This document discusses the chromatography methods used to analyze BSN and related impurities. It evaluates four different chromatography columns and mobile phase compositions to achieve separation of BSN and six impurities (Impurities A-E). The Ascentis Express C18 column with a mobile phase of 70% Buffer A and 30% Buffer B achieved acceptable separation, with retention times reported for each component. Linearity and accuracy tests were also performed for method validation.

2. BSN STRUCTURE
• 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-
4-yl]benzene-1-sulfonamide monohydrate
• ASSAY
• RELATED SUBSTANCES
Manpreet kaur et al(2012)

4. Column use FOR ASSAY FOR RELATED SUBSTANCES
Column name
Inertsil ODS 3V Zodiac C18 Inertsil ODS-3 Ascentis express
C18
mode of
chromatography Reversed Phase Reversed Phase Reversed Phase Reversed Phase
operating pH 2-10 2-10 2-9 2-10
particle size 5um 5um 5um 2.7um

5. Mobile phase Buffer: Acetonitrile (60:40)
Column temperature 40° c
Flow rate 2.0ml/min
Injection volume 10 μl
Wavelength 220nm

6. Mobile phase Buffer: Acetonitrile (45:55)
Column temperature 40° c
Flow rate 2.0ml/min
Injection volume 20 μl
Wavelength 205nm

10. Impurity E Impurity A Impurity D
Observation: - Impurity B eluting at the retention time of BSN. ImpurityC is not eluted till 30min
Sample RetentionTime
Impurity E 1.378
Impurity D 1.620
Impurity B 4.621
BSN 4.654
ImpurityA 12.651
ImpurityC Not Eluted

11. Time(minute) %A %B
0.0 90 10
20.0 90 10
30.0 10 90
40.0 10 90
50.0 90 10
60.0 90 10

12. Sample Retention Time
Impurity E 24.051
Impurity D 24.741
Impurity B 26.210
BSN 26.185
Impurity A 27.334
Impurity C 28.700

13. Time(minute) %A %B
0.01 90 10
12.0 90 10
15.0 10 90
20.0 10 90
30.0 90 10

14. Time(minute) %A %B
0.00 70 30
17.0 70 30
22.0 30 70
25.0 30 70
27.0 70 30
30.0 70 30

15. Sample RetentionTime
Impurity E 2.052
Impurity D 2.420
Impurity B 12.955
BSN 14.393
ImpurityA 20.544
ImpurityC 22.545

16. Time(minute) %A %B
0.00 70 30
17.0 70 30
22.0 40 60
25.0 40 60
27.0 70 30
30.0 70 30

17. Sample RetentionTime
Impurity E 1.723
Impurity D 2.058
Impurity B 12.442
BSN 13.880
ImpurityA 21.109
ImpurityC 24.179

18. Name Concentration % RetentionTime
Lin level 1 50 1.721
Lin level 2 80 1.720
Lin level 3 100 1.722
Lin level 4 120 1.720
Lin level 5 150 1.719

23. Name Concentration % RetentionTime
Lin level 1 50 2.456
Lin level 2 75 2.455
Lin level 3 100 2.455
Lin level 4 125 2.456
Lin level 5 150 2.456

25. Name % Recovery %RSD
Accuracy level 1 101.1
Accuracy level 2 100.7 0.8
Accuracy level 3 100.7