Isolation and characterization of novel degradation ... · S-1 Isolation and characterization of...

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Isolation and characterization of novel degradation products in Cilnidipine by LC-QTOF-MS/MS, LCMSn,

2D-NMR and FTIR

Ch Krishnam Raju1,2 , J V Shanmukha Kumar*1, Sanath Kumar Goud2

1Department of chemistry,K L University, Guntur, Andhra Pradesh-522502, India.

2Analytical Research and Development, United States Pharmacopeial Convention – India (P) Ltd., Plot No. D6 & D8, IKP, Genome

Valley, Shameerpet, Hyderabad, Telangana-500078, India.

List of figures:

1. HPLC chromatogram ofCIL degradation products S-6

2. UPLC chromatogram of mixed sample solution of CIL with degradants S-7

3. Final purity of CD1 S-8

4. Final purity of CD2 S-8

5. Final purity of CD3 S-9

6. Chiral purity of CIL S-9

7. High resolution mass spectrum of CIL S-10

8. Mass accuracy of CIL S-11

Electronic Supplementary Material (ESI) for New Journal of Chemistry.This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2017

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9. High resolution MSMS Spectrum of CIL S-12

10. MSnspectrum of CIL S-13

11. Mass fragmentation pathway of CIL S-14

12. High resolutionmass Spectrum of CD1 S-15

13. Mass accuracy of CD1 S-16

14. High resolutionMSMS Spectrum of CD1 S-17

15. MSn spectrum of CD1 (a) S-18

16. MSn spectrum of CD1 (b) S-19

17. Mass fragmentation pathway of CD1 S-20

18. High resolutionmass Spectrum of CD2 S-21

19. Mass accuracy of CD2 S-22

20. High resolutionMSMS Spectrum of CD2 S-23

21. MSn spectrum of CD2 (a) S-24

22. MSn spectrum of CD2 (b) S-25

23. Mass fragmentation pathway of CD2 S-26

24. High resolution mass Spectrum of CD3 S-27

25. Mass accuracy of CD3 S-28

26. High resolution MSMS Spectrum of CD3 S-29

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27. MSn spectrum of CD3 S-30

28. Mass fragmentation pathway of CD3 S-31

29. High resolution mass Spectrum of KD1 S-32

30. Mass accuracy of KD1 S-33

31. GCMS Spectrum of KD2 S-34

32. GCMS Library match spectrum for KD2 S-35

33. High resolution mass Spectrum of KD3 S-36

34. Mass accuracy of KD3 S-37

35. High resolution mass Spectrum of KD4 S-38

36. Mass accuracy of KD4 S-39

37. 1H NMR Spectrum of CD1 S-40

38. 13C NMR Spectrum of CD1 S-41

39. DEPT90 NMR Spectrum of CD1 S-42

40. DEPT135 NMR Spectrum of CD1 S-43

41. COSY NMR Spectrum of CD1 S-44

42. TOCSY NMR Spectrum of CD1 S-45

43. HSQC NMR Spectrum of CD1 S-46

44. HMBC NMR Spectrum of CD1 S-47

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45. NOESY NMR Spectrum of CD1 S-48

46. ROESY NMR Spectrum of CD1 S-49

47. ROESY NMR Zoomed Spectrum of CD1 S-50

48. 1H NMR Spectrum of CD2 S-51

49. 13C NMR Spectrum of CD2 S-52

50. DEPT90 NMR Spectrum of CD2 S-53

51. DEPT135 NMR Spectrum of CD2 S-54

52. COSY NMR Spectrum of CD2 S-55

53. TOCSY NMR Spectrum of CD2 S-56

54. HSQC NMR Spectrum of CD2 S-57

55. HMBC NMR Spectrum of CD2 S-58

56. NOESY NMR Spectrum of CD2 S-59

57. ROESY NMR Spectrum of CD2 S-60

58. ROESY NMR Zoomed Spectrum of CD2 S-61

59. 1H NMR Spectrum of CIL S-62

60. 13C NMR Spectrum of CIL S-63

61. DEPT90 NMR Spectrum of CIL S-64

62. DEPT135 NMR Spectrum of CIL S-65

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63. COSY NMR Spectrum of CIL S-66

