Gerhard K. E. ScribaFriedrich Schiller University Jena, Pharmaceutical Chemistry
Philosophenweg 14, 07743 Jena, [email protected]
CE Enantioseparations and Application to the Determination of the Stereoisomeric Purity of Drugs
2
Introduction Mechanistic aspects Examples of selector combinations Determination of chiral purity
Levomepromazine Dextromethorphan
Conclusions
Outline
3
Why chiral analysis of drugs?
different pharmacological activities different toxicological profiles different pharmacokinetic properties
Enantiomers should be considered different entities.
Enantiomers: The same thing – only different?
Drug Activity eutomer Activity distomer
Penicillamine (S): antiarthritic (R): toxic
Ethambutol (S,S): tuberculostatic (R,R): causes blindness
Cetirizine (R): antihistaminic (S): inactive
DOPA (S): antiparkinsonian (R): agranulocytoxic
Ketamine (S): anesthetic/analgesic (R): hallucinogenic
4
Top ten best selling non-peptide drugs in 2016
# Product (company) API Form US $ billions
1 Harvoni TM (Gilead Sciences) ledipasvirsofosbuvir
enantiomerenantiomer
9.081
2 Revlimid TM (Celegene) lenalidomide racemate 6.974
3 Xarelto TM (Bayer) rivaroxaban enantiomer 5.392
4 Lyrica TM (Pfizer) pregabalin enantiomer 4.966
5 Advair TM / Seretide TM
(GlaxoSmithKline)fluticasonesalmeterol
enantiomerracemate
4.252
6 Sovaldi TM (Gilead Sciences) sofosbuvir enantiomer 4.001
7 Tecfidera TM (Biogen) dimethyl fumarate achiral 3.968
8 Januvia TM (Merck & Co) sitagliptin enantiomer 3.908
9 Truvada TM (Gilead Sciences) emtricitabinetenofovir
enantiomerenantiomer
3.566
10 Crestor TM (AstraZeneca) rosuvastatin enantiomer 3.401
5
Enantioseparation in HPLC versus CE
HPLC CE
Rcplxµ
Scplxµ
KRKS
Sfreeµ R
freeµ
Enantioseparation KS ≠ KR
Enantioseparation KS ≠ KR
Rcplx
Scplx µµ ≠
KRKS
Sfreev R
freev
6
Enantiomer separation by capillary electrophoresis
+
+ +
anode cathodedetector
EOF+ +
+
+
KS ≠ KR
Rcplx
Scplx
µµ ≠
Chromatographic principle:
]C[SK1
]C[SKScplxf
]C[RK1
]C[RKRcplxf
SR +
µ+µ−
+
µ+µ=µ−µ=µ∆
Electrokinetic principle:
Rcplxµ
Scplxµ
KRKS
Sfreeµ R
freeµ
7
Enantioseparation of Aly-Tyr by cyclodextrins
40/47 cm, 50 µm fused-silica capillary, 50 mM sodium phosphate buffer, 25 kV
pH 3.5pH 2.5
18 20
DD
LL
14 16
LL
DD
β-CD
LL
DD
12 14
pH 3.5pH 2.5
DD
LL
18 19[min]
DM-β-CD
pH 3.5pH 2.5
11 12
DDLL
15 20 25
LL
DD
TM-β-CD
8
pH-dependent enantiomer migration order
pH 3.5pH 2.5
[min]
Ala-Tyr
[min]
LL
DD
12 14
DD
LL
18 19
DDLL
14 16 18
DDLL
28 30
Asp-PheOMe
40/47 cm, 50 µm fused-silica capillary, 50 mM sodium phosphate buffer, 20 mg/mL DM-β-CD, 25 kV
