Pyrrolizidine alkaloids (PAs) are secondary plant metabolites produced by a number of plants from the Asteraceae (Compositae), Boriginaceae and Fabaceae (Leguminosae) families. Many of these alkaloids have been shown to be highly toxic, causing hepatic veno-occlusive disease (VOD), liver cirrhosis and ultimately death. PAs may have also mutagenic and carcinogenic potential. Amongst livestock, cattle and horses are especially susceptible to the toxic effects of the PAs. Humans may also be at risk by the consumption of milk of livestock fed with PA-contaminated forage. At RIKILT - Institute of Food Safety a (semi)quantitative method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of PAs in animal feeds has been developed and validated. This method comprises 40 macrocyclic PAs (including tertiary amines and N-oxides) representative for ragwort species. The method has been used for the analysis of 147 forage samples collected in 2006-2008