Acquity uplc pump system

For KCF18 pharmacokinetic analysis, a set of mice was employed. Pharmacokinetic analysis was performed according to the method of a previous study [27 (link)]. In brief, the mice were intraperitoneally (i.p.) administered one dose of KCF18 (0.6 mg/kg). Serial samples of blood were obtained through submandibular vein puncture before dosing (time 0) and at another 5 time points (i.e., 0.5, 1, 2, 4, 8, and 12 h) after KCF18 administration. Blood samples were centrifuged (3000 rpm, 10 min) immediately after collection. Then, plasma samples were collected and stored (−80 °C) until analysis. The plasma concentrations of KCF18 were analyzed through liquid chromatography–tandem mass spectrometry (Xevo TQ-S with Acquity UPLC pump system; Waters, Milford, MA, USA). Data were analyzed with MassLynx 4.1 Software (version 4.1, SCN 714; Waters) to facilitate the pharmacokinetic analysis of KCF18.
The KCF18 in vivo half life, as our data demonstrated, was estimated to be 2 h. Detailed information is provided in Section 3.1.

Mass spectrometry for anion exchange LC-MS/MS and GC-EI-MS were carried out using the methods described in ref. 12 (link) and detailed in Dataset S5. Reverse-phase LC-MS/MS and GC-chemical ionization (CI)-MS had the following changes: Samples for reverse-phase liquid chromatography-tandem mass spectrometry were analyzed by an ACQUITY UPLC pump system (Waters) coupled with Waters XEVO TQ-S ultra-performance liquid chromatography tandem mass spectrometry (Waters) by the method described in ref. 12 (link). Samples for gas chromatography-electron ionization-mass spectrometry were analyzed by an Agilent 7890B GC system (Agilent) coupled to an Agilent 7010B triple quadrupole gas chromatography-electron ionization-mass spectrometer with an autosampler (CTC PAL) (Agilent). An Agilent VF5ms GC column, 30 m × 0.25 mm × 0.25 m with 10-m guard column was used. One microliter of the derivatized sample was injected with helium carrier gas at a flow rate of 1.2 mL⋅min⁻¹. The oven temperature gradient was: 40 °C (1-min hold), increased at 40 °C/min to 150 °C, then a 10 °/min to 250 °C, then a 40 °C/min to 320 °C, and finally held at 320 °C for 4.5 min. CI was used, and the mass scan range was 150 amu to 650 amu with step size 0.1 amu. The ionization source temperature was set at 300 °C, and the transfer line temperature was 300 °C.

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.