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The analytical solvent gradient was followed by a 16 min re-equilibration gradient to prep the column before the next sample injection that went as follows: 0-0.05 min held at 99% B at 0.25 mL per min, 0.05-1 min from 99% B to 50% B and 0.25 mL per min to 0.1 mL per min, 1-11 min held at 50% B and 0.1 mL per min, 11–11.05 min from 50% B to 0% B at 0.1 mL per min, 11.05–14 min held at 0% B at 0.1 mL per min, 14–14.05 min held at 0% B and increased flow rate from 0.1 mL per min to 0.25 mL per min, and 14.05–16 min held at 0% B and 0.25 mL per min