4.5.1. Liquid Chromatography Separation Conditions

Shimadzu liquid chromatography equipped with Kinetex C18 column (2.6 µm particle size, 100 × 3.00 mm, Phenomenex, Torrance, CA, USA) was used. The mobile phases were mobile phase A, 0.5 mM ammonium fluoride, 5 mM ammonium formate and 0.1% formic acid in water; mobile phase B, 0.5 mM ammonium fluoride, 5 mM ammonium formate and 0.1% formic acid in methanol. Samples were eluted using a gradient at a flow rate of 0.45 mL/min throughout the 16 min run-time at 40 ◦C with injection volume of 10 µL. The gradient conditions were optimized as follows: 10% B from 0.01 to 0.5 min, 10–50% B from 0.5 to 10 min, 100% from 10–11 min, 10% 11–13.5 and 0% B from 13.5 to 16 min.
