UPLC-ESI-MS characterization was performed using an ACQUITY UPLC H-Class Bio System (Waters® Corp., Milford, MA, USA). The separation was conducted using an ACQUITY UPLC® HSS T3 130 Å column (1.8 µm, 2.1 × 50 mm, Waters® Corp., Milford, MA, USA) with a column temperature of 35 °C. For HAEPa, we used an isocratic elution, using a binary system consisting of 20% ammonium hydroxide in water to 0.05% (A) and 80% acetonitrile (B); and for EAcE we used a binary system consisting of ammonium hydroxide in water to 0.05% (A) and acetonitrile (B). We used a gradient elution of 0–2 min 90% A, 2–4 min 80% A, 4–6 min 50% A, 6–8 min 20% A, and 8–9 min 90% A. Then, 5 μL of the samples and standards at 100 ppm concentration were injected with a flow rate of 0.4 mL/min, and methanol was used as blank solvent.
Detection was performed using an ACQUITY QDa detector mass spectrometer (Waters Corp., Milford, MA, USA) with an electrospray ionization interface (ESI); the voltage of the capillary was set to −1.0 kV for the negative-ion mode (ESI-). The data were processed using Waters Empower™ 3 software (Waters Corp., Milford, MA, USA). A mass scan acquisition was programmed at 50 to 1250 Da and a selected ion recording (SIR) for each targeted mass was selected [51 (link)].
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