20260216_Muhammad

Liquid chromatography–mass spectrometry (LC–MS) analyses were performed using a Vanquish UHPLC system coupled to a Q Exactive HF quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI) source. Chromatographic separation was achieved on a Kinetex EVO C18 reversed-phase column (50 × 2.1 mm, 1.7 µm particle size, 100 Å pore size; Phenomenex, Torrance, USA). The column temperature was maintained at 25 °C and the autosampler at 14 °C. The mobile phases consisted of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B), both LC–MS grade. The flow rate was set to 0.5 mL min⁻¹ and the injection volume was 5 µL. The gradient elution program was as follows: initial conditions at 5% B; linear increase to 50% B at 8.0 min; linear increase to 99% B at 10.0 min; held at 99% B until 13.0 min; returned to 5% B at 13.1 min and re-equilibrated at 5% B until 16.0 min. The total run time was 16 min. Data-dependent acquisition (DDA) was performed in positive ion mode. Full MS scans were acquired over an m/z range of 150–1500 at a resolution of 30,000 (at m/z 200), followed by MS/MS scans at a resolution of 15,000 on the top 5 most intense precursor ions per cycle. Fragmentation was performed using stepped normalized collision energies (NCE) of 20, 25, and 30. Source parameters in positive ion mode were set as follows: spray voltage 3.5 kV, capillary temperature 250 °C, probe heater temperature 400 °C, sheath gas 50 (arbitrary units), auxiliary gas 12 (arbitrary units), sweep gas 1 (arbitrary units), and S-lens RF level 50.