*4.9. High-Performance Liquid Chromatography and Mass Spectrometry*

Before the analysis, the samples were mixed with an equal part of acetonitrile and centrifuged. High-performance liquid chromatography and mass spectrometry (HPLC-MS) was carried out using the system, consisting of the CBM-20 control unit, two LC-2020AD pumps, SIL-30AC auto sampler, and CTO-20AC column thermostat, using the SPD-M20A detector and LCMS-2020 mass spectrometer with ESI source (Shimadzu, Kyoto, Japan).

Chromatographic fractionation was conducted using YMC-Pack Pro C18 column, 150 × 3 mm (YMC, Kyoto, Japan) at 40 ◦C, with 0.1% formic acid solution in water and acetonitrile gradient from 5% to 95%.

Mass spectra were recorded from *m*/*z* 10 up to 500 *m*/*z* at 350 ◦C and ±4500 V using N2. Mass spectrometry analysis was carried out using both the positive and negative ionization modes. The data were analyzed using LabSolutions LC/MS software (Shimadzu, Japan).
