Analytical Method Development: Are we solving the right ... · Analytical Method Development: Are...

Imad Haidar Ahmad 7Feb2018

Merck

[email protected]

Analytical Method Development: Are we solving the right

problem?

A systematic approach to select an appropriate RPLC column and to

optimize separation.

Work done during my time at Novartis pharmaceuticals and was

approved for external use.

Increase the chances of successful optimizing of analytical

methods using ACD labs Recommendation to follow before, while, and after method

optimization using ACD labs.

3

Take Home Message

For a successful separation, you need 1. Careful selection of a library of column to

screen 2. Protocol for best candidate column selection 3. Use of ACD labs to optimize the separation

The fist two steps are very essential for the success of ACD labs optimization. Have you ever had ACD fail in optimizing a separation?

4

Take Home Message

For a successful separation, you need • Careful selection of a library of column to

screen • Protocol for best candidate column selection • Use of ACD labs to optimize the separation

5

Importance of column choice

General Resolution Equation (Purnell Eq.)

𝑅𝑠 ≈𝑁

4×(𝛼 − 1)

𝛼×

𝑘′

1 + 𝑘′

• k’: is the retention coefficient

• N: is the number of theoretical plates

• : is the selectivity factor

The column is the heart of chromatography

Haidar Ahmad, Imad "Necessary analytical skills and knowledge for identifying, understanding, and

performing HPLC troubleshooting." Chromatographia 80, 5 (2017): 705-730.

6

Importance of column choice

General Resolution Equation (Purnell Eq.)

𝑅𝑠 ≈𝑁

4×(𝛼 − 1)

𝛼×

𝑘′

1 + 𝑘′

• k’: is the retention coefficient

• N: is the number of theoretical plates

• : is the selectivity factor

The column is the heart of chromatography

Haidar Ahmad, Imad "Necessary analytical skills and knowledge for identifying, understanding, and

performing HPLC troubleshooting." Chromatographia 80, 5 (2017): 705-730.

Optimized by ACD labs

Analyst job

7

Our approach to select columns using HSM (hydrophobic subtraction model)

Retention mechanism – relation between retention and descriptors

Coulombic κ’ C

H-bond basicity α’ B

H-bond acidity β’ A

Steric hindrance σ’ S

Hydrophobic η’ H

solute stationary phase interaction type

parameters

Retention contributions from (hydrophobicity – steric hindrance + H-Bond acidity & basicity + columbic interactions)

8

Hydrophobic Subtraction Model

Journal of Chromatography A, 961 (2002) 171–193

5 unknowns

67 equations

κ’

α’

β’

σ’

η’

Solutes with known

9

Our approach

www.hplccolumns.org or http://www.usp.org/app/USPNF/columnsDB.html

http://www.hplccolumns.org/

http://www.usp.org/app/USPNF/columnsDB.html

http://www.usp.org/app/USPNF/columnsDB.html

10

Choosing columns of different selectivity using HSM model

2

21

2

21

2

21

2

21

2

21

))(*83())(*143(

))(*30())**(*100())(*13(

CCBB

AASSHHFS

Fs is essentially the distance separating 2 columns in a 5 dimensional

parameter space. It is the basis for ranking the similarity and

difference between two columns.

• Fs < 3 are considered excellent matches (equivalent)

• Fs : 5-10 are considered reasonable matches (similar)

• 10 < Fs < 50 start becoming different

• Fs > 50 are considered orthogonal (very different)

Visual aid for HSM model to select the right stationary phase

Max value

Min value

11

12

Buckets of columns with similar selectivity

13

Buckets of columns with similar selectivity

1. excel C18 amid

2. discovery amid C16 3. X select CSH phenyl-Hexyl 4. X bridge shield RP18

5. Xselect CSH C18

1. SymmetryShield C18 2. Ascentis RP-Amide (supelco 3. Symmetry Shield C8 (waters) 4. Ascentis Express RP-Amide

(supelco) 5. poroshell 120 bonus RP 6. halo RP amid 7. Ascentis Express Rp-Amide

1. Zorbax SB CN

2. Zorbax SB -C3 3. Precision CN (MAC-MOD) 4. Discovery CN supelco 5. ultra PFP (Restek) 6. XSelect CSH Fluoro-

Phenyl

1. Zorbax Extend C18 (Agilent)

2. Halo C18 (Waters) 3. Ascentis C18/supelco

4. kinetx C18 (phenomenex)

