1.0 ml/min gave good resolution with
no shouldering; therefore, 1 ml/min was used for further study.
The retention time of vitamin D2 was 12.30 ± 0.50 min at 1 ml/
min flow rate of solvent system acetonitrile:methanol:chloroform
(88:8:4) and total run time per sample was 15 min (Fig. 1).