Agilent masshunter quantitative analysis b 06

The released N-glycans were quantitatively profiled on Chip-QQQ-MS using MRM method in positive mode. The dry-gas (N₂) temperature and flow rate were 225 °C and 11 l min⁻¹, respectively. The RF voltage amplitude of the high-pressure and low-pressure ion funnels were 150 and 200 V, respectively. The dwell time was set as 10–50 ms. All of the raw data were processed using Agilent MassHunter Qualitative Analysis B.06.00 and Agilent MassHunter Quantitative Analysis B.06.00 software. The optimized method was validated for linearity, sensitivity, recovery and repeatability. The abundance of each N-glycan composition was relatively quantified based on the peak areas of their MRM chromatograms and expressed as a percentage of summed peak areas for total N-glycans within each IgG samples.

The Agilent 6540 Ultra High Definition (UHD) Accurate-Mass Q-TOF LC/MS system from Agilent Technologies (Santa Clara, CA, USA) was used for mass spectrometry analysis. The compounds in 10 μL were injected and separated on an ACE C18 HPLC column at a flow rate of 1.0 mL/min and column temperature of 40°C, by using the same gradient from the mobile phases of (A) 0.1% formic acid in water and (B) methanol as described for HPLC. The MS parameters were set as follows: electrospray ionization source (ESI) in a negative mode, nitrogen (N₂) as drying gas, flow rate at 8 L/min, gas temperature at 300°C, nebulizer at 40 psi, sheath gas temperature at 350°C, flow rate of sheath gas at 8 L/min, capillary voltage at 4.0 kV, end plate offset at -500 V, fragmentor at 150 V, skimmer at 65 V, Oct RF Vpp at 600 V, scan range of 100-1700 m/z, and collision energy at 25 V for MS and 45 V for MS/MS, respectively. The data was analyzed on Agilent MassHunter Qualitative Analysis B.06.00 and Agilent MassHunter Quantitative Analysis B.06.00 from Agilent Technologies (Santa Clara, CA, USA).

UPLC-MS/MS profiling was performed on Agilent 6,540 Ultra High Definition (UHD) Accurate-Mass Q-TOF LC/MS system from Agilent Technologies (Santa Clara, CA, United States). The samples (2 μL) were separated on a BEH C18 UPLC column at a flow rate of .4 mL/min, column temperature at 40°C, and with a gradient from solvents of .1% formic acid in water (A) and .1% formic acid in acetonitrile B) as follows: 2%–35% B at 0–10 min, 35%–75% B at 10–18 min, and 75%–100% B at 18–22.1 min. The MS instrument was configured as follows: electrospray ionization source (ESI) for both positive and negative modes, nitrogen (N2) as drying gas, flow rate at 8 L/min, gas temperature at 300°C, nebulizer at 40 psi, sheath gas temperature at 350°C, flow rate of sheath gas at 8 L/min, capillary voltage at 3.0 kV, end plate offset at −500 V, fragmentor at 150 V, skimmer at 65 V, Oct RF Vpp at 600 V, scan range of 100–1,700 m/z, and collision energy at 25 V for MS and 45 V for MS/MS, respectively. The data were analyzed with Agilent MassHunter Qualitative Analysis B.06.00 and Agilent MassHunter Quantitative Analysis B.06.00 from Agilent Technologies (Santa Clara, CA, United States).