analytical method development: are we solving the right ... · analytical method development: are...
TRANSCRIPT
Imad Haidar Ahmad 7Feb2018
Merck
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
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
Journal of Chromatography A, 961 (2002) 171–193
’ ’ ’ ’ ’
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
Importance of corner points
Journal of Chromatography A, 961 (2002) 171–193
CBASHresolution '''*''
20
Importance of corner points
Journal of Chromatography A, 961 (2002) 171–193
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
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
24
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
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
Poor retention, peak
shape, or asymmetry
out
Columns missing
more than 2 peaks
30
Run initial gradient
Adjust gradient so that last
peak elutes ~ comparable tR
out
Poor retention, peak
shape, or asymmetry
out
Columns missing
more than 2 peaks
Run 3 gradients (of different
slopes) out
Can’t see almost all
peaks
31
Run initial gradient
Adjust gradient so that last
peak elutes ~ comparable tR
out
Poor retention, peak
shape, or asymmetry
out
Columns missing 2
peaks or more
Run 3 gradients (of different
slopes) out
Can’t see almost all
peaks
Compare # of resolved peaks
,w1/2, and asymmetry out
Can’t see almost all
peaks
32
Run initial gradient
Adjust gradient so that last
peak elutes ~ comparable tR
out
Poor retention, peak
shape, or asymmetry
out
Columns missing 2
peaks or more
Run 3 gradients (of different
slopes) out
Can’t see almost all
peaks
Compare # of resolved peaks
,w1/2, and asymmetry out
Can’t see almost all
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
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
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
8.49IHA140715_01 #13 BlankQCN forced - degradation Channel_AmAU
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
8.49IHA140715_01 #13 BlankQCN forced - degradation Channel_AmAU
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.