64. TOCSY NMR Spectrum of CIL S-67

65. HSQC NMR Spectrum of CIL S-68

66. HMBC NMR Spectrum of CIL S-69

67. NOESY NMR Spectrum of CIL S-70

68. 1H NMR Spectrum of CD3 S-71

69. 13C NMR Spectrum of CD3 S-72

70. DEPT90 NMR Spectrum of CD3 S-73

71. DEPT135 NMR Spectrum of CD3 S-74

72. COSY NMR Spectrum of CD3 S-75

73. TOCSY NMR Spectrum of CD3 S-76

74. HSQC NMR Spectrum of CD3 S-77

75. HMBC NMR Spectrum of CD3 S-78

76. IR Spectrum of CD1 S-79

77. IR Spectrum of CD2 S-80

78. IR Spectrum of CD3 S-81

79. Prep HPLC chromatogram for the isolation of CD3 S-82

80. Prep HPLC chromatogram for the isolation of CD1 and CD2. S-83

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Figure 1: HPLC chromatograms Base degradation (a), Acid degradation (b), Photolytic degradation (c), Oxidative degradation (d),Heat degradation (e),Humid degradation (f)

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Figure2:UPLC chromatogram of mixed sample solution of CIL with degradants.

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Figure3:Final purity of CD1

Figure4:Final purity of CD2

Pea

k1 -

15.9

91

Pea

k2 -

17.4

30C

D-I

- 18.

221

Pea

k4 -

19.0

00P

eak5

- 19

.952

AU

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

Pea

k1 -

13.3

70

Pea

k2 -

16.1

52

Pea

k3 -

18.1

90C

D-II

- 18

.979

Pea

k5 -

20.9

06P

eak6

- 21

.031

Pea

k7 -

22.0

67

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

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Figure5:Final purity of CD3

Figure6: Chiral purity of CIL

CD

3 - 2

9.10

6P

eak2

- 30

.299

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00

CIL

-Isom

er-1

- 5.

404

CIL-

Isom

er-2

- 6.

370

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

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Figure7:High resolution mass spectrum of CIL

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Figure 8:Mass accuracy of CIL

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Figure9: High resolution MSMS Spectrum of CIL

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Figure 10: MSn spectrum of CIL

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H2N

O OO

OO

NO2C27H29N2O7

+, 493.1969Nitrogen rule: Even, RDBE: 14.5

CIL

C27H27N2O6+, 475.1864

Nitrogen rule: Even, RDBE: 15.5

N

O OO

O

NO2

-H2O

C9H9+, 117.0699

Nitrogen rule: Even, RDBE: 5.5

-C18H20N2O7

N

HOHO O

OO

NO2

C18H19N2O7+, 375.1187

Nitrogen rule: Even, RDBE: 10.5

-C9H10

N

HO OH2

OO

NO2

C15H13N2O6+, 317.0768

Nitrogen rule: Even, RDBE: 10.5

-C3 H

6 O

N

HO

O

NO2

C14H11N2O4+, 271.0713

Nitrogen rule: Even, RDBE: 10.5

-CH2 O

2

-C3H8O2

-C9H8

N

H2O

O

C14H12NO2+, 226.0863

Nitrogen rule: Odd, RDBE: 9.5

N

O

OO

NO

C24H21N2O5+, 417.1445

Nitrogen rule: Even, RDBE: 15.5

OH2

N

HO

OO

NOC15H13N2O5

+, 301.0819Nitrogen rule: Even, RDBE: 10.5

OH2

-CHN

O 4

HN

O

C14H12NO+, 210.0913Nitrogen rule: Odd, RDBE: 9.5

-C10H

8 NO4

N

HO

O

NO

C14H11N2O3+, 255.0764

Nitrogen rule: Even, RDBE: 10.5

-CH2 O

2

Figure 11: Mass fragmentation pathway of CIL

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Figure 12:High resolution mass spectrum of CD1

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Figure13: Mass accuracy of CD1

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Figure14:High resolution MSMS Spectrum of CD1

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Figure15:MSn Spectrum of CD1 (a)

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Figure16:MSn Spectrum of CD1 (b)

