Analytical method UHPLC-ESI-MS/MS
Extracts of Paulownia leaves, flowers, fruits and twigs were analyzed by UHPLC-ESI-MS/MS, using a Thermo Ultimate 3000RS (Thermo FischerScientific, MS, USA) UHPLC system, quipped with a charged aerosol detector (CAD), a diode array detector, and coupled with a Bruker Impact II (Bruker Daltonics GmbH, Bremen, Germany) Q-TOF mass spectrometer. Samples were chromatographed on a an ACQUITY HSS T3 column (2.1 × 150 mm, 1.8 µm; Waters, MA, USA) maintained at 55° C, the injection volume was 2.0 µL. A 22 min gradient elution program was applied, from 2 to 45% of solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formic acid in Milli-Q water); the flow rate was 0.500 mL min-1. UHPLC–MS/MS analysis was performed in negative ion mode, the scanning range was from m/z 80 to 2000. The following settings were applied: capillary voltage was 3 kV, dry gas flow was 6 L min−1, dry gas temperature was 200° C, nebulizer pressure was 0.7 bar, collision RF was 750 V, transfer time was 100 ms, prepulse storage time was 10 ms. Two precursor ions of intensity over 2000 counts were fragmented in each scan. Depending on the m/z of a fragmented ion, the value of collision energy was set automatically in the range from 2.5 to 80 eV. The internal calibration of the acquired data was performed with sodium formate, introduced to the ion source via a 20 µL loop at the beginning of a separation. Constituents of the extracts were determined by external calibration, using authentic standards when possible. The UV detection at λ = 330 nm was used for quantitation and semi-quantitation of phenolic compounds. The content of verbascoside was determined on the basis of the following standard curve (y = 4.90599x - 1.97683; R² = 0.9999). The same standard curve was also used for semi-quantitation of other phenylethanoids, and hydroxycinnamic acid derivatives. Flavone glycosides were determined on the basis of a standard curve of apigenin-7-O-glucuronopyranosyl-(1-2)-glucurono-pyranoside (y = 6.89810x - 19.60755; R² = 0.9997). Apart from phenolics, two iridoids were additionally quantified. 7-hydroxytomentoside was determined on the basis of UV chromatograms (λ = 248 nm), with a standard curve y = 4.02230x - 1.07988; R² = 0.9995. Extracted ion chromatograms (EIC) were used for quantitative analyses of catalpol, on the basis of the following standard curve y = -41.329553x2 + 17210.064761x + 12019.481295; R² = 0.9972).