Thermo vanquish

Metabolites in the colonic contents were extracted with methanol and quantified using an ultra-high performance liquid chromatography/mass spectrometry (UHPLC-MS) system (25 (link)) on a Thermo Vanquish (Thermo Fisher Scientific, United States) as previously detailed (26 (link)) and a Thermo Orbitrap Exploris 120 mass spectrometer (Thermo Fisher) using data collection from both positive and negative ion modes as previously described (27 (link)).
PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted in R software to analyze and screen metabolites. Potential biomarkers were selected based on VIP scores >1 in OPLS-DA. Simultaneously, metabolites were screened further and those with FC >2 and p-value <0.05 in student’s t-test were selected.
Differential metabolites from optimized data underwent substance enrichment analysis in R software with reference to the KEGG database to determine host pig metabolic pathways.

LCMS grade
water and acetonitrile
were purchased from Fisher Scientific (Pittsburgh, PA). Capillary
columns with photopolymerized integrated frits and emitter tips were
fabricated in-house from fused silica capillary (Polymicro Technologies,
Phoenix, AZ) with dimensions 17.5 cm × 50 μm and packed
with 3 μm Atlantis T3 C18 particles (Milford, MA) as previously
described.^{30 (link)} Flow through the column was
delivered by a Thermo Vanquish (Thermo Fisher Scientific, Waltham,
MA) LC pump and autosampler connected to a stainless steel tee which
acted as a flow splitter. The split was a 50 μm × 100 cm
open capillary. The column was operated at a flow rate of 125 nL/min
with an injection of 4 nL split from the bulk flow of 175 μL/min
and injection of 6 μL. The flow rate was determined based on
the experimental dead time and used to determine a split ratio of
1:1400. Mobile phase A was 0.1% formic acid in water. Mobile phase
B was 0.1% formic acid in acetonitrile. The gradient was as follows:
0–1 min, 0% B; 12 min, 98% B; 14 min, 98% B; 14.1–25
min, 0% B.

The samples used for the metabolomics assay were consistent with the transcriptome, both being P. ternata leaves. Using an ACQUITY UPLC ® HSS T3 (2.1 150 mm, 1.8 m) (Waters, Milford, MA, USA) chromatographic column, the Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid phase system operates at a flow rate of 0.25 mL/min, a column temperature of 40°C, and an injection volume of 2 µL (Liu et al., 2020b (link); Fu et al., 2021 (link)).