5. Accucore C18 (Thermo) 6. Zorbax Eclipse XDB-C18

(Agilent)

7. TSKgel ODS - 100 S (TOSOH)

8. kinetx C18 (phenomenex)

9. ULTracore super C18 (ACT)

10. Xbridge C18- waters

11. Zorbax SB C18 80 A(Agilent)

12. TSKgel ODS-80T 13. poroshell SB c18

14. MacMod precision c18

15. Zorbax Eclipse plus (C18) Polaris Amide C18

Zorbax bonus C18

14

Average parameters for each bucket

FS C1

C1 0 C2

C2 24 0 C3

C3 259 242 0 C4

C4 151 135 112 0 C5

C5 61 45 201 92 0 C6

C6 30 22 245 136 45 0

2

21

2

21

2

21

2

21

2

21

))(*83())(*143(

))(*30())**(*100())(*13(

CCBB

AASSHHFS

~ 1 week

Select best

column

Column library

Approach for simple separations (few peaks)

Peak width, tailing, number

of components resolved,

relative retention of columns

. . .

15

For a very challenging separation

16

Example: an impurity method more than 30 peaks – will be shown in the next slides

For a very challenging separation

17

Example: an impurity method more than 30 peaks – will be shown in the next slides

18

Importance of corner points


’ ’ ’ ’ ’

Ketoprofen -0.589 0.296 -0.044 0.546 0.005

Nitrobenzene -0.579 0.322 -0.009 0.01 -0.036

’ ’ ’ ’ ’

Ketoprofen -0.589 0.296 -0.044 0.546 0.005

Nitrobenzene -0.579 0.322 -0.009 0.01 -0.036

19



CBASHresolution '''*''

20



CBASHresolution '''*''

B Selectivity

(k’ketoprophen/K’Nitrobenzene)

Intersil -0.0255 1.09

Symmetry -0.0289 1.09

SB-100 0.0064 1.02

SB-90 0.0093 1.06

SB-300 0.0761 1.32

Eclipse -0.0325 1.09

YMC 17 -0.0128 1.02

YMC 16 -0.0105 1.01

YMC 17 -0.0099 1.02

Discovery 0.0163 0.96

’ ’ ’ ’ ’

Ketoprofen -0.589 0.296 -0.044 0.546 0.005

Nitrobenzene -0.579 0.322 -0.009 0.01 -0.036

Haidar Ahmad, I. Chromatographia, just accepted.

Our column library

21

22

Haidar Ahmad, I. Chromatographia, just accepted.

Take Home Message



24

Take Home Message



1’ priority should be: separating the peaks of interest.

2’ priority: peak shape, pH and T range, etc.

Useless !

column:

great performance but

Not separating my peaks

N

CH3

CH3

N

NH2

OH

O

N

OH

NH2

OH

O

NH

OF

F

F

N

NO

CH3

CH3

NH

CH3

O

O

CH3

NH

HO

NH

OH

NH

O OCH3

NH

OH

NH

OCH3

NH

NOO

NH

NH

NH

CH3

NH

OH

CH3

N

CH3

N NH

NH

OH

NH

OO

CH3

OH

NH

CH3

O

N

O

CH3

O

NH2

NH

O

N

NH

NH

O

25

Structures of

indoles, obtained

from Sigma Aldrich

10 mobile phase A

10 mobile phase B

6 columns

Column screening

27

Column

#

A1B1 (pH 3.5 : methanol) A1B2(pH 3.5 : ACN) A2B1 (pH 5 : methanol) A2B2(pH 5 : ACN) # of

peaks W1/2 Asy

tR

(last peak)

# of

peaks W1/2 Asy

tR

(last peak)

# of

peaks W1/2 Asy

tR

(last peak)

# of

peaks W1/2 Asym

tR

(last peak)

