Trial no. |
UPLC
Conditions |
|
Remark |
1 |
Column |
Inertsil ODS-3, 50 mm × 2.1 mm, 2 μm particles |
Separation of unknown impurity (RT 3.18) from NS is poor |
Mobile phase |
Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2)
Solvent B: Acetonitrile and water in the ratio 90:10 v/v |
Flow rate |
1.0 mL min-1ww |
Gradient |
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 |
2 |
Column |
Waters AQUITY BEH C8, 50 mm × 2.1 mm, 1.7 μm particles |
Separation of unknown impurity (RT 3.18) from NS is poor |
Mobile phase |
Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2)
Solvent B: Acetonitrile and water in the ratio 90:10 v/v |
Flow rate |
1.0 mL min-1 |
Gradient |
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 |
3 |
Column
Mobile phase
Flow rate
Gradient |
Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles |
NS Peak is strongly retained and tailing of NS is more (1.8) |
Solvent A: 0.01 M KH2PO4 buffer, tetra hydro furan and methanol in the ratio 67: 23: 10 (v/v/v), pH adjusted to 4.0 with ortho phosphoric acid.
Solvent B: Acetonitrile and water in the ratio 90:10 v/v |
0.6 mL min-1 |
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 |
NS Peak is strongly retained and tailing of NS is more (1.9) |
4 |
Column |
Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles |
Mobile phase |
Solvent A: 0.01 M ammonium acetate buffer pH adjusted to 7.0 with aqueous ammonia solution
Solvent B: Acetonitrile and water in the ratio 90:10 v/v |
Flow rate |
1.0 mL min-1 |
Gradient program |
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 |
5 |
Column |
Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles |
Separation of unknown impurity (RT 3.18) from NS is satisfactory and tailing for both SS and NS peaks were less than 1.3 |
Mobile phase |
Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2)
Solvent B: Acetonitrile and water in the ratio 90:10 v/v |
Flow rate |
1.0 mL min-1 |
Gradient program |
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 |