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C27H31N2O10+, 543.1973

Nitrogen rule: Even, RDBE: 13.5CD1

O O

O

O

O

O

O

NO2

NH2HO

C26H31N2O8+, 499.2075

Nitrogen rule: Even, RDBE: 12.5

O O

O

O

O

NO2

NH2HO

C26H29N2O6+, 465.2020

Nitrogen rule: Even, RDBE: 13.5

HO

O

O

NO2

HON

NH2

NO2

O

O

OO

OO

H

C18H21N2O8+, 393.1292

Nitrogen rule: Even, RDBE: 9.5

NH

NO2

OOH

OO

H

C17H21N2O6+, 349.1394

Nitrogen rule: Even, RDBE: 8.5

C23H21N2O4+, 389.1496

Nitrogen rule: Even, RDBE: 13.5

HO

O

NO2

N

C14H16NO+, 214.1226Nitrogen rule: Odd, RDBE: 7.5

H2O

N

N

C12H14N+, 172.1121Nitrogen rule: Odd, RDBE: 6.5

C12H11+, 155.0855

Nitrogen rule: Even, RDBE: 7.5

C26H30N2O7•+, 482.2048

Nitrogen rule: Even, RDBE: 13.0

O O

O

O

O

NO2

NH2

NH2

OO

H

C7H12NO2+, 142.0863

Nitrogen rule: Odd, RDBE: 1.5

HN

NO2

O

C15H17N2O3+, 273.1234

Nitrogen rule: Even, RDBE: 8.5

HN

NO2

C14H17N2O2+, 245.1285

Nitrogen rule: Even, RDBE: 7.5

C9H9+, 117.0699

Nitrogen rule: Even, RDBE: 5.5

-C18H22N2O10

-CO2

-H2O2

-OH.

-C 12H 13NO5

-C2H2O -C2H5NO

-C3H8O2

-C3 H

10O3

-C9H10O-C10H10O2

-C9H10O2

-CO2-C11H9NO6

-C2H4O3-C3H4O4

O

NO2

N

C14H11N2O4+, 271.0713

Nitrogen rule: Even, RDBE: 10.5

HO

NO2

N

C13H11N2O3+, 243.0764

Nitrogen rule: Even, RDBE: 9.5

HO

O

O

NO2

N

C23H21N2O5+, 405.1445

Nitrogen rule: Even, RDBE: 14.5

HO

Figure17: Mass fragmentation pathway of CD1

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Figure18:High resolution mass Spectrum of CD2

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Figure19:Mass accuracy of CD2

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Figure20:High resolution MSMS Spectrum of CD2

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Figure21: MSn spectrum of CD2 (a)

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Figure 22: MSn spectrum of CD2 (b)

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C27H31N2O10+, 543.1973

Nitrogen rule: Even, RDBE: 13.5CD2

O O

O

O

O

O

O

NO2

NH2 OH

C9H9+, 117.0699

Nitrogen rule: Even, RDBE: 5.5

C26H31N2O8+, 499.2075

Nitrogen rule: Even, RDBE: 12.5

O O

O

O

O

NO2

NH2 OH

-CO2

C26H29N2O6+, 465.2020

Nitrogen rule: Even, RDBE: 13.5

HO

O

O

NO2

N

OH

-H 2O2

C23H21N2O5+, 405.1445

Nitrogen rule: Even, RDBE: 14.5

O

O

NO2

N

OH

-C3H10O3

O O

O

O

NH2

OH

C18H22NO5+, 332.1492

Nitrogen rule: Odd, RDBE: 8.5

-C9H9NO5

O

C9H9O+, 133.0648Nitrogen rule: Even, RDBE: 5.5

-C9H13NO4

C8H9+, 105.0699

Nitrogen rule: Even, RDBE: 4.5

-CO

-C18H22N2O10

C27H29N2O9+, 525.1868

Nitrogen rule: Even, RDBE: 14.5

O O

O

O

O

O

O

NO2

NH2

-H2O

C26H29N2O7+, 481.1969

Nitrogen rule: Even, RDBE: 13.5

O O

O

O

O

NO2

NH2

-CO2

C18H19N2O7+, 375.1187

Nitrogen rule: Even, RDBE: 10.5

H2O

O

O

O

O

NO2

N

-C9H10O2

O

NO2

N

C14H11N2O4+, 271.0713

Nitrogen rule: Even, RDBE: 10.5

HO

-C4H8O3

C26H27N2O8+, 495.1762

Nitrogen rule: Even, RDBE: 14.5

O O

O

O

O

O

NO2

NH2

-CH2O

C15H12NO6+, 302.0659

Nitrogen rule: Odd, RDBE: 10.5

O O

O

O OH

O

NH2

-C11H15NO2

C13H10NO4+, 244.0604

Nitrogen rule: Odd, RDBE: 9.5

O O

OH

O

NH2

-C2H2O2

NH2

O

OO

C10H18NO3+, 200.1281

Nitrogen rule: Odd, RDBE: 2.5

-C17H11NO6

NH2

OO

C7H12NO2+, 142.0863

Nitrogen rule: Odd, RDBE: 2.5

-C3H6O

C18H23N2O10+, 427.1347

Nitrogen rule: Even, RDBE: 8.5

O O

HO

O

O

O

O

NO2

NH2 OH -C9H8

Figure 23: Mass fragmentation pathway of CD2

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Figure24:High resolution mass Spectrum of CD3

