Aquity uplc protein beh c4 column

Samples were analysed using a 1290 Infinity II UHPLC system coupled to a 6550 iFunnel Q-TOF mass spectrometer with an electrospray ionization source (Agilent, Waldbronn, Germany). Aliquots of the sample (0.75 µg) were injected onto a reversed phase C4 column (Aquity UPLC Protein BEH C4 Column, 300 Å, 1.7 µm, 2.1 mm, 100 mm, Waters, Manchester, UK) and the column temperature was maintained at 80 °C. Mobile phase A was 0.1% (v/v) formic acid in water, and mobile phase B was 0.1% (v/v) formic acid in ACN. The flow rate was 0.5 mL/min. The complete run consisted of 1 min isocratic elution (20% solvent B), followed by a 40 min gradient (20% to 32% solvent B), a subsequent purge step (quick rise to isocratic elution at 95% solvent B), and a final 5 min reequilibration step (quick decrease to isocratic elution at 20% solvent B).
Mass spectrometer settings included a mass range of m/z 600 to m/z 3,200; nebulizer: 2.8 bar, drying gas flow: 14 L/min; drying gas temperature: 290 °C; sheath gas flow: 12 L/min; sheath gas temperature: 375 °C; fragmentor voltage: 400 V. Spectra were acquired at 2 spectra/s. Internal mass calibration was achieved by continuously spraying of internal reference compounds through the reference nebulizer.
Data acquisition was controlled with the MassHunter software (Agilent, Santa Clara, CA, USA).

We injected 1 μl of the candidate RM into an LC-MS system using the AQUITY UPLC Protein BEH C4 column (1.7 μm, 2.1 mm diameter × 100 mm length, Waters, USA), mobile phase A: 0.1% formic acid/H₂O, B: 0.1% formic acid/acetonitrile, flow rate: 0.2 ml/min, column temperature: 60°C, and gradient condition: 5–15 %B in 15 min. The mass spectrometer was operated under the following conditions: capillary voltage: 4500 V, nebulizer gas: 1.2 bar, dry gas: 6 L/min, isCID: 30 eV, quadrupole ion energy 4 eV, collision energy: 8 eV, mass range: m/z 500–3000, and spectra rate: 3 Hz. The mass spectrometer was calibrated using ESI-L Low concentration tuning mix (G1969-85000, Agilent Technologies, USA).