Quantification of Active Principles and Pigments in Leaf Extracts of Isatis tinctoria by HPLC/UV/MS

Abstract An HPLC method has been developed and validated for the quantification of the pharmacologically active principles tryptanthrin (1), 1,3-dihydro-3-[(4-hydroxy-3,5-dimethoxyphenyl)methylene]-2H-indol-2-one (indolinone) (3), indirubin (4), α-linolenic acid (2), and indigo (5), an isomer of indirubin, in extracts from the traditional anti-inflammatory plant ISATIS TINCTORIA (woad). The chromatographic separation was performed on a C-18 column with a linear gradient of acetonitrile in water containing 0.1 % formic acid. The method combines UV and electrospray MS detection in the positive ion mode for the detection of the alkaloids, with a switch to the negative mode for the analysis of α-linolenic acid. The method was applied to the analysis of woad extracts obtained by supercritical fluid (SFE) CO 2 extraction, and by pressurized liquid extraction (PLE) with dichloromethane and methanol, respectively. While the highest concentration of α-linolenic acid was found in the SFE extract (7.43 %), the concentrations of tryptanthrin , indolinone, indirubin and indigo were the highest in the dichloromethane extract (0.30, 0.035, 2.48 and 0.84 %, respectively). Compound 3 was not detected in the methanolic extract and only traces of compounds 1, 4 and 5 and low amount of α-linolenic acid (0.39 %) were present in this extract.