Available online at www.sciencedirect.com Journal of Chromatography A, 1190 (2008) 63–73 Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography Gerold Jerz a , Tanja Skotzki a , Kathrin Fiege a , Peter Winterhalter a , Slawomir Wybraniec b,∗ a Institute of Food Chemistry, Technische Universit¨ at Braunschweig, Schleinitz-Strasse 20, 38106 Braunschweig, Germany b Faculty of Analytical Chemistry, Institute C-1, Department of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland Received 4 December 2007; received in revised form 19 February 2008; accepted 21 February 2008 Available online 5 March 2008 Abstract The first preparative fractionation of betalain pigments by means of ion-pair high-speed counter-current chromatography (IP-HSCCC) from berry extracts of Phytolacca americana (Phytolaccaceae) is presented. A novel HSCCC solvent system consisting of 1-butanol–acetonitrile–water (5:1:6, v/v/v) was applied using ion-pair forming trifluoroacetic acid at low concentration (0.7%, v/v). Affinity of polar betacyanins and betaxanthins to the organic stationary phase of the biphasic HSCCC solvent mixture was considerably improved. Partitioning coefficient values and influence of increasing trifluoroacetic acid additions to the biphasic solvent mixture were measured for all identified betacyanins and betaxanthins. Gentle separation by IP-HSCCC of the injected pigment extract (900 mg) yielded sufficient amounts of the principal pigments 15S-betanin/15R-isobetanin. The pure epimers separated by C 18 -HPLC were immediately studied by one- and two-dimensional NMR. In the recovered fractions, minor concentrated betacyanins and betaxanthins were significantly enriched by IP-HSCCC and were detected for the first time in the extracts of P. americana. IP-HSCCC and C 18 -HPLC were shown to be complementary techniques in the isolation procedure of recovering minor concentrated, highly polar and chemically instable betacyanins and betaxanthin from complex plant matrices. Altogether, identification of 17 betalains was achieved by HPLC-diode array detection-electrospray ionization MS/MS in the HSCCC fractions with their respective isomers, also resulting in the tentative elucidation of betacyanins with novel salicylic acid substitution pattern in the berry extracts of P. americana. © 2008 Elsevier B.V. All rights reserved. Keywords: Betanin; Betalains; Betacyanins; Betaxanthins; Ion-pair counter-current chromatography; IP-HSCCC; HPLC-DAD-ESI-MS/MS; NMR; Phytolacca americana; Phytolaccaceae 1. Introduction High-speed counter-current chromatography (HSCCC) has been proved to be a very useful technique for preparative iso- lation of natural compounds from plant extracts [1,2]. In this study, a separation of betalains, from the extracts of dark violet pokeberry fruits – Phytolacca americana – (Phytolaccaceae) is presented. Because of high levels of saponins, the Phytolacca species were proposed as biological molluscicides for the control of bilharziosis transmitting snails in Ethiopia [3,4]. On the other hand, betalains, having strong antioxidant properties, are arising ∗ Corresponding author. Tel.: +48 12 628 2707; fax: +48 12 628 2036. E-mail address: swybran@chemia.pk.edu.pl (S. Wybraniec). as interesting chemopreventive natural compounds [5–8]. There- fore, in connection to their good colorant properties, a growing interest in betalains has been noticed during the past decade [9,10] and some of these compounds have not been identified yet. Betalains (red-violet betacyanins and yellow-orange betax- anthins) are water soluble plant pigments. Betacyanins can be considered as condensation products of betalamic acid with cyclodopa or its O-glycosylated (in most cases 5- O-glucosylated) derivatives. Frequent esterification of the O-glycosides with acids such as ferulic, p-coumaric or malonic acid was also confirmed [11,12]. Closely related betaxanthins are condensation products of betalamic acid with amines or amino acids. Betalains exist in two diastereomeric forms dif- fering by the configuration of the C-15 carbon (Fig. 1) [11,12]. 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.093