Positive and negative ion calibration solutions

High-resolution LC−MS analysis was performed on a ThermoFisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. 1 μL extract was injected and separated using a water-acetonitrile gradient on an Agilent Zorbax Hilic Plus column (150 mm × 2.1 mm, particle size 1.8 μm) maintained at 40°C with a flow rate 0.3 mL/min. HPLC grade solvents were purchased from Fisher Scientific. Solvent A: 0.1% formic acid in water; Solvent B: 0.1% formic acid in acetonitrile. Analytes were separated using a gradient profile as follows: 2 min (95% B) → 20 min (50% B) → 22 min (10% B) → 25 min (10% B) → 27 min (95% B) →30 min (95% B). Mass spectrometer parameters: spray voltage 3.0 kV, capillary temperature 380°C, prober heater temperature 300°C; sheath, auxiliary, and spare gas 60, 20, and 2, respectively; S-lens RF level 50, resolution 240,000 at m/z 200, AGC target 3 × 106. The instrument was calibrated with positive and negative ion calibration solutions (ThermoFisher). Each sample was analyzed in positive and negative modes with m/z range 100–700. As a reference for betaine, betaine hydrochloride was purchased from Sigma-Aldrich. HRMS (ESI) m/z: calculated for C₅H₁₂NO₂⁺, [M + H]⁺: 118.0863, found: 118.0860.

LC−MS was performed on a Thermo Fisher Scientific Vanquish UHPLC system coupled with a Thermo Q-Exactive HF hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. Mass spectrometer parameters were used as spray voltage 3.5 kV, capillary temperature 380 °C, probe heater temperature 400 °C; 60 sheath flow rate, 20 auxiliary flow rate, and one spare gas; S-lens RF level 50, resolution 240,000, AGC target 3 × 10⁶. The instrument was calibrated weekly with positive and negative ion calibration solutions (Thermo Fisher). Each sample was analyzed in negative and positive ionization modes using an m/z range of 100 to 800.