We analyzed the known copepodamides (A to H) and two additional copepodamides sharing the same molecular scaffold with m/z (mass/charge ratio) of 658 and 686 in negative mode. All chemicals were purchased from Thermo Fisher and used as received. Copepodamides were separated by gradient elution on a high-performance liquid chromatography (Agilent 1200 series with a C18 column, 150 mm × 2.1 mm thermostated to 50°C) coupled to a triple quadrupole MS (Agilent 6410). The gradient went from 5 to 83% B during 18 min. Eluent A consisted of 35:35:30 methanol/acetonitrile/water, and eluent B consisted of 2-propanol. Both eluents were supplemented with 0.2% formic acid and 0.1% ammonia. We designed a multiple reaction monitoring method in negative mode with diagnostic fragments: m/z 432.2 for copepodamides A to C; m/z 430.2 for copepodamides D to F and the two new copepodamides with m/z 659 and 686 (34); m/z 124.0 for copepodamides G and H; fragmentor voltage 250 V; collision energy 44 for A to F, 659 and 686 and 40 for G and H. The ion source was operated at 300°C and 4500 V in negative mode with a nitrogen flow of 6 liters min−1 at 25 psi. Concentrations were determined by comparing with authentic standard available from previous work (14), assuming equal ionization efficiency for the individual copepodamides. Copepodamides for dose-response experiments were isolated from frozen C. finmarchicus, as described in (14). C. finmarchicus is a dominating copepod with geographical distribution overlapping with both Skeletonema and Pseudo-nitzschia.

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