Hss t3 column

An UltiMate 3000 system (Thermo Scientific, Massachusetts, MA, USA) was used for separation, applying an Acquity HSS T₃ column (2.1 mm ID × 100 mm, 1.8 μm) maintained at 30 °C. The analytes were separated by applying an aqueous solution containing 0.1% formic acid (v/v)-ammonium acetate (5 mmol/L) as eluent (A) and methanol as eluent (B). Gradient elution was performed as follows: 0.00 min~2.00 min, 10.0~40.0% (B); 2.00 min~7.00 min, 40~95.0% (B); 7.00 min~7.01 min, 95~10% (B); 7.01 min to 10.00 min, 10% (B). The flow rate was set to 0.30 mL/min, while 5.0 μL of sample solution was injected.

Serum samples were collected and immediately frozen at −80°C until metabolite extraction. Total metabolite content was extracted from 0.05 g of each of the serum samples, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, performed using the ultrahigh-performance liquid chromatography (UHPLC) system (1290, Agilent Technologies) with an ultraperformance (UPLC) HSS T3 column (2.1 mm by 100 mm, 1.8 μm) coupled to the Q Exactive system (Orbitrap MS; Thermo). MS raw data were filtered if the following criteria were met: (i) metabolites present in less than 50% of all samples in a group were removed, and (ii) missing values were replaced by half of the minimum values found by default in the data set. Peaks were annotated against an in-house MS/MS database constructed using HMDB, Metlin, and Mona. Finally, the relative content of each peak was determined by the normalization method of peak area to generate percentage value for each metabolite.

For sample preparation for metabolomics study, serum (50 μL) was thoroughly mixed with methanol (200 μL, containing 30 μg/mL chlorophenylalanine, internal standards). Then, the sample was centrifuged at 12000 r/min at 4°C for 15 min. The obtained supernatant was used for metabolomics study.
For UPLC-MS condition and data analysis, the serum metabolites profiling was performed on Ultimate 3000 UPLC system (Thermo Fisher Scientific) coupled with an Orbitrap Elite Mass Spectrometer (Thermo Fisher Scientific).
The metabolites were chromatographically separated on a Hss T3 column (100 mm × 2.1 mm, 1.8 μm, ACQUITY UPLC) at a flow rate of 0.3 mL/min for 17 min. Buffer A consisted of 0.1% formic acid in water and buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was set as follows: 0–2 min, 95% A; 2–12 min, 5% A; 12–15 min, 5% A; and 15–17 min, 95% A.
For metabolite identification and pathway analysis, the collected data were processed by Compound Discover 2.0 (Thermo Fisher Scientific) to identify potential biomarkers according to the online database (HMDB, KEGG, m/z cloud). The preprocessed data was imported into SIMCA-P software (Umetrics, Umea, Sweden) for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). MetaboAnalyst 4.0 was used for pathway analysis.