Simultaneous Determination of Indolic Compounds in Plant Extracts by Solid-Phase Extraction and High-Performance Liquid
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Simultaneous Determination of Indolic Compounds in Plant Extracts by Solid-Phase Extraction and High-Performance Liquid Chromatography with UV and Fluorescence Detection Tadeusz Pilipczuk & Natalia Dawidowska & Barbara Kusznierewicz & Jacek Namieśnik & Agnieszka Bartoszek
Received: 24 November 2014 / Accepted: 20 January 2015 # Springer Science+Business Media New York 2015
Abstract A high-performance liquid chromatographic method with UV and fluorescence detection (HPLC-DAD-FLD) was developed for simultaneous determination of indolic compounds in plant material. Indole-3-carbinol (I3C), indole-3-acetic acid (I3AA), indole-3-acetonitrile (I3ACN), and 3,3′-diindolylmethane (DIM) were used as representative compounds that cover a wide spectrum of indole structures occurring in nature. For concentration and purification of the analytes, a solid-phase extraction (SPE) pretreatment was used. The separation utilized a Zorbax XDB-C8 reversed-phase column, acetonitrile-water containing 0.01 % formic acid gradient and UV (280 nm) in series with fluorescence (ex. 280 nm; em. 360 nm) detection. Good linearities of calibration curves were found within the ranges of 0.1– 100 nmol/mL for I3C and DIM, 0.4–100 nmol/mL for I3AA and I3ACN for UV detection and 0.003–10 nmol/mL for I3C, 0.003–5 nmol/mL for DIM, and 0.02–10 nmol/mL for I3AA and I3ACN for fluorescence detection. The lowest detection limits (LOD) for UV detection were 0.03 nmol/mL for I3C, 0.1 nmol/mL for I3AA and I3ACN, and 0.04 nmol/mL for DIM, while for fluorescence detection were 0.001 nmol/mL for I3C and DIM, and 0.006 nmol/mL for I3AA and I3ACN. The usefulness of HPLC-DAD-FLD method with SPE
T. Pilipczuk (*) : N. Dawidowska : B. Kusznierewicz : A. Bartoszek Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdansk University of Technology, G. Narutowicza 11/12, 80-233 Gdansk, Poland e-mail: [email protected] J. Namieśnik Department of Analytical Chemistry, Faculty of Chemistry, Gdansk University of Technology, G. Narutowicza 11/12, 80-233 Gdansk, Poland
pretreatment is illustrated by the analysis of indolic compounds in extracts from different parts of Brassica vegetables. Keywords Indolic compounds . HPLC-DAD-FLD . SPE . Savoy cabbage . Brussels sprouts . Auxins
Introduction Indolic compounds are widespread throughout the plant kingdom. Plants can synthesize indoles by several independent biosynthetic pathways. Most of them start from tryptophan resulting in such secondary metabolites as indole-3-pyruvic acid, tryptamine, indole-3-acetaldoxime, indole-3-acetamide, or indolic glucosinolates (Kobayashi et al. 1993; Mashiguchi et al. 2011; Sugawara et al. 2009). There are also pathways independent of tryptophan in which biosynthesis of indole-3acetic acid (I3AA) begins with indole-3-glycerol phosphate (Ouyang et al. 2000; Zhang et al. 2008) or indole (Mano and Nemoto 2012; Sitbon et al. 2000). Diversified structures and physicochemical properties of indoles are behind their varied biological activities. Indolic phytohormon
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