Analysis of ingenol and its conjugates in some species of the Euphorbia genus by ultra-high performance liquid chromatography-tandem mass spectrometry using isotope dilution method

Ingenol is a chemically and biologically important plant-derived diterpenoid compound, first isolated in 1968 from the seed oil of a leafy shrub Croton tiglium belonging to the Euphorbiaceae plant family. Ingenol is the key precursor for the synthesis of ingenol mebutate (angelate), used to treat actinic keratosis. Although, its total synthesis was published lately, isolation from natural raw material is still used for its industrial scale production. Therefore, a rich source of ingenol seems to be of great economic importance. We have developed an ultra-high performance liquid chromatography-tandem mass spectrometry method for screening of ingenol in 38 species of the Euphorbia genus. The highest ingenol concentration (547 mg.kg^-1 of dry weight) was found in the lower leafless stems of E. myrsinites. Moreover, three ingenol conjugates (two known: 3-O-deca(2'Z,4'E)-dienoyl ingenol, 20-O-acetyl-3-O-deca(2'Z, 4'E)-dienoyl ingenol and one new: 3-O-octa (2'Z,4'E)-dienoyl ingenol) were successfully identified in E. myrsinites by means of accurate mass determination.