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).