Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, and their 1,2-unsaturated derivatives, which contain the retronecine, heliotridine, or otonecine type of the necine base, have raised concern due to their ability to form hepatotoxic intermediates and exhibit serious toxic effects. Several hundred individual pyrrolizidine alkaloids and their N-oxides have been identified mostly using liquid chromatography coupled with mass spectrometry, although the number of available reference standards is limited.In this work, characteristic fragment ions and their abundance in the mass spectra of different PAs were used to reveal typical fragmentation patterns for various classes of PAs that can be further employed to distinguish monoesters (retronecine, heliotridine type), open chain diesters and macrocyclic diesters, and corresponding N-oxides.Fragment ions at m/z 120 and 138 were found in all types of PAs with a different relative abundance. Additional observation of fragment ions at m/z 94 and 156 was found to be typical for monoester PAs esterified at position C9 of the necin base, whereas fragment ions at m/z 111 and 172 were characteristic for monoester N-oxides. Fragment ions at m/z 180 and 220 were found to be typical for open chain diesters with esterification at C7 with acetic and angelic acid, respectively, whereas fragment ions at m/z 214 and 254 were characteristic for the respective N-oxides. For the 3'-acetyl PA monoester or open chain diester derivatives, characteristic fragment ions were observed after loss of the acetyl moiety ([M+H](+)-60), whereas for macrocyclic diesters and their N-oxides, fragment ions due to the neutral loss of CO were found ([M+H](+)-28).