The 20 min LC-MS experiment started by ramping the mobile phase B from 40% to 43% within 2 min, to 50% within 2.1min, to 54% within 12min to 70% within 12.1min and finally 99% within 18min. It was decreased to 40% within 18.1min and kept there until 20min to re-equilibrate the column. The injection volume was 5μL.