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]-2 H-indol-2-one (indolinone) (3), indirubin (4), alpha-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 alpha-linolenic acid. The method was applied to the analysis of woad extracts obtained by supercritical fluid (SFE) CO2 extraction, and by pressurized liquid extraction (PLE) with dichloromethane and methanol, respectively. While the highest concentration of alpha-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 alpha-linolenic acid (0.39%) were present in this extract.

FT‐Raman spectroscopy—a rapid and reliable quantification protocol for the determination of natural indigo dye in Polygonum tinctorium

“…This discrepancy might be the result of the complex and extensive sample preparation necessary for HPLC-UV analysis of the plant material. [25] Because of the strong absorption patterns of chlorophyll at the used wavelength for UVdetection (606 nm), it is hardly necessary to separate it prior to the indigo analysis. Therefore, ASE with methanol was applied before indigo extraction with ethyl acetate.…”

A comprehensive metabolite profiling of Isatis tinctoria leaf extracts

“…Negative aspects of compound interaction in plant extracts 29 2.3 The role of plant extracts in the pharmacopoeia, and aspects of quality control 31 Definitions by the European Pharmacopoeia 31…”

Development and full validation of an UPLC-MS/MS method for the determination of an anti-allergic indolinone derivative in rat plasma, and application to a preliminary pharmacokinetic study

“…A HPLC-ESIMS assay for quantification of indolinone in Isatis extracts has been previously reported by us [13], but this method was not applicable to the dosage in biological fluids. We here describe the development and validation, according to FDA guidance for industry [14,15], of an UPLC-MS/MS assay for dosage of indolinone in lithium heparinized rat plasma, and its application in a preliminary pharmacokinetic study in Sprague-Dawley male rats (2 mg/kg BW i.v.…”