pH 2.5 pH 3.5
K [M-1] µc [cm2V-1s-1]x 105
K [M-1] µc [cm2V-1s-1]x 105
LL 96 7.10 35 3.12
DD 114 7.32 40 3.43
pH 2.5 pH 3.5
K [M-1] µc [cm2V-1s-1]x 105
K [M-1] µc [cm2V-1s-1]x 105
LL 73 4.50 43 0.56
DD 84 4.65 50 0.71
9
Complexation equilibria of chargeable analytes
µHB+ ≠ µHB+⋅CDand
µHB+⋅CD1 ≠ µHB+⋅CD2and / or
K+1 ≠ K+2and / or
Kn1 ≠ Kn2
Enantiomer 1 and 2
2eff
1effSµµ
=
± H+
± CD
± CD
± H+
⋅+⋅+
+−+
+⋅
+
++
+
⋅⋅+
⋅⋅µ+µ=µ
]CD[K]CD[KlogpKpH
CDHBHBeff
na
]CD[K]CD[K
11
101
11
µB·CD = 0µB = 0
µHB+·CDµHB+
B-CDB
HB+-CDHB+
pKa
Kn
K+
pKa/c
10
Complexation-induced pKa shift
K+1 > K+2
Kn1 > Kn2
pH ↓
µf µc
pH ↑
µc µf
+
+
+
+
⋅
+
⋅
−
>
>
CDHB
HB
n
CDHB
HBpKpK
µµ
KK
µµ
10 ac/a
Enantiomer 1Enantiomer 2
pKa
pH →
µf
µc
0pKa/c
± H+
± CD
± CD
± H+
µB·CD = 0µB = 0
B-CDB
HB+-CDHB+
pKa
Kn
K+
pKa/c
µHB+·CDµHB+ µHB+·CD
pKa/c
11
Enantioseparation of Ala-Tyr by DM-β-CD
9.5 10.0
LL
DD
[min]
9 mol/L 60 mol/L
16 17[min]
9 mol/L 60 mol/L
16 17
LL
DD
[min]21 22
LL
DD
[min]
pH 2,2
pH 3,8
2.0 2.5 3.0 3.5 4.0 4.5 5.0pH
µf
µc
μ
Parameter Ala-TyrDD LL
µHB+ [10-9m2s-1V-1] 15,88 ± 0,07
µHB⋅CD+ [10-9m2s-1V-1] 6,68 ± 0,06 6,55 ± 0,06
µHB+ / µHB⋅CD+ 2,38 ± 0,02 2,42 ± 0,03K+ [M-1] 165 ± 7 139 ± 6Kn [M-1] 18,5 ± 1,5 15,0 ± 1,5K+ / Kn 9,0 ± 0,6 9,2 ± 0,6pKa 3,12 ± 0,01pKa/c 4,07 ± 0,03 4,08 ± 0,03
12
Enantioseparation of Asp-PheOMe by DM-β-CD
Parameter Asp-PheOMeDD LL
µHB+ [10-9m2s-1V-1] 15,66 ± 0,08
µHB⋅CD+ [10-9m2s-1V-1] 5,89 ± 0,09 5,84 ± 0,10
µHB+ / µHB⋅CD+ 2,66 ± 0,04 2,68 ± 0,05K+ [M-1] 141 ± 6 116 ± 5Kn [M-1] 114 ± 7 94 ± 6K+ / Kn 1,23 ± 0,06 1,23 ± 0,06pKa 2,99 ± 0,01pKa/c 3,08 ± 0,02 3,08 ± 0,02
9 mol/L 60 mol/L
10 11
LL
DD
[min]18 19
LL
DD
[min]
9 mol/L 60 mol/L
20 21
LLDD
[min]12 13
LL
DD
[min]
pH 2,2
pH 3,0
2.0 2.5 3.0 3.5 4.0 4.5 5.0pH
µf
µc
μ
13
Dexamfetamine
Treatment of attention deficit hyperactivity disorders Impurities from chiral starting materials or synthetic intermediates Achiral impurities from synthesis of racemic amphetamine followed by
fractional crystallization with L-(+)-tartaric acid
S-amphetamine(dexamphetamine)
R-amphetamine
1S,2S-(+)-norpseudoephedrine 1R,2S-(–)-norephedrine
phenylacetone oximephenylacetone
CH3
NH2
CH3
NH2
CH3
NH2
CH3
NH2
OH OH
CH3
N
OH
CH3
O
14
Dexamphetamine CE assays
1 – dexamphetamine 2 – levoamphetamine 3 – norpseudoephedrine 4 – norephedrine5 – phenylacetone 6 – phenylacetone Z-oxime 7 – phenylacetaone E-oxime
10 12 14 16 18 20 22[min]
IS
1
2
3 456
7
MEEKC-CD0.5% ethyl acetate, 1.5% SDS, 3.5% 1-butanol, 2.5% 2-propanol, 92.0% 50 mM sodium phosphate, pH 3.0, 20 °C, –14 kV 5.5% S-β-CD
Single CD