1 20 (1) 0.11 1.09 14 18 (2) 0.15 1.10 15 19(0) 0.11 1.08 13 18 (2) 0.13 1.1 14

2 20 (1) 0.13 1.06 15 21 (1) 0.11 1.06 16 20 (1) 0.14 1.36 15 21(1) 0.11 1.1 14

4 20 (0) 0.12 1.03 14 19 (0) 0.12 1.64 15 19 (0) 0.12 1.05 14 19(0) 0.12 1.1 14

6 17 (2) 0.14 1.07 16 20 (0) 0.09 1.01 13 19(2) 0.10 0.99 15 20(0) 0.10 1.01 13

7 20(0) 0.09 1.05 16 21(0) 0.11 1.03 16 20(1) 0.09 1.05 13 21(1) 0.11 1.02 14

8 21(0) 0.09 1.02 13 19(1) 0.11 1.03 15 20(0) 0.10 1.01 14 19(1) 0.10 1.02 13

9 20(3) 0.2 1.01 15 19(2) 0.17 0.89 15 19(1) 0.21 1.02 16 19(2) 0.3 1.02 13

10 20(1) 0.09 1.01 14 20(2) 0.08 1.05 13 19(0) 0.09 1.09 13 20(2) 0.10 1.05 14

11 20(2) 0.09 0.94 14 20(1) 0.11 0.90 14 19 0.11 0.88 14 20(1) 0.11 0.88 14

12 17(2) 0.12 0.99 14 19(1) 0.11 1.01 14 19(3) 0.12 1.02 15 19(0) 0.11 1.01 14

13 19(1) 0.12 1.17 15 19(1) 0.11 1.04 14 19(1) 0.12 1.04 14 19(1) 0.12 1.04 14

14 19(2) 0.12 1.37 14 18(1) 0.12 1.08 14 19(2) 0.12 1.09 14 19(2) 0.12 1.09 14

28

Run initial gradient out

Poor retention, peak

shape, or asymmetry

29

Run initial gradient

Adjust gradient so that last

peak elutes ~ comparable tR

out


shape, or asymmetry

out

Columns missing

more than 2 peaks

30




out


shape, or asymmetry

out

Columns missing

more than 2 peaks

Run 3 gradients (of different

slopes) out

Can’t see almost all

peaks

31




out


shape, or asymmetry

out

Columns missing 2

peaks or more


slopes) out


peaks

Compare # of resolved peaks

,w1/2, and asymmetry out


peaks

32




out


shape, or asymmetry

out

Columns missing 2

peaks or more


slopes) out


peaks

Compare # of resolved peaks

,w1/2, and asymmetry out


peaks

Pick your best column (check

reproducibility on different batches)

33

12

13

14

15

16

17

18

19

20

21

22

0 2 4 6 8 10 12 14 16

Nu

mb

er

of

pe

aks

ob

serv

ed

(in

clu

din

g co

-elu

tin

g p

eaks

)

Column number

Condition 1

Condition 2

Condition 3

Condition 4

21.35 21.40 21.50 21.60 21.70 21.80 21.90 22.00 22.10 22.20 22.30 22.40 22.50 22.60 22.70 22.80 22.85

-278

-250

-200

-150

-100

-50

0

50

100

144IHA141023_01 #406 [modified by haidaim1] sample_to_column_2 Channel_AmAU

min

18 -

21.8

20

19 -

22.1

03

WVL:215 nm

34

12

13

14

15

16

17

18

19

20

21

22

0 2 4 6 8 10 12 14 16

Nu

mb

er

of

pe

aks

ob

serv

ed

(in

clu

din

g co

-elu

tin

g p

eaks

)

Column number

Condition 1

Condition 2

Condition 3

Condition 4

35

0

1

2

3

4

0 2 4 6 8 10 12 14 16

Nu

mb

er

of

co-e

luti

ng

pai

rs o

f p

eak

s

Column number

Condition 1

Condition 2

Condition 3

Condition 4

21.35 21.40 21.50 21.60 21.70 21.80 21.90 22.00 22.10 22.20 22.30 22.40 22.50 22.60 22.70 22.80 22.85

-278

-250

-200

-150

-100

-50

0

50

100

144IHA141023_01 #406 [modified by haidaim1] sample_to_column_2 Channel_AmAU

min

18 -

21.8

20

19 -

22.1

03

WVL:215 nm

36

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 2 4 6 8 10 12 14 16

Pe

ak w

idth

Column number

Condition 1

Condition 2

Condition 3

Condition 4

37

12

13

14

15

16

17

18

19

20

21

22

0 2 4 6 8 10 12 14 16

Nu

mb

er

of

pe

aks

ob

serv

ed

(in

clu

din

g th

e u

nfu

lly r

eso

lved

pea

ks)

