Agilent 1260 hplc 6520 qtof ms instrument

HPLC analyses were carried out with an Apollo C18 column (5 μm, 4.6 mm × 250 mm, Alltech, Deerfield, IL, USA) on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). Briefly, the column was developed with solution A (water with 0.1% trifluoroacetic acid) and acetonitrile at a flow rate of 0.8 mL/min. The percentage of acetonitrile was changed using the following gradient: 0–5 min, 10%; 5–25 min, 10%–90%; 25–30 min, 90%–100%; 30–35 min, 100%. The detection wavelength was 320 nm. LC-MS analyses were performed on an Agilent 1260/6460 Triple-Quadrupole LC/MS system (Santa Clara, CA, USA) with an electrospray ionization source. HR-ESI-MS was performed on an Agilent 1260 HPLC/6520 QTOF-MS instrument (Santa Clara, CA, USA). NMR spectra were recorded at room temperature on a Bruker-500 NMR spectrometer (Billerica, MA, USA).

Analytical HPLC was performed with a Dionex CarboPac™ PA1 BioLC™ column (4 × 250 mm, Thermo Fisher Scientific, USA) on a Shimadzu HPLC system (Shimadzu, Japan). For the detections of ADP-d,d-manno-heptose and ADP-β-d-mannose, the column was developed with solvent A (ddH₂O) and solvent B (1.0 M CH₃COONH₄) at a flow rate of 1.0 mL/min. The percentage of solvent B was changed using the following gradient: 0–5 min, 15%; 5–30 min, 15%–45%; 30–32 min, 45%–90%; 32–38 min, 90%; 38–40 min, 90%–15%; and 40–45 min, 15%. To detect ADP-l,d-manno-heptose, the percentage of solvent B was changed using the following gradient:0–5 min, 24%; 5–30 min, 24%–27%; 30–32 min, 27%–90%; 32–38 min, 90%; 38–40 min, 90%–24%; and 40–45 min, 24%. To detect ADP-d-glycero-β-d-altro-heptose, the percentage of solvent B was changed using the following gradient: 0–5 min, 15%; 5–30 min, 15%–30%; 30–40 min, 30%–45%; 40–42 min, 45%–90%; 42–52 min, 90%; and 52–53 min, 90%–15%; and 53–63 min, 15%. The detection wavelength was set as 254 nm.
HRMS was performed on an Agilent 1260 HPLC/6520 QTOF-MS instrument with an electrospray ionization source. NMR spectra were recorded at room temperature on a Bruker-500 NMR.

A direct heat CO₂ incubator (Forma™ 311), Varioskan® Flash Microplate Multimode Readers, and NanoDrop One UV-Vis spectrophotometer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A LightCycler PCP was purchased from Roche (CA, USA). A Centrifuge 5417R was obtained from Eppendorf (Hamburg, Germany). Agilent 1260 HPLC/6520 QTOF-MS instrument, Eclipse Plus C18 chromatography column, and Cary Eclipse fluorescence spectrophotometer were obtained from Agilent Technologies, Inc. (Agilent, Milford, MA, USA). Ultra-High-Performance Liquid Chromatography System and ACQUITY UPLC HSS C18 column (100 × 2.1 mm, 1.7 μm) were from Waters (Milford, MA, USA). The X500 QTOF Mass spectrometer system was purchased from AB Sciex Pte. Ltd. (MA, USA).