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Figure 25: Mass accuracy of CD3

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Figure26:High resolution MSMS Spectrum of CD3

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Figure27: MSn spectrum of CD3

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-C3H6O

Figure28: Mass fragmentation pathway of CD3

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Figure29:High resolution mass Spectrum of KD1

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Figure30: Mass accuracy of KD1

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Figure31: GCMS spectrum of KD2

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Figure32: GCMS Library match spectrum for KD2

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Figure 33:High resolution mass Spectrum of KD3

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Figure 34: Mass accuracy of KD3

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Figure 35:High resolution mass Spectrum of KD4

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Figure36: Mass accuracy of KD4

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Figure37: 1H NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure38:13C NMR Spectrum of CD1 (126 MHz, DMSO-d6, 298K)

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Figure39:DEPT90 NMR Spectrum of CD1 (126 MHz, DMSO-d6, 298K)

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Figure 40: DEPT135 NMR Spectrum of CD1 (126 MHz, DMSO-d6, 298K)

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Figure 41: COSY NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure 42: TOCSY NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure 43: HSQC NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure 44: HMBC NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure45: NOESY NMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure46:ROESYNMR Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure47: ROESY NMR Zoomed Spectrum of CD1 (500 MHz, DMSO-d6, 298K)

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Figure48: 1H NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure49:13C NMR Spectrum of CD2 (126 MHz, DMSO-d6, 298K)

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Figure 50:DEPT90 NMR Spectrum of CD2 (126 MHz, DMSO-d6, 298K)

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Figure 51:DEPT135 NMR Spectrum of CD2 (126 MHz, DMSO-d6, 298K)

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Figure 52: COSY NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure 53: TOCSY NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure 54: HSQC NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure55: HMBC NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure56: NOESY NMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure57:ROESYNMR Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure58: ROESY NMR Zoomed Spectrum of CD2 (500 MHz, DMSO-d6, 298K)

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Figure59: 1H NMR Spectrum of CIL (500 MHz, DMSO-d6, 298K)

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Figure60:13C NMR Spectrum of CIL (126 MHz, DMSO-d6, 298K)

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Figure61:DEPT90 NMR Spectrum of CIL (126 MHz, DMSO-d6, 298K)

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Figure62:DEPT135 NMR Spectrum of CIL (126 MHz, DMSO-d6, 298K)

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Figure63: COSY NMR Spectrum of CIL (500 MHz, DMSO-d6, 298K)

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Figure64: TOCSY NMR Spectrum of CIL(500 MHz, DMSO-d6, 298K)

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Figure65: HSQC NMR Spectrum of CIL(500 MHz, DMSO-d6, 298K)

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Figure66: HMBC NMR Spectrum of CIL(500 MHz, DMSO-d6, 298 K)

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Figure67: NOESY NMR Spectrum of CIL(500 MHz, DMSO-d6, 298K)

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Figure68: 1H NMR Spectrum of CD3 (500 MHz, DMSO-d6, 298K)

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Figure69:13C NMR Spectrum of CD3 (126 MHz, DMSO-d6, 298K)

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Figure70:DEPT90 NMR Spectrum of CD3 (126 MHz, DMSO-d6, 298K)

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Figure71:DEPT135 NMR Spectrum of CD3 (126 MHz, DMSO-d6, 298K)

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Figure72: COSY NMR Spectrum of CD3 (500 MHz, DMSO-d6, 298K)

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Figure73: TOCSY NMR Spectrum of CD3(500 MHz, DMSO-d6, 298K)

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Figure74: HSQC NMR Spectrum of CD3(500 MHz, DMSO-d6, 298K)

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Figure75: HMBC NMR Spectrum of CD3(500 MHz, DMSO-d6, 298 K)

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Figure76:IRSpectrumofCD1

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Figure77:IRSpectrumofCD2

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Figure78:IR Spectrum of CIL

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Figure 79: Prep HPLC chromatogram for the isolation of CD3.

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Figure 80: Prep HPLC chromatogram for the isolation of CD1 and CD2.