105 7.5 12.5[min]
IS
1
23 4
100 mM sodium phosphate pH 2.520 °C, 25 kV10 mg/mL HDAS-β-CD
15 20 25 30[min]
IS
1
2 34
56
7
Dual CD50 mM sodium phosphate pH 3.0 20 °C, –10 kV80 mg/mL SBE-β-CD25 mg/mL S-β-CD
15
Analysis of dexamfetamine sulfate sample
12 14 16 18 20 22 24 26 [min]
1
2IS
1 2 IS
5
67
1 – dexamphetamine 2 – levoamphetamine 3 – norpseudoephedrine 4 – norephedrine5 – phenylacetone 6 – phenylacetone Z-oxime 7 – phenylacetaone E-oxime
40.2/35 cm, 50 mm ID fused-silica capillary, 50 mM sodium phosphate, pH 3.080 mg/mL SBE-β-CD, 25 mg/mL S-β-CD–10 kV, 20°C, 200 nm5 mg/mL dexamphetamine sulfate , 70 mg/mL ephedrine (IS)
16
Dexamfetamine sulfate assay comparison
HDAS-β-CD, heptakis-(2,3-di-O-acetyl-6-O-sulfo)-β-CDSBE-β-CD, sulfobutylether-β-CDS-β-CD, sulfated β-CD
Single CD Dual CD CD-mediated MEEKCSelector HDAS-β-CD SBE-β-CD, S-β-CD S-β-CD
Buffer phosphate buffer, pH 2.5 phosphate buffer, pH 3.0 microemulsion in phosphate buffer, pH 3.0
Range 0.06 – 5.0 % 0.05 – 1.0%0.05 – 5.0% (R-AM)
0.1 – 1.0% to 0.5 – 1.0%0.1 – 5.0% (R-AM)
LOD 0.02 – 0.03% 0.01 – 0.02% 0.05 – 0.2%
Precision < 6.7% < 7.5% < 8.2%
Comments only charged impurities
expensive CD
charged and uncharged impuritiesexpensive CD
charged and uncharged impuritiesinexpensive CD
17
Separation of Met(O) peptide diastereomers
pH range: pH 2.5 – 9.5
CDs: β-CD, γ-CD, CM-β-CD, SBE-β-CD, S-β-CD
Crown ethers: 15-crown-5, 18-crown-6, Kryptofix 21, Kryptofix 22
no separation
no separation
(partial) separationCM-β-CD, S-β-CD
H3C NH
HN
NH
HN
NH
HN
O
O
O
O
O
O
NH2
COOH
SCH3O
NH2
NH2
NH
NO2
NO2
18
Met(O) peptide separation - crown ethers
ac-KEM(O)KK-Dnp
Experimental conditions: 40/50.2 cm, 50 µm ID FS capillary, 50 mM Tris-HCl, pH 8.020 kV, 20 °C, 214 nm, 10 mg/mL S-β-CD, 10 mM crown ether
Abs
orba
nce
[mA
U]
11 12 130
1
2
3
4
Time [min]11 12 13 14
0
1
2
3
Time [min]12 13 14
0
1
2
3
4
Time [min]17 18 19 20
0
1
2
Time [min]19 20 21
0
1
2
3
Time [min]
O
O
O
O
O O
OO
O
OO
NH
O
O
HN
O O
NHO
O
HNO
15-crown-5 18-crown-6 Kryptofix 21 Kryptofix 22
19
Met(O) reductase assay
Enzyme incubation: 50 mM Tris-HCl, pH 8.0, 20 mM DTT, 15 µg/mL Msr, 160 µM ac-KIFM(O)K-Dnp, 37 °C
Separation conditions: 45/55.2 cm, 50 µm ID FS capillary, 50 mM Tris-HCl, pH 7.85,14.3 mg/mL S-β-CD, 5 mM 15-crown-5, 25 kV, 21.5 °C, 214 nm
6 8 10 12-1
0
1
2
3
4
5
6
Abso
rban
ce [m
AU]
Time [min]
Fmoc
-β-A
la
KIF
MK
-Dnp
KIF
M-R
(O)-
K-D
npK
IFM
-S(O
)-K
-Dnp
blankno enzyme
hMsrAhMsrB2
6 8 10 120
2
4
6
8
Abso
rban
ce [m
AU]
Time [min]
3 min
10 min
15 min
Fmoc
-β-A
la
KIF
MK
-Dnp
KIF
M-R
(O)-
K-D
npK
