The gradient system consisting of water with 0.1% formic acid and methanol with 0.1% formic acid was used.
A gradient system consisting of solvents A (0.1 % formic acid in water) and B (0.1 % formic acid in acetonitrile) was employed.
The mobile phase of the gradient system consisted of cyclohexane (A) and EtOAc (B).
The chromatographic system consisted of a Shimadzu UFLC binary gradient system (Shimadzu, Reinach, Switzerland) with pumps LC-30AD, vacuum degasser DGU-20A, thermostated column compartment CTO-20 and autosampler SIL-30A.
Chromatographic conditions consisted of a binary gradient system composed of 3% formic acid in water (A) and acetonitrile (B), varied according to the following gradient: t0, A = 68%; t1 = 4.4 min, A = 0%; t2 = 6 min, A = 0% isocratic; t3 = 7 min, A = 68%; t4 = 8 min, A = 68% isocratic.
After a simple solvent precipitation procedure, separation was achieved using an amide column (2.1 mm by 50 mm, 2.7 μm; Advanced Materials Technology, Wilmington, DE, USA) with a binary gradient solvent system consisting of 2.5 to 10 mM acetonitrile-ammonium acetate at pH 3.5.
The binary gradient system of water (mobile A) and acetonitrile (mobile B) containing 0.1% formic acid served as mobile phase.
HPLC was performed with the following binary gradient system: solvent A, water with 0.1% formic acid and solvent B, 100% methanol with 0.1% formic acid.
The mobile phase used was a binary gradient system, with phase A comprising 10 m m-sodium sulphate containing 0·02%% (v/v) phosphoric acid (pH 2·75) and phase B comprising methanol acetonitrile (70 30, v/v).
The binary gradient elution system consisted of 0.001% phosphoric acid in water (A) and 0.001% phosphoric acid in acetonitrile (B).
