AUTHENTICATION OF PLANT MATERIAL BY FINGERPRINT ANALYSIS 123 Copyright © 2006 John Wiley & Sons, Ltd. Phytochem. Anal. 18: 123–132 (2007) DOI: 10.1002.pca Phytochemical Analysis Phytochem. Anal. 18: 123–132 (2007) Published online 15 December 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pca.960 A New Method for the Authentication of Plant Samples by Analyzing Fingerprint Chromatograms MARKO OBRADOVIÇ, 1 * SIMONA STRGULC KRAJSEK, 2 MARINA DERMASTIA 2 and SAMO KREFT 1 1 Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000 Ljubljana, Slovenia 2 Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 100, SI-1000 Ljubljana, Slovenia Received 12 April 2005; Revised 24 August 2006; Accepted 30 August 2006 Abstract: Chemical analysis by high-performance liquid chromatography or capillary electrophoresis of plant pulverized samples, juices or extracts is an excellent method for the authentication of medicinal plant species and their products, particularly when morphological authentication is not possible. In the conventional procedure, chromatograms are integrated and the heights or areas of several peaks are used in a supervised pattern recognition method to confirm the authenticity of the product. We propose a new section approach in analysing chromatograms, where chromatograms are split into sections, which are described by four variables (number of peaks in the section, average retention time of peaks in the section, total area of peaks in the section and average area of peaks in the section), and these variables are then used in statistical analysis. The method is especially useful when the peaks on the chromatogram are not well separated and it is not easy to link individual peaks on one chromatogram with corresponding peaks on other chromatograms. In comparison with the standard procedure, our approach in analyzing chromato- graphic data of willow-herb (Epilobium and Chamaenerion spp.) extracts was more objective, gave better results and was also easier to perform. Copyright © 2006 John Wiley & Sons, Ltd. Keywords: Capillary electrophoresis; chromatogram; plant sample authentication; multivariate analysis; Epilobium; Chamaenerion. Phytochemical Analysis * Correspondence to: M. Obradoviç, Faculty of Pharmacy, University of Ljubljana, Askerçeva 7, SI-1000 Ljubljana, Slovenia. E-mail: marko.obradovic@ffa,uni-lj.si INTRODUCTION A reliable pharmacological or clinical study must employ well-authenticated plant material. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE) or thin-layer chromatography (TLC) are the most commonly used analytical methods for the chemical authentication of medicinal products (Barakat et al., 1997; Blanquer et al., 1998; Schaneberg et al., 2003; Xie et al. 2006), food products (Goodall et al., 1995; Gonzáles et al., 2001; Alonso-Salces et al., 2004) and in the manufacturer of an individual product (Welsh et al., 1996). If the peaks on chromatograms are well separated and retention times are very reproducible, such that corresponding peaks on different chromato- grams unambiguously represent the same substance, a plant sample can be authenticated by a chemical fingerprinting method based on the presence or ab- sence of a limited number of peaks (Schaneberg et al., 2003). In such cases no statistical procedure is required and it is not necessary for the substances represented by these peaks to be known. For the differ- entiation of a large number of similar samples by multivariate statistical analysis, discriminant analysis was found to be efficient (Goodall et al., 1995; Gonzáles et al., 2001). In this approach a large number of variables (peaks) are required. As with the limited peak number method, there must be no doubt that an individual peak on a chromatogram represents the same substance as the equivalent peak on another chromatogram, but again, the substances represented by these peaks need not be known. Unambiguous identification of all peaks in all chromatograms can be a difficult task, even using retention times, absorption spectra and multiple internal standards. There is always some bias involved in the selection of peaks. Frequently retention times can vary by perhaps up to 1 min, which is sometimes equal to the distance between peaks (Goodall et al., 1995). Willow-herbs (Epilobium), which are frequently used in folk medicine, were used in the present study as model plants. Pharmacological studies of willow-herb extracts have confirmed their antimicrobial (Battinelli et al., 2001), anti-inflammatory (Hiermann et al., 1991), analgesic (Tita et al., 2001) and anti-tumour (Voynova et al., 1991 ) activities. The retarding effect of willow- herbs on the growth of epithelial cells of human prostate was also proven (Ducrey et al., 1997; Vitalone et al., 2001). The 16 willow-herb species growing in Slovenia are members of the genera Chamaenerion and Epilobium (Ravnik, 1999; Strgulc Krajsek and Jogan, 2006). The genus Chamaenerion comprises species C. angustifolium and C. palustre. The genus Epilobium is divided into two sections (Haussknecht, 1884): section Schizostigma comprising species E. hirsutum, E. parviflorum, E. montanum and E. collinum; and