Table 2 Different trials for method development for Tolvaptan

Different column

1

Column- Phenomenex Luna C 18 250 × 4.6 mm (10 µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 1 mL/min Inj. Volume: 3 µL Time: 10 min Diluents: Acetonitrile and water as 90:10

Peak shape is adequate but a short Retention Time required

Figure 2

2

Column: Phenomenex Luna C18 250 × 4.60 mm (5 µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 1 mL/min Inj. Volume: 10 µL Time: 10 min Diluents: Acetonitrile and water as 90:10

Peak splitting (peak shape was not adequate)

3

Column: Eclipse plus C18 100 × 4.60 mm (3.5 µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.5 mL/min Inj. Volume- 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Peak shape was not adequate, tailing factor > 2

4

Column: ACE Excel C18 100 × 2.1 mm (2 µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow- 0.2 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Peak shape not adequate, theoretical plate < 2000 main peak starts with another peak, no clear separation

Different mobile phase with different flow rate

5

M.P: Acetonitrile and Methanol in 95:5

Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.1 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates are too low (lot lesser than 2000)

Figure 3

6

M.P –Acetonitrile and Methanol in 95:5

Column- Ace Excel Super C18 Temp- 25 °C Flow-0.3 mL/min Inj. Volume- 3 µL Time- 5 min Diluents- Acetonitrile and water as 90:10

Retention Time is too high

7

M.P: Acetonitrile and Methanol in 95:5

Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.5 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates are too low (less than 2000)

8

M.P: 0.1%TFA in Acetonitrile and methanol in ratio 95:5

Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.1 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Peak not detected

Figure 4

9

M.P: 0.1%TFA in Acetonitrile and methanol in ratio 95:5

Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.3 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates are too low

Different temperature and flow rate

10

Column: ACE Excel Super C18 100 × 2.1 mm (2 µm)

M.P: Acetonitrile and water (95:5) Temp: 45 °C Flow: 0.5 mL/min Inj. Volume- 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are too low

Figure 5

11

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P: Acetonitrile and water ( 95:5) Temp: 45 °C Flow: 0.3 mL/min Inj. Volume: 3 µL Time- 5 min Diluents- Acetonitrile and water as 90:10

Theoretical plate and resolution are too low

12

Column- ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P –Acetonitrile and water ( 95:5) Temp: 45 °C Flow: 0.2 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are good

13

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P: Acetonitrile and water (95:5) Temp: 45 °C Flow: 0.1 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are good but RT is not adequate

14

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.5 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are too low

Figure 6

15

Column: ACE Excel Super C18 100 × 2.1 mm (2 µm)

M.P:Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.3 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates are less than 2000 but resolution is good

16

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.2 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are good and can be adopted in the present method

17

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)

M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.1 mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and water as 90:10

Theoretical plates and resolution are good but RT is not adequate