Journal of Chromatography A, 1319 (2013) 166–171 Contents lists available at ScienceDirect Journal of Chromatography A j our nal homep age: www.elsevier.com/locate/chroma Short communication Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby Mariana N. Vieira a,b,∗ , Suzana G. Leitão a , Paula C.C. Porto a , Danilo R. Oliveira a , Shaft Corrêa Pinto a,c , Raimundo Braz-Filho d , Gilda G. Leitão e a Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, CCS, Bl. A 2 o andar, Ilha do Fundão 21941-590, RJ, Brazil b Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany c Curso de Farmácia/Campus UFRJ-Macaé, Rua Aluisio da Silva Gomes, 50, Granja dos Cavaleiros, Macaé 27930-560, RJ, Brazil d Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes 28013-602, RJ, Brazil e Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, CCS, Bl. H, Ilha do Fundão 21941-590, RJ, Brazil a r t i c l e i n f o Article history: Received 22 May 2013 Received in revised form 10 October 2013 Accepted 12 October 2013 Available online 22 October 2013 Keywords: Indole alkaloids Aspidosperma pH-zone-refining countercurrent chromatography a b s t r a c t Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200 mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3- aricine (circa 17 mg), isoreserpiline (ca. 22 mg) and 3-reserpiline (ca. 40 mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2 h. Two of them – 3-aricine and isoreserpiline – were isolated and identified for the first time in this species. © 2013 Elsevier B.V. All rights reserved. 1. Introduction High-speed countercurrent chromatography (HSCCC) is an hydrodynamic preparative technique based on the distribution coefficient (K) of substances between the two phases of a biphasic solvent system, where one of them is the stationary phase and the other acts as mobile phase [1,2]. Specially for the case of ionizable molecules like organic acids and bases, a method proposed by Ito [3,4] allows the separation process also according to pK a values and hydrophobicity of the substances. For the separation of alka- line organic compounds, this method consists on the addition of a basic retainer to the stationary phase and an acidic eluter to the mobile phase [4]. pH-Zone refining CCC shows some advantages when compared to conventional CCC, for example the increase of the sample loading capacity, the high concentration of the fractions and the possibil- ity of monitoring the analysis by measuring the pH value of each ∗ Corresponding author. Tel.: +49 17656523379; fax: +49 55 21 2562 6413. E-mail addresses: mnvieira87@gmail.com, m.neves-vieira@tu-bs.de (M.N. Vieira). fraction collected. On the other hand, one obvious disadvantage is that the analyte must be ionic (or ionizable) [4]. Furthermore, such approach in does not allow continuous operation, due to the fact that the system has to be re-established again after each run. Nevertheless, this method was successfully applied to the separa- tion of several types of alkaloids [5–8], including those of the indole type, which are also the major components of Aspidosperma rigidum Rusby [9,10]. Species of Aspidosperma genus (Apocynaceae) are generally trees found in Central and South America, commonly known in the North of Brazil as “Carapanaúba”, which means mosquito’s tree [11]. In this region, the teas made from its barks are popularly used to treat several diseases [12–14]. To date, only nine alkaloids were described for A. rigidum. extracts: 3-reserpiline, burnamine, picraline, caboxine A, caboxine B, isocaboxine, (-) carapanaubine, isocarapanaubine and haplocidine [10,15]. Based on the ethnopharmacological background and on the lack of recent studies about this plant, the aim of the present work was to apply the modern technique of CCC in the pH- zone refining mode for the separation of the components present in the dichloromethane extract of Carapanaúba (A. rigidum Rusby). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.044