Acquity uplc csh c18

Re-suspended samples were injected at 3 µL and 5 µL for ESI positive and negative modes respectively, onto a Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id; 1.7 µm particle size) with an additional Waters Acquity VanGuard CSH C18 pre-column (5 × 2.1 mm id; 1.7 µm particle size) maintained at 65 °C was coupled to a Vanquish UHPLC System. To improve lipid coverage, different mobile phase modifiers were used for positive and negative mode analysis^{101 (link)}. For positive mode 10 mM ammonium formate and 0.1% formic acid were used and 10 mM ammonium acetate (Sigma–Aldrich) was used for negative mode. Both positive and negative modes used the same mobile phase composition of (A) 60:40 v/v acetonitrile:water (LC-MS grade) and (B) 90:10 v/v isopropanol:acetonitrile. The gradient started at 0 min with 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). A flow rate of 0.6 mL/min was used. For data acquisition a Q-Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer was used with the following parameters: mass range, m/z 100–1200; MS¹ resolution 60,000: data-dependent MS² resolution 15,000; NCE 20, 30, 40; 4 targets/MS¹ scan; gas temperature 369 °C, sheath gas flow (nitrogen), 60 units, aux gas flow 25 units, sweep gas flow 2 units; spray voltage 3.59 kV.

Instruments used in this study included an ultrahigh-performance liquid chromatograph (UPLC) (Waters 2D UPLC, Waters, USA), a high-resolution mass spectrometer (Q Exactive, Thermo Fisher Scientific, USA), chromatographic column: ACQUITY UPLC CSH C18 (1.7 μm, 2.1*100 mm, Waters, USA), a low-temperature ultracentrifuge (Centrifuge 5430, Eppendorf), a vortex (QL-901, Qilin Beier instrument, China), a water purifier (Milli-Q Integral, Millipore Corporation, USA), and a refrigerated vacuum concentrator (Maxi Vacbeta, GENE COMPANY). Reagents used in this study included LC–MS-grade (Thermo Fisher Scientific, USA) methanol (A454-4), acetonitrile (A996-4), isopropanol (A461-4), ammonium formate (17843-250G, Honeywell Fluka, USA), and formic acid (50,144–50 ml, DIMKA, USA) and water purified by a water purifier.

To detect lipids, 2 µL aliquots of sample solution was injected onto a reversed phase Waters Acquity UPLC CSH C18 (100 mm × 2.1 mm, 1.7 μm) maintained at 60 °C by gradient elution. Mobile phase A was water: ACN (6:4), and mobile phase B was isopropanol: ACN (9:1), both containing 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.3 mL/min, with the elution gradient as follows: 0–4.0 min, 15% B; 4.0–5.0 min, 15–48% B; 5.0–22.0 min, 48%–82% B; 22.0–23.0 min, 82–99% B; 23.0–24.0 min, 99% B; 24.0–24.2 min, 99%–15% B; 24.2–30.0 min, 15% B [24 (link)].
The source and ion transfer parameters applied were as followed: spray voltage 3.5 kV (positive) and 3.0 kV (negative). For both ionization modes, the sheath gas, aux gas, the capillary temperature and the heater temperature were maintained at 35, 15 (arbitrary units), 325 °C and 300 °C, respectively [24 (link)].