The HPLC system consisted of a Shimadzu LC-20AD solvent delivery pump, a DGU-20A3 degasser, a CTO-20A column oven, a SPD-M20A photodiode array detector, and a SIL-20A auto sampler (Shimadzu, Kyoto, Japan). The mass spectrometer was a Shimadzu LCMS-2020 single quadrupole equipped with a ESI source interface. At the end of data collection, the chromatograms were processed with the software LCMS Solution (Shimadzu, Kyoto, Japan).
For chromatographic analysis, an YMC-ODS C18 column (250 mm × 4.6 mm, 5 μm) was used. HPLC separation was performed using a linear gradient and a flow rate of 1.0 mL/min. The column temperature was 35°C. The mobile phase consisted of acetonitrile (A) and water containing 0.1% formic acid (B) using the elution gradient 5-25% A at 0-35 min, 25-35% A at 35-65 min, 35-50% A at 65-80 min, 50-70% A at 80-90 min, 70-5% A at 90-95 min and 5% A at 95-100 min. Detection wave-length was set at 254 nm. The ESI-MS spectra were acquired in both negative and positive modes scanning from 100 to 800. The typical ion source parameters were as follows: ESI probe temperature 350°C, CDL temperature 280°C, heat block temperature 320°C, ESI probe voltage 4.5 kV, detector voltage 1.5 kV, and nebulizing gas flow 1.5 L/min.
For chromatographic analysis, an YMC-ODS C18 column (250 mm × 4.6 mm, 5 μm) was used. HPLC separation was performed using a linear gradient and a flow rate of 1.0 mL/min. The column temperature was 35°C. The mobile phase consisted of acetonitrile (A) and water containing 0.1% formic acid (B) using the elution gradient 5-25% A at 0-35 min, 25-35% A at 35-65 min, 35-50% A at 65-80 min, 50-70% A at 80-90 min, 70-5% A at 90-95 min and 5% A at 95-100 min. Detection wave-length was set at 254 nm. The ESI-MS spectra were acquired in both negative and positive modes scanning from 100 to 800. The typical ion source parameters were as follows: ESI probe temperature 350°C, CDL temperature 280°C, heat block temperature 320°C, ESI probe voltage 4.5 kV, detector voltage 1.5 kV, and nebulizing gas flow 1.5 L/min.