Unraveling the Ergot Alkaloid and Indole Diterpenoid Metabolome in the Species Complex Using LC-HRMS/MS Diagnostic Fragmentation Filtering.

The plant parasitic fungus sensu lato produces sclerotia containing toxic ergot alkaloids and uncharacterized indole diterpenoids in grasses including cereals. The aim of this study was to detect as many peptide ergot alkaloids and indole diterpenoids in ergot sclerotia as possible by using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS) approach and applying filtering of diagnostic fragment ions for data extraction. The sample set consisted of 66 sclerotia from four different geographic locations in southeastern Norway as well as Saskatchewan, Canada. The host plants included both wild grasses and important cereal grains such as rye. DNA sequencing showed that the sclerotia were from three species, i.e., sensu stricto (s.s.), , and (former genotypes G1, G2, and G2a, respectively). All sclerotia from cereal grains were from s.s. Diagnostic fragment filtering was based on detecting specific product ions in MS/MS data sets that are well-conserved across the different ergot alkaloid subgroups and indole diterpenoids of the paspaline/paxilline type. The approach extracted mass spectra from 67 peptide ergot alkaloids (including C-8 epimers and lactam variants) and five indole diterpenoids. In addition, three clavines were detected by using targeted analysis. The sum of the peak areas for ergot alkaloids, which have been assigned as "major" analogues by the European Food Safety Authority (ergometrine, ergosine, ergotamine, α-ergocryptine, ergocornine, ergocristine, and their 8- epimers), accounted for at least 50% of the extracted total ergot alkaloid metabolome. Univariate and multivariate statistical analyses showed that several of the alkaloids were specific for certain species within the species complex and could be used as chemotaxonomic markers for species assignment.