Product ion spectra from the protonated molecules of anabaenopeptin A (3) and B (4) acquired at a 40 eV laboratory-frame collision energy

The numbers in parentheses beside each dashed line are nominal masses corresponding to the side of each formal cleavage site on the chemical structure for anabaenopeptin A. The italicized numbers are nominal masses of actual fragment ions that were generated with hydrogen rearrangement(s). The data were acquired by using a Synapt G2 quadrupole/time-of-flight (Q-TOF) tandem mass spectrometer (Waters Co., Manchester, UK) equipped with an Acquity UPLC system (Waters) and an ESI source. Samples were injected into a reversed-phase column (ACQUITY UPLC BEH C18 1.7 µm, 2.1×50 mm, at 25°C) and eluted with an isocratic solvent system (solvent A: 0.1% formic acid in water; Solvent B: acetonitrile; 30% B) at a flow rate of 0.2 mL/min for short LC-MS/MS runs. The ions generated at positive ESI capillary (3 kV) under atmospheric pressure went into the vacuum system through the sampling cone (15 V). The ions were transferred to the quadrupole mass analyzer for isolation of precursor ions of interests (i.e., the protonated molecules of analytes). The isolated precursor ions were subjected to low-energy CID (40 eV laboratory-frame collision energy) with Ar (99.9999%, 0.4 mL/min) at the trap collision cell (2.5×10⁻² mbar). Ions went through the collision cell and the following ion optics were mass analyzed at the orthogonal TOF analyzer for the recording of a high-resolution (approx. 20,000 FWHM) externally calibrated product ion spectrum in each second. In post-run data processing, a few spectra at the retention time of each analyte were combined and converted to a centroid spectrum. The precursor ion peak in each combined spectrum was used as an internal lock mass for recalibration to ensure ppm-level mass accuracy.