Accela ultra hplc system

LC-MS analysis was carried out on a model Accela ultra HPLC system coupled to a Q Exactive Orbitrap Mass Spectrometer and a CTC PAL autosampler (all from ThermoFisher, San José, CA, USA). Mixtures were injected onto a Kinetex C18 column (150 × 3 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) with a Phenomenex UHPLC C18 pre-column (3.0 mm i.d.). Gradient elution was performed at a flow rate of 300 μL/minute using a linear gradient of 10 mM NH₄OH in H₂O to 10 mM NH₄OH in MeOH. Mass analysis was performed under positive and negative ESI in full scan mode. Quantitation was performed with Xcalibur^™ software by extracting the protonated analyte masses within 5 ppm of the respective monoisotopic masses except for chavibetol which was monitored in negative mode at its deprotonated monoisotopic mass. We found that the sensitivity using tSIM was similar to that using full scan mode and since we intended to search for potential unknown metabolites, the only possible way to achieve that was to use full scan mode.

HPLC analysis was carried out on a model Accela ultra HPLC system (Thermo Fisher, San Jose, CA) consisting of a Kinetex C18 column (150 × 3 mm, 2.6 μm) coupled to a UV/VIS photo diode array detector (Thermo Scientific, San Jose, CA). Gradient elution was performed at a flow rate of 250 μL/minute using a linear gradient with MeOH (A) against 5mM ammonium bicarbonate in water (B) starting at 20% A, holding for 1 minute then linear gradient to 60% A over 13 minutes linear gradient, back to 20% A over 2 minutes, then holding for 8 minutes for equilibration. Detection was performed from 300-600 nm and data were predominantly extracted from the 488 nm trace.