Uhplc q exactive hf x system

Approximately 50 mg of the sample was accurately weighed and grinded with 400 µL of extract buffer (methanol: water = 4:1 (v: v)) at −10°C for 6 min (50 Hz), followed by ultrasound treatment (40 KHz) at 5°C for 30 min. The extract was further stored at −20°C for 30 min and centrifuged at 13,000 rpm at 4°C for 15 min. Then the supernatants were filtrated before conducting instrumental analysis.
The UHPLC-Q Exactive HF-X system (Thermo Scientific, USA) was employed to analyze the metabolites of Z. nitidum seed embryos. The chromatographic column conditions were as follows: ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm, Waters, Milford, USA); column temperature, 40°C; mobile phase A, 95% water + 5% acetonitrile (containing 0.1% formic acid); mobile phase B, 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid); and injection volume, 3 μL. For mass spectrometric detection, samples were ionized via ESI. The irons were scanned using both positive and negative modes at a resolution of 60,000 and scan range of 70–1,050 m/z. The flow rates of sheath gas and aux gas were 50 and 13 arb, respectively, and the temperature was 425°C.

LC-MS analysis was performed using the UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific. Fecal samples were weighed. The metabolites were extracted using a 400 µL methanol:water (4:1 v:v) solution with 0.02 mg/mL L-2-chlorophenylalanine. The mixture was allowed to settle at −10°C and treated with a Wonbio-96c high-throughput tissue crusher (Shanghai Wanbo Biotechnology) at 50 Hz for 6 min, followed by ultrasound at 40 kHz for 30 min at 5°C. The samples were placed at −20°C for 30 min to precipitate proteins. The supernatant was carefully transferred to sample vials for LC-MS analysis after centrifugation at 13,000 g at 4°C for 15 min. All samples were stored at 4°C during the period of analysis. A Thermo UHPLC-Q Exactive HF-X mass spectrometer was used to collect the mass spectrometric data, which was performed in Data Dependent Acquisition mode. The detection was carried out over a mass range of 70–1050 m/z. We used orthogonal partial least-squares discrimination analysis to visually discriminate between groups. The differential metabolites discriminating between groups were identified by orthogonal projections to latent structures discriminant analysis (variable importance plot [VIP] >2, P<0.05). The Kyoto Encyclopedia of Genes and Genomes Pathway database was used to carry out pathway analyses.