Thermo scientific vanquish uhplc system

Liquid chromatography-mass spectrometry system consisted of a Thermo Scientific Vanquish UHPLC system (ThermoFisher Scientific, CA, USA) with Poroshell 120 EC-C18 (2.1 x 100 mm, 2.7 μm) column (Agilent) and a triple ToF 5600+ mass spectrometer system (Triple ToF MS) (SCIEX, Foster City, CA, USA). Triple TOF MS, equipped with a DuosprayTM ion source, was used to complete the high resolution experiment.

Two-gram homogenized sample taken from the total leaves sample were mixed with 5 mL of ethanol and subjected to the sequential steps—vortexing and sonication—to obtain the concentrated (20 mg/mL) extract. Prepared samples were subjected to liquid chromatography-mass spectrometry with a Thermo Scientific Vanquish UHPLC system (ThermoFisher Scientific, CA, USA) with Poreshell 120 PFP (2.1 × 100 mm, 2.7 μm), column (Agilent), and a triple ToF 5600⁺ mass spectrometer system (Triple ToF MS) (SCIEX, Foster City, CA, USA) with a Duospray^TM ion source and universal gradient conditions. The system was calibrated before the original experiment with the blank samples and 18 alkaloid standards. Molecular weights and molecular breakdown information detected from the system were collected through the information-dependent acquisition (IDA) scan method in LC-QTOF (HRMS, high-resolution mass spectrometer). Finally, all the peaks for the given standards and similar predictable alkaloids component peaks were obtained and were annotated with Metlin (https://metlin.scripps.edu/) and isotope MS. All of the experimental procedures were conducted at the Korean Medicine Clinical Trial Center. The multivariate analysis of the data was conducted using R (https://www.r-project.org/).

Mass spectral experiments were performed using a Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer equipped with a Thermo Scientific Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). HILIC-MS analyses of PSP toxins were carried out with a 5 µm TSK-gel Amide-80 column (250 mm × 4.6 mm i.d.) (Tosoh Bioscience LLC, Montgomeryville, PA, USA). Mobile phases consisted of (A) 100% DI MilliQ filtered water and (B) 95% acetonitrile to 5% water (v/v), both containing 0.1% formic acid and pH adjusted to 5.5 with ammonium formate (VWR International LLC., Radnor, PA, USA). LC conditions consisted of 65% (B) at 0.8 mL/min with column temperature at 25 °C and 5 µL injection volumes. Targeted SIM analyses were performed in positive ionization mode with targeted m/z including 300, 282, 396, 316, and 298 m/z. Each NRC PSP toxin standard was analyzed before 50 µL were combined with 12 mg of cysteine, incubated in a water bath at 70 °C for 30 min, and then analyzed again. Conversion efficiency was calculated per toxin as the percent decrease in intensity of the starting toxin’s prominent ion following L-cysteine processing.