UPLC-MS/MS analysis of flavones from plants and enzyme reaction products

For analysis of flavone compounds from plant materials, leaves or flowers were harvested and lyophilized before extracting with methanol⁵². For acid hydrolysis, samples were treated an equal volume of 1 M HCl⁵³. Analyses were performed using an ACQUITY ultra performance liquid chromatography system (UPLC I-class, Waters, USA) with an ACQUITY UPLC HSS C18 column (1.7 μm, 100 × 2.1 mm i. d.; Waters); the flow rate was 0.4 mL/min and operated at 35 °C. The mobile phase consisted of water: formic acid (999:1, v/v; eluent A) and acetonitrile (eluent B). 1 μL of the sample extract was injected, and the elution program was 0 min, 5% B; 6 min, 45% B; 7 min, 90% B, and isocratic with 5% B (7.1–10 min). Absorbance values at 350 nm by a UV detector were used for the quantification of flavones and luteolin was used as the standard. All samples were analyzed in triplicate.

To confirm the products of plant metabolites and the enzyme reactions, samples were analyzed via LC-MS/MS analysis. The UPLC separation conditions were the same as those described above. The ESI-MS/MS analysis performed using a Xevo TQ-MS mass spectrometer (Waters). The compounds were acquired in both positive-ion (PI) and negative-ion (NI) mode. The detection conditions were as follows: desolvation temperature: 400 °C; desolvation gas (N₂) rate, 800 L/h; cone gas flow, 50 L/h; cone voltage and capillary voltage were 30 V and 3 kV, respectively, for PI, and −60 V and 2 kV, respectively, for NI. Mass spectra were recorded across an m/z range of 100–1000, and MassLynx version 4.1 was used for system control and data processing.