Agilent 1260 uplc dad 6530 esi q tof ms

The determination of molecular masses and structural identification of the isolated flavonoids were carried out in a liquid chromatography-mass spectrometry (LC-MS), and the model is Agilent 1260 UPLC-DAD-6530 ESI-QTOF MS (Agilent, California, USA). MassHunter B0.05.0 workstation was utilized for assay development. The samples were separated using an Agilent Poroshell 120 EC-C₁₈ column (100 mm × 3.0 mm, 2.7 μm). The flow rate was 0.4 mL min⁻¹ and the detection wavelength was 255 nm. The column temperature was 35 °C with an injection volume of 5 μL. The mobile phase consisted of 0.1% formic acid in water (phase A) and acetonitrile (phase B). The elution conditions were as follows: 0–15 min, 15–25% B; 15–25 min, 25–100% B. MS analysis was performed in negative mode. Nebulizer pressure, 50 psi; N₂ drying gas flow, 10 mL min⁻¹; N₂ drying gas temperature, 350 °C; capillary voltage, 3500 V; fragmentor voltage, 150 V; scanning range, 50–1500 m/z.

To identify anthocyanin molecules, ~2.0 g petals were ground in liquid nitrogen into powder and suspended in 20 mL methanol:formic acid (9 : 1) for extraction three times by ultrasonication. The extracts were concentrated to 5 mL and purified with C18 Sep‐pak solid phase extraction (500 mg/8 mL, GRACE, USA), with a mixed solution of methanol:water:formic acid (4 : 5 : 1) as eluent (10 mL). Elution solutions were subjected to Agilent 1260 UPLC‐DAD‐6530 ESI‐QTOF MS (Agilent Technologies, Palo Alto, CA) equipped with Agilent Poroshell 120 SB‐Aq C18 column (4.6 × 100 mm, 2.7 μm). LC–ESI–ToF–MS/MS analyses were carried out in accordance with a method described previously (Zhao et al., 2011 (link)). MS data were managed with the MassHunter B0.05.0 Workstation.