Ultimate 3000 ultra high performance liquid chromatograph

LC-MS/MS analysis was conducted on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph (UHPLC) coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). Chromatographic separation was achieved with an ACQUITY Amide BEH column (1.7 μm, 2.1 × 150 mm) maintained at 30 °C (Waters, Milford, MO). Mobile phase compositions for solvents A and B consisted of ACN/H₂O (95:5, v/v) and (50:50, v/v) respectively, with 10 mM ammonium acetate. The gradient profile had a flow rate of 0.4 mL min⁻¹ and ramped from 0.1 to 30% B in 5 min, from 30 to 90% B in 0.1 min, held at 90% B for 1 min, dropped from 90 to 0.1% B in 0.4 min, and held 0.1% B for 1.5 min. Total chromatographic run time was 8.0 min. The injection volume was 2 μL. All analytes were detected using positive ionization mode and SRM. Electrospray ionization (ESI) source conditions are included in supporting information (SI) Table S1. Collision energies and RF lens voltage were optimized for each reference standard and shown in SI Table S2 along with the precursor to product ion transitions. Data collection and analysis was done using Xcalibur 4.2 Qual and Quan Browser (Thermo Scientific, San Jose, CA).