Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra‐high Performance Liquid Chromatography Tandem Mass Spectrometry

Béres T., Dragull K., Pospíšil J., Tarkowská D., Dančák M., Bíba O., Tarkowski P., Doležal K., Strnad M.

Keywords: ingenol; ultra‐high performance liquid chromatography; mass spectrometry; isotope‐dilution method; Euphorbia genus
Abstract: Introduction – Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol‐3‐angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. Objective – To develop a rapid and reliable method for quantification of ingenol in various plant extracts. Methodology – Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra‐high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) after methanolysis and solid‐phase extraction (SPE) purification. The 18O–labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. Results – The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. Conclusion – The newly established UHPLC–MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation.
DOI: 10.1002/pca.2711 IEB authors: Karel Doležal, Jiří Pospíšil, Miroslav Strnad, Danuše Tarkowská