Waters acquity uplc system

The LC-MS was performed on Water’s ACQUITY UPLC^TM system (Serial No # F09 UPB 920 M; Model code # UPB; Waters Corp., Milford, MA) equipped with a binary solvent delivery system, an auto sampler, column manager, and a tunable MS detector (Serial No # JAA 272; Synapt; Waters, Manchester, UK). Chromatographic separation was performed on a Water’s ACQUITY UPLC^TM BEH C18 (100.0 × 2.1 mm ×1.7 μm) column at 40 ± 5 °C. The mobile phase used for UPLC analysis consisted of 0.5% formic acid (A) and acetonitrile (B), at a pH of 2.7, which was degassed before analysis, and the gradient elution was 2 to 100% (B) in 40 min at a flow rate of 0.5 mL/min and 25 °C. The injection volume was 20 μL. The column and auto sampler were maintained at 40 ± 5 °C and 25 ± 5 °C, respectively, and the pressure of the system was set to 15000 psi.

Waters ACQUITY UPLC^TM system from Waters Corp., MA, USA, hyphenated with a binary system of solvent delivery and tuneable MS detector (Synapt; Waters, Manchester, UK), was used to perform the process of UHPLC whereas Waters ACQUITY UPLC^TM BEH C-18 column with dimension, i.e. 2.1 mm × 100 mm, 1.7 µm was utilized for chromatographic separation. The UHPLC mobile phase conditions were as follows: degassed LC-MS grade solvent, i.e. Acetonitrile (85 %): 10 mM Ammonium Formate (15 %): Formic Acid (0.1 %), v/v/v, with isocratic elution, flow rate of 0.25 mL/min sample injection volume of 10 µl/run and a total chromatographic run time of 5.0 min.

Chromatographic analysis was conducted on Waters Acquity^TM UPLC System, and a Waters Acquity^TM UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) (Waters Corp., Milford, MA, USA) chromatography column was used at a temperature of 50 °C and a flow rate of 0.4 mL/min. The injection sample volume was 5 μL. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B), and the gradient eluting conditions were as follows: 0–2 min, 5–13% A; 2–11 min, 13–25% A; 11–18 min, 25–45% A; 18–20 min, 45–70% A;20–23 min, 70–100% A.
The Waters SynaptTM G2-Si MS^E mass spectrometry system (Waters Corporation, Milford, MA, USA) was used to collect data in both positive and negative ion modes (ESI+, ESI−). The optimized conditions were as follows: ion source temperature 110 °C, desolvation gas temperature 350 °C, cone gas flow rate 50 L/h. In positive ion detection mode, the capillary voltage was 2.8 kV, the sample cone voltage was 20 V, the extraction cone voltage was 4.0 V, and the desolvation gas flow rate 800 L/h. In the negative ion detection mode, the capillary voltage was 2.2 kV, the sample cone voltage was 40 V, the extraction cone voltage was 4.0 V, and the desolvation gas flow was 600 L/h. Leucine enkephalin solution was used as the mass lock solution. The data acquisition interval was 0.2 s/scan, and the mass scanning range was 50–1200 Da.