Ionpac guard column ag11

Other phosphorylated metabolites (e.g., sugar phosphate, 2PG, and PEP) and nucleotide sugars (ADPG and UDPG) were analyzed using an anion-exchange LC-MS/MS method described in Alonso et al. (2010) (link) with slight modifications. Metabolites were reconstituted in 100 μL of water from lyophilized extract, and 10 μL of extracts was injected into an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled with a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). Metabolites were separated by an IonPac AS11 analytical column (2 × 250 mm, Dionex) equipped with an IonPac guard column AG11 (2 × 50 mm, Dionex) at a flow rate of 0.35 mL min⁻¹. A multi-step gradient was applied with mobile phase A (0.5 mM KOH) and mobile phase B (75 mM KOH): 0–2 min, 100% A; 2–4 min, 100%–93% A; 4–13 min, 93%–60% A; 13–15 min, 0% A; 15–17 min, 100% A. The KOH concentration was suppressed by a post-column anion self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific), with a current of 50 mA and flow rate of 3.5 mL min⁻¹. An IonPac ATC-3 Anion Trap Column (4 × 35 mm), conditioned with 2M KOH, was used to remove contaminant ions from KOH solvents. Mass spectra were acquired using MRM in negative ESI mode. Parent-product ion transitions for metabolites were described in Supplemental Table S2.