Column number

Condition 1

Condition 2

Condition 3

Condition 4

Column

#

A1B1 (pH 3.5 :

methanol) A1B2(pH 3.5 : ACN)

A2B1 (pH 5 :

methanol) A2B2(pH 5 : ACN)

# of

peaks W1/2 Asy

# of

peaks W1/2 Asy

# of

peaks W1/2 Asy

# of

peaks W1/2 Asym

2 21(1) 0.13 1.06 21 (1) 0.11 1.06 21(1) 0.14 1.36 21(1) 0.11 1.1

7 21(1) 0.09 1.05 21(0) 0.09 1.05 21(0) 0.11 1.02

8 21(0) 0.09 1.02

38 38

0.2 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.3

-323

-250

-200

-150

-100

-50

0

50

100

150

200

250

300

350

400

450

526

1 - IHA141023_01 #411 [modified by haidaim1] sample_to_column_8 Channel_A

2 - IHA141023_01 #442 [modified by haidaim1] sample_to_column_7 Channel_AmAU

min

2

1

1 -

2.1

33

2 -

3.0

20 3 -

4.2

30

4 -

4.7

20

5 -

7.0

33

6 -

7.8

63

7 -

8.7

63

8 -

9.3

90 9

- 9

.683

10 -

9.7

90

11 -

10.0

57

12 -

10.7

77

13 -

11.1

40

14 -

11.3

23

15 -

11.9

30

16 -

12.1

43

17 -

12.2

63

18 -

12.4

23

19 -

12.6

57

20 -

12.9

80

21 -

13.4

80

WVL:215 nm

39

Take Home Message



3 columns

~ 2 weeks

Select best

column

Build model

using ACD

lab

~ 1-2 week

Column library

WHY ACD LAB?

If column fails,

select another

column

Our approach . . .

Peak width, tailing, number

of components resolved,

relative retention of columns

. . .

40

Over 30 peaks

Many partially co-eluting peaks.

Degradation products had very similar spectra (MS needed)

ACD LAB: how does it work? Why is it essential?

41

Solvent B, %

7065605550454035302520151050

Colu

mn T

em

pe

ratu

re,

°C

25

30

35

40

45

50

55

60

65

70

750.81

0.78

0.74

0.71

0.68

0.64

0.61

0.58

0.54

0.51

0.47

0.44

0.41

0.37

0.34

0.30

0.27

0.24

0.20

0.17

0.14

0.10

0.07

0.03

0.00

41

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 27.5 28.8 30.0 31.3 32.5 33.8 35.0 36.3 37.5 38.8 40.0

-100

0

100

200

300

400

500

600

700IHA140715_01 #13 BlankQCN forced - degradation Channel_AmAU

min

1 -

0.9

93

2 -

1.0

96

3 -

1.3

94

4 -

1.4

17

5 -

1.7

31

6 -

5.5

14

7 -

7.5

62

8 -

11.8

17

9 -

12.7

88

10 -

T2-T

7 -

13.7

60

11 -

14.1

53

12 -

14.4

34

13 -

15.0

00

14 -

15.6

07

15 -

16.1

94

16 -

16.4

02

17 -

16.6

56

18 -

17.4

12

19 -

18.1

98

20 -

18.6

70

21 -

19.1

81

22 -

19.9

07

23 -

20.6

92

24 -

21.5

39

25 -

T1 -

22.0

52

26 -

22.7

35

27 -

23.2

61

28 -

24.6

78

29 -

25.1

63

30 -

26.2

80

31 -

26.3

77

32 -

26.7

59

33 -

28.8

28

34 -

31.8

47

35 -

32.1

40

36 -

32.2

53

37 -

32.6

25

38 -

36.1

07

39 -

36.9

62

40 -

37.0

96

0.53 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.28

-2.2

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.7IHA140715_01 #13 BlankQCN forced - degradation Channel_AmAU

min

1 -

0.9

93

2 -

1.0

96 3

- 1

.394

4 -

1.4

17

5 -

1.7

31

6 -

5.5

14

7 -

7.5

62

Drug A impurity method example

11.42 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 14.20 14.40 14.60 14.80 15.00 15.20 15.40 15.60 15.88

