Journal of Chromatography A, 1100 (2005) 116–125 Batch immunoextraction method for efficient purification of aromatic cytokinins Eva Hauserov´ a a , Jana Swaczynov´ a a , Karel Doleˇ zal a,∗ , Ren´ e Lenobel a , Igor Popa a , Mari´ an Hajd ´ uch b , David Vydra b , Kvˇ etoslava Fuksov´ a c , Miroslav Strnad a a Laboratory of Growth Regulators, Palack´ y University and Institute of Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc, Czech Republic b Laboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine, Palacky University and University Hospital in Olomouc, Puskinova 6, 775 20 Olomouc, Czech Republic c Institute of Nuclear Medicine, The First Faculty of Medicine, Charles University, CZ 212 08 Prague, Czech Republic Received 22 June 2005; received in revised form 8 September 2005; accepted 9 September 2005 Available online 26 September 2005 Abstract A range of benzylaminopurines naturally occur in plants and exhibit high biological activity. Others have been synthesized, such as 6-(2-hydroxy- 3-methoxybenzylamino)purine riboside (2OH3MeOBAPR), which has shown interesting anti-cancer activity under in vitro conditions. In order to study the biological activity of this interesting compound in more detail, a rapid and highly efficient method for its purification from complex samples (e.g. blood and plant extracts) is needed. Therefore, we prepared monoclonal antibodies against 2OH3MeOBAPR. The antibody had undetectable cross-reactivity with all natural isoprenoid cytokinins, but relatively high cross-reactivity with aromatic cytokinins as well as some synthetic di- and tri-substituted 6-benzylaminopurines and the corresponding ribosides. The antibody also showed strong responses and specificity in enzyme-linked immunoassays (ELISAs). In addition, it was used to prepare, for the first time, an immunoaffinity sorbent with high specificity and capacity for aromatic cytokinins. A batch immunoextraction method was then developed and optimized for the purification of 2OH3MeOBAPR from murine blood samples. The high efficacy and simplicity of this method (in off-line combination with HPLC-MS) for the isolation of target analytes from biological material is demonstrated in this study. © 2005 Elsevier B.V. All rights reserved. Keywords: Antibody; 6-Benzylaminopurine; Batch immunoextraction 1. Introduction Cytokinins are an important group of plant growth regula- tory substances [1,2]. They promote, in the presence of auxin, cell division in plant tissue cultures and affect a wide range of biological processes, including seed germination, bud differ- entiation, branching, chlorophyll and starch production, plant- pathogen resistance, apical dominance and leaf senescence. These compounds are derived from adenine, substituted at the N 6 -position with an isoprenoid or aromatic side-chain. They mainly occur endogenously as free bases, nucleosides, gluco- sides and nucleotides, and are often present at very low concen- trations (pmol g -1 fresh weight). Moreover, some of the cytokinin-derived compounds are specific inhibitors of cyclin-dependent kinases [3] and exhibit ∗ Corresponding author. Tel.: +420 585634940. E-mail address: dolezal@risc.upol.cz (K. Doleˇ zal). interesting therapeutic effects against various types of diseases, especially cancer [4,5]. In our laboratory, more than 40 deriva- tives of 6-benzyladenosine, with various substituents on the benzyl ring, have been recently prepared, characterized and tested in different plant and animal systems [6]. To study the most promising compounds in more detail, a rapid and highly efficient method for purifying them from complex samples (e.g. blood and plant extracts) is urgently needed. It is known that immunoaffinity purification methods, which are based on antibody-antigen interactions, can be highly spe- cific for their respective target analytes [7,8]. This approach can provide selective sample enrichment, and thus greatly enhance detection limits of trace analyses. Off-line configurations for such methods include immunofiltration with non-immobilized antibodies or membrane strips with antibody (or antigen)- immobilized zones and immunoaffinity extraction using diverse types of immunosorbents. For immunoaffinity-based extrac- tions, the immunosorbent is often packed into a disposable 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.09.020