Pyrrolizidine alkaloids (PAs) are potentially carcinogenic plant metabolites. They occur mainly in plants of the Boraginaceae, Asteraceae and Fabaceae families. They contain a pyrrolizidine core and make up a large group of heterocyclic alkaloids mainly derived from the 4 Necin bases platynecine, retronecine, heliotridin and ontonecin. PAs are hepatotoxic if they carry a 1,2-double bond as well as an esterified side chain which is a structural prerequisite for their hepatic activation. Exposure to PAs in food, beverages or phytopharmaceuticals, is a possible long-term concern for human health. Based on available data the Panel on Contaminants in the Food Chain (CONTAM) have proposed a list of PAs to be monitored in foodstuffs. Since some of the analytes are isomers that can’t be distinguished by different mass, they have to be separated chromatographically. LC-MS/MS is the standard method for determination of PAs. However, separation of these compounds often poses a challenge. SFC offers complementary chromatographic selectivity to RP-LC and an advantage for separation of stereoisomers, shown in the development of a separation method for determination of 34 PAs including 5 Lycopsamin and 2 Senecionin isomers.