Simultaneous Determination of Indolic Compounds in Plant Extracts by Solid-Phase Extraction and High-Performance Liquid Chromatography with UV and Fluorescence Detection

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 pretreatment is illustrated by the analysis of indolic compounds in extracts from different parts of Brassica vegetables.