry 67 (2006) 1151–1159 PHYTOCHEMISTRY