Zorbax eclipse plus c18 chromatography column

Plasma metabolites were acquired using Agilent 1290 ultra-performance liquid chromatography (UPLC) equipped with 6540 Q-TOF/MS. Zorbax Eclipse plus C18 chromatography column (1.8 μm, 3.0 × 100 mm; Agilent) was adapted for chromatography separation. Acetonitrile (solvent B) and water (solvent A) with 0.1% formic acid were used as the mobile phase with a gradient program as follows: 0–2 min, 5% B; 2–4 min, 5% B to 40% B; 4–5 min, 40 to 50% B; 5–10 min, 50% B; 10–15 min, 50 to 70% B; 15–19 min, 70 to 80% B. The flow rate was 0.8 ml/min, and the injection volume was 5 μl. The column temperature was 40°C. MS was performed in ESI and positive ion mode. The MS parameters were as follows: the mass range was from 100 to 1700; voltage was 3.5 kV; dry gas flow rate was 5 l/min; nebulizer gas was set at 40 psig; capillary temperature was 350°C; internal reference was used for the real-time correction to ensure the stability and accuracy of instrument. Quality control (QC) samples were obtained by pooling 20 µl aliquots of all plasma samples for assessing the quality of the metabolomics workflow. All of the samples were analyzed randomly.

Furthermore, to conduct metabolomics analysis, we employed a UHPLC-HESI-Q-Exactive Plus Orbitrap-MS instrument, which was equipped with a ZORBAX Eclipse Plus C18 chromatography column (2.1 × 100 mm², 1.8 μm; Agilent Technologies, Santa Clara, CA, USA). The optimized gradient consisted of water (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B), with the following gradient: 0–2.5 min, 2–2% B; 2.5–5 min, 2–40% B; 5–12 min, 40–100% B; 12–16 min, 100–100% B; 16–16.1 min, 100–2% B; 16.1–19 min, 2–2% B. The Compound Discoverer 3.2 was employed to preprocess mass spectrometry data, which involved identifying peaks, matching peaks, and correcting retention time. The software used for conducting OPLS-DA and PCA analysis was SIMCA-P 14.1 (Umetrics, AB, Malmo, Sweden). Endogenous metabolites were identified by comparing primary and secondary mass spectrometry fragmentation information using the KEGG and HMDB databases. Furthermore, the composition was verified using MS2 spectroscopy with the internal metabolite fragment spectral library. MetabolAnalyst 5.0 facilitated the analysis of metabolic impacts associated with ALI after RRFJ prophylactic therapy, with metabolic pathways having an influence value >0.1 considered significant.