-0.83

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00


min

8 -

11.8

17

9 -

12.7

88

10 -

T2-T

7 -

13.7

60

11 -

14.1

53

12 -

14.4

34

13 -

15.0

00

14 -

15.6

07

15.17 15.50 15.75 16.00 16.25 16.50 16.75 17.00 17.25 17.50 17.75 18.00 18.25 18.50 18.75 19.00 19.25 19.50 19.75 20.00 20.25 20.50 20.75 21.00 21.37

-3.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.7IHA140710_01 #16 QCN Channel_AmAU

min

11 -

15.7

81

12 -

16.3

71

13 -

16.8

68

14 -

17.5

79

15 -

18.4

08

16 -

18.8

89

17 -

19.3

58 1

8 -

19.9

82 19 -

20.7

14

19.51 19.75 20.00 20.25 20.50 20.75 21.00 21.25 21.50 21.75 22.00 22.25 22.50 22.75 23.00 23.25 23.50 23.75 24.00 24.25 24.50 24.75 25.00 25.25 25.50 25.70

-3.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.7IHA140710_01 #16 QCN Channel_AmAU

min

18 -

19.9

82 19 -

20.7

14

20 -

21.7

78

21 -

T1 -

22.1

26

22 -

22.3

91

23 -

22.9

42

24 -

23.4

93

25 -

24.4

85

26 -

24.8

82

27 -

25.1

40

42

Column with extreme parameter values

43

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 27.5 28.8 30.0 31.3 32.5 33.8 35.0 36.3 37.5 38.8 40.0

-100

0

100

200

300

400

500

600

700IHA140715_01 #13 BlankQCN forced - degradation Channel_AmAU

min

1 -

0.9

93

2 -

1.0

96

3 -

1.3

94

4 -

1.4

17

5 -

1.7

31

6 -

5.5

14

7 -

7.5

62

8 -

11.8

17

9 -

12.7

88

10 -

T2-T

7 -

13.7

60

11 -

14.1

53

12 -

14.4

34

13 -

15.0

00

14 -

15.6

07

15 -

16.1

94

16 -

16.4

02

17 -

16.6

56

18 -

17.4

12

19 -

18.1

98

20 -

18.6

70

21 -

19.1

81

22 -

19.9

07

23 -

20.6

92

24 -

21.5

39

25 -

T1 -

22.0

52

26 -

22.7

35

27 -

23.2

61

28 -

24.6

78

29 -

25.1

63

30 -

26.2

80

31 -

26.3

77

32 -

26.7

59

33 -

28.8

28

34 -

31.8

47

35 -

32.1

40

36 -

32.2

53

37 -

32.6

25

38 -

36.1

07

39 -

36.9

62

40 -

37.0

96

Drug A impurity method example

11.42 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 14.20 14.40 14.60 14.80 15.00 15.20 15.40 15.60 15.88

-0.83

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00


min

8 -

11.8

17

9 -

12.7

88

10 -

T2-T

7 -

13.7

60

11 -

14.1

53

12 -

14.4

34

13 -

15.0

00

14 -

15.6

07

44

45

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

0 5 10 15 20 25 30

Re

lati

ve r

ete

nti

on

tim

e

peak number

Column 1

Column 2

column 3

column 4

column 5

column 6

Overlay of peak tR for six columns

46

Things that helped success building of the model in the ACD software

Use 3 gradients x 3 temperatures rather than 2x2.

Make sure you calculate your dead volume and delay volume experimentally.

In each gradient, make sure the last peak elutes close to the end of the gradient (i.e. maximize peak capacity)

• Interpolation rather than extrapolation

• Better (i.e. more precise) predictions

• If the optimized gradient is multistep, have your peaks away from the point where the gradient slope changes.

47

Conclusion

Take home message

A systematic approach to build a column library using the HSM.

Step by step approach for selecting best column.

Example of a successful separation.

Analytical Method Development: Are we solving the right ... · Analytical Method Development: Are...

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