Table 1

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

Table 1: Results from different method development trials.