Uplc beh amide column

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed using an Ultra High Performance Liquid Chromatography (UHPLC) system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo) (Xue et al., 2021 (link)). The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra in information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo). Metabolites were detected and analyzed in both positive- and negative-ion conditions to obtain the total ion current of the metabolites. After raw data were filtered and denoised, the final data set containing the peak number, sample name and normalized peak area information was imported into the SIMCA16.0.2 software package (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate analysis. Data were scaled and logarithmically transformed to minimize the impact of noise and high variance of the variables. Principal component analysis (PCA) was performed to visualize the distribution and grouping of the samples. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed in Metaboanalyst 5.0.

After grinding with liquid nitrogen 25 mg of CK, R, S, Ri and Fm root samples were extracted in 500 μL solution containing methanol:water (3:1). Samples were mixed for 30 s using a vortex, homogenized at 35 hz for 4 min and sonicated for 5 min in an ice-water bath, these steps were repeated three times. Next, samples were incubated for 1 h at -40 °C and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant was transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples. Each treatment was repeated six times.
LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mM ammonium acetate and 25 mM ammonia hydroxide in water (pH = 9.75) and acetonitrile. The column and auto-sampler temperature were set at 30 °C and 4 °C respectively, and the injection volume was 3 μL. The QE HFX mass spectrometer was used to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the acquisition software (Xcalibur, Thermo).
The relative abundance of metabolites was calculated using the following formula = 100 × (Peak area of class/Total peak area of metabolites).

Metabolomics profiling was performed in tissues of septic rats and sham control by using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo, United States). The QE HFX mass spectrometer was used to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur, Thermo, United States). The resulting three-dimensional data involving the peak number, sample name, and normalized peak area were used as the input for performing principal component analysis (PCA) in the SIMCA14 software package (Umetrics, Umea, Sweden). The annotated differently expressed metabolites were illustrated as a Volcano plot. Chord diagram was applied for biomarker metabolites depicting distributions and links between potential metabolites. The expression levels of the significantly changed metabolites and metabolic pathway analysis between septic rats and sham group were analyzed by a heatmap and a bubble plot generated by TBtools (Chen et al., 2020 (link)), respectively.

Untargeted metabolomics analysis was conducted as previously described [20 (link)]. Briefly, metabolic extracts were obtained from fecal samples following methanol-assisted protein precipitation and then analyzed by an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column. The raw data were converted to the mzXML format using ProteoWizard and processed with R package xcms [21 ], for peak detection, extraction, alignment, and integration. The in-house MS2 database was applied for metabolite identification. The OPLS-DA was performed using the statistics function prcomp in R (version 4.0.2). In order to avoid overfitting, a permutation test (200 permutations) was performed. The discriminative metabolites between the two groups were identified with variable importance in the projection (VIP) > 1 (determined by OPLS-DA) and P < 0.05 (determined by Student’s t test). The KEGG database (http://www.genome.jp/kegg/) and MetaboAnalyst database (http://www.metaboanalyst.ca/) were used for pathway enrichment analysis.