Acquity uplc csh c18 vanguard precolumn

The lipid analysis was conducted by UPLC-QToF MS (Agilent 6530 QToF MS coupled with an Agilent 1290 UPLC system; Agilent, Los Angeles, CA, USA). Dried samples were reconstituted with 100 μL of a methanol/toluene mixture (9:1 v/v) and then injected into the UPLC system using an Acquity UPLC SCH C18 column (2.1 × 100 mm, 1.7 μm) combined with an Acquity UPLC CSH C18 VanGuard precolumn (2.1 × 5 mm, 1.7 μm) (Waters, Milford, MA) for separation. The gradient conditions and solvents (A: 60:40 (v/v) acetonitrile:water and B: 90:10 (v/v) isopropanol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid in both mobile phases) were adopted and modified from previous studies [27 (link)]. The information was acquired in positive ion mode. The MS condition was established following a previous study using auto MS/MS data-dependent acquisition mode and scan mode with proper modifications [28 (link)].

For LC-MS analysis, dried samples were re-dissolved in 300 μl acetonitrile:water (80:20; v/v). All measurements were carried out on a Q Exactive HF mass spectrometer coupled with a Vanquish LC system (Thermo Scientific). A sample volume of 5 μl were separated on a Waters Acquity UPLC CSH C₁₈ column (100 × 2.1 mm; 1.7 μm) coupled to an Acquity UPLC CSH C₁₈ VanGuard precolumn (5 × 2.1 mm; 1.7 μm). The column was maintained at 65 °C at a flow rate of 0.6 ml min^− 1. The mobile phases consisted of A: acetonitrile:water (60:40, v/v) with ammonium formate (10 mM) and formic acid (0.1%) and B: 2-propanol:acetonitrile (90:10, v/v) with ammonium formate (10 mM) and formic acid (0.1%). The 15 min separation was conducted under the following gradient: 0 min 15% B; 0–2 min 30% B; 2–2.5 min 48% B;2.5–11 min 82% B; 11–11.5 min 99% B; 11.5–12 min 99% B; 12–12.1 min 15% B; 12.1–15 min 15% B. Orbitrap MS instrument was operated in electrospray ionization (ESI) in positive mode with the following parameters: mass range 60–900 m/z; spray voltage 3.6 kV, sheath gas (nitrogen) flow rate 60 units; auxiliary gas (nitrogen) flow rate 25 units, capillary temperature 320 °C, full scan MS1 mass resolving power 120,000, data-dependent MSMS (dd-MSMS) 4 scans per cycle, dd-MSMS mass resolving power 30,000. Thermo Xcalibur 4.0.27.19 was used for data acquisition and analysis [73 (link)].

The non-polar extracts were reconstituted in 100 μl methanol:toluene (9:1). Untargeted lipidomics analysis was adapted from reference (66 (link)). Briefly, lipid separation was accomplished by chromatography on an ACQUITY UPLC CSH C18 column (1.7 μm, 100 × 2.1 mm) equipped with an ACQUITY UPLC CSH C18 VanGuard pre-column (1.7 μm, 5 × 2.1 mm) (Waters) using the same LC-HRMS instrument as for untargeted metabolomics analysis. The column temperature was set at 65°C and the mobile phase flow rate at 0.6 ml/min. Mobile phases (A) 60:40 (vol/vol) acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%) and (B) 90:10 (vol/vol) isopropanol:acetonitrile with ammonium formate (10 mM) and formic acid (0.1%) were mixed according to the following gradient program: 0 min, 15% (B); 0–2 min, 30% (B); 2–2.5 min, 48% (B); 2.5–11 min, 82% (B); 11–11.5 min, 99% (B); 11.5–12 min, 99% (B); 12–12.1 min, 15% (B); and 12.1–15 min, 15% (B). The sample temperature was maintained at 4°C. Peak picking, lipid MS2 annotation, and data normalization by the LOESS algorithm were performed following the protocol for lipid analysis using MS-DIAL (version 4.48) (67 (link), 68 (link)).