UHPLC-MS-Based Metabolite Profiling

Chromatographic separation on a Vanquish UHPLC system, with a SeQuant ZIC pHILIC column (150 × 2.1 mm i.d., 5 μm) coupled to a QE-HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA)^{27 (link)}, was performed with a mobile phase that consisted of (A) 10 mmol/L AcONH₄ in water (pH 9.8) and (B) ACN. The gradient was as follows: 0.0–1.0 min (90% B), 1.0–15.0 min (30% B), 15.0–18.0 min (30% B), 18.0–19.0 min (90% B), 19.0–29.0 min (90% B), and then initial conditions were maintained for 5 min to equilibrate the column. The flow rate was 250 μL/min, and the injection volumes were set to 2 μL. All samples, including a QC sample that contained equal amounts of each sample, were analyzed by negative and positive electrospray ionization in full scan MS mode with a mass range of 70–1050 m/z, while the rest setting of the MS parameters was the same as those for C18. The acquired raw files were processed using Compound Discoverer 2.1, as previously described.