4.6. UPLC-DAD-MS/MS Analysis

The Waters UPLC system (Waters ACQUITY UPLC H-class Plus) coupled with a diode array detector (DAD) and a Thermo Finnigan LCQ Advantage ion-trap mass spectrometer (San Jose, CA, USA) was used for analysis at the Metabolomics Core Facility of the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan. The extracts were separated by UPLC with a column (ACQUITY UPLC BEH, 1.7 μm C18, 2.1 mm × 100 mm) at the flow rate of 0.45 mL/min. The mobile phase consisted of water containing 0.1% (v/v) formic acid (FA) (mobile phase A) and methanol containing 0.1% (v/v) FA (mobile phase B), and separations were performed using the following gradients: 10% B from 0 to 0.5 min, 29% B from 0.5 to 4.5 min, 35% B from 4.5 to 8.5 min, 45% B from 8.5 to 11.5 min, 49% B from 11.5 to 11.6 min, 100% B from 11.6 to 14 min, 10% B from 14 to 16 min. The sample injection volume was 10 μL. Eluting peaks were monitored by 330 nm. The chemical structures of these four compounds (namely demethylwedelolactone, wedelolactone, luteolin and apigenin) were elucidated by spectroscopic analysis. Electrospray ionization mass spectrometry data were collected with a LCQ Advantage mass spectrometer (Thermo Finnigan, San Jose, CA, USA) and nuclear magnetic resonance spectra were recorded with Advance 500 and 300 MHz FT nuclear magnetic resonance spectrometers (Bruker, Bremen, Germany) at 500 MHz (¹H) and 75 MHz (¹³C).