Multiple reaction monitoring (MRM) scans were performed for each standard by selecting the protonated molecular ion [M + H]+1 (parent ion) with the first mass filter quadrupole (Q1). The product ions, yielded by collision-induced dissociation, were scanned by the third quadrupole (Q3). Unambiguous Q1/Q3 pair transitions were obtained by manually optimizing the declustering potential to get characteristic product ions of ANTX and PHE (Table (Table2).2). Standard curves were produced with anatoxin-a fumarate obtained from Tocris Bioscience (USA) and analyzed in line with unknowns. All samples were analyzed on the same run to minimize any day to day instrument variability. The instrument limit of detection was 1 pg of ANTX and the limit of quantification 3.5 pg on column.