Zorbax rrhd extend c18 column

Ion pair LC-MS/MS analysis was performed with LC separation on a Zorbax RRHD Extend-C18 column (150 mm × 2.1 mm, 1.8 μm particle size, Agilent Technologies), and using gradient of solvent A (10 mM tributylamine and 15 mM acetic acid in 97:3 water:methanol) and solvent B (10 mM tributylamine and 15 mM acetic acid in methanol) according to the manufacturer’s instructions (MassHunter Metabolomics dMRM Database and Method, Agilent Technologies).

LC-MS analysis was conducted using an Agilent 1290 Infinity LC system containing a Zorbax RRHD Extend C18 column (2.1 mm × 150 mm; Agilent) coupled to an Agilent Accurate Mass 6230 TOF as previously described [41 (link)]. The mobile phase consisted of solvent A (5 mM tributylamine (TBA), 5.5 mM acetic acid in 97% ddH₂O 3% methanol) and solvent B (5 mM TBA, 5.5 mM acetic acid in methanol) run at a flow rate of 0.25 mL/min with the following gradient: 0–3.5 min, 0% B; 4–7.5 min, 30% B; 7.5–8 min, 35% B; and 12–16 min, 99% B; followed by a 5 min equilibration period at 0% solvent B prior to injection of the next sample. Dynamic mass axis calibration was accomplished by continuous infusion of a reference mass solution. ESI capillary and fragmentor voltages were set at 4000 V and 125 V respectively. The nebulizer pressure was set to 45 psig and nitrogen drying gas was set to a flow rate of 8 L/min. The drying gas temperature was maintained at 325°C. The MS acquisition rate was 1.5 spectra/ sec and m/z data ranging from 50–1100 was stored. Data was analyzed using Profinder B.08.00 software, and ions were assigned as specific metabolites based on mass accuracy within 5 parts per million (ppm) and retention times within 1 min of those determined for chemical standards.