IFM
-S(O
)-K
-Dnp
StereospecificityTime course hMsrA
20
Analytical Quality by Design (AQbD)
Method design
Method control
Life cyclemanagement
Methodevaluation
Selection of technique
Selection of experimental conditions
Definition of ATP
Multifactor experimental
design
Risk assessment
Definition of method control
strategy
Continuous verification
Continuous improvement
Knowledge management
Method operable design region
21
Levomepromazine
Levomepromazine is a chiral antipsychotic phenothiazine drug No test for enantiomeric purity is described in pharmacopeias
Levomepromazine
N
S
NCH3
CH3
OCH3
CH3
Dextromepromazine
N
S
NCH3
CH3
OCH3
CH3
Levomepromazinesulfoxide
N
S
NCH3
CH3
OCH3
CH3
O
15 20 25 30time [min]
Experimental conditions: 40/50.2 cm, 50 µm ID fused-silica capillary; 20 °C; 20 kV110 mM sodium citrate, pH 4.0; 30 mg/mL HP-γ-CD
Analytical target profile Determination of
dextromepromazine with precision and accuracy of ≤ 15 % at the 0.1 % level and ≤ 10 % at > 0.1 % levels
The diastereomers of levomepromazine sulfoxide should not be separated allowing the determination with precision and accuracy of ≤ 15 % at the 0.1 % level and ≤ 10 % at > 0.1 % levels
22
Selector screening
CD CD conc. (mg/mL)
Polarity of voltage t1 t2 α RS
Migration order
sulfated α-CD 2 + 12.06 13.10 1.086 4.03 Levo > Dexsulfobutyl-α-CD 30 - 7.15 7.28 1.018 0.91 Levo > Dexsulfopropyl-α-CD 30 - 15.44 16.40 1.062 2.64 Levo > Dex(2-hydroxy-3-N,N,N-triethylamino)propyl-β-CD
30 + 16.56 16.77 1.013 0.64 Levo > Dex
carboxymethyl-β-CD 30 + 21.41 21.58 1.008 0.31 Dex > Levosuccinyl-β-CD 2 + 12.89 13.49 1.047 1.30 Levo > Dexsulfated β-CD 2 - 5.40 5.59 1.035 0.45 Levo > Dexsulfobutyl-β-CD 30 - 6.63 6.71 1.012 1.22 Levo > Dexsulfopropyl-β-CD 10 - 21.91 23.20 1.059 1.89 Levo > Dexγ-CD 2 + 7.90 8.07 1.022 1.03 Levo > Dexcarboxymethyl-γ-CD 2 + 11.24 11.98 1.066 2.25 Dex > Levohydroxypropyl-γ-CD 2 + 7.63 8.09 1.060 2.83 Dex > Levosuccinyl-γ-CD 2 + 7.48 7.65 1.023 0.96 Levo > Dexsulfated γ-CD 2 - 3.42 3.55 1.038 1.15 Levo > Dex
Experimental conditions: 40/50.2 cm, 50 µm id fused-silica capillary, 50 mM sodium phosphate buffer, pH 2.5, 25 kV, 20 °C, detection at 253 nm
23
Defining the knowledge space – initial screening
Fractional factorial resolution V+ design 2-level design, +1 and -1 x = 2m-g
m = number of variables, g = number of generated factors x = 25-1 = 24 = 16 experiments plus 3 center points = 19 experiments
Varia
ble
1
Variable 2
Variables HP-γ-CD concentration: 1 - 60 mg/mL Citric acid buffer concentration 25 - 200 mM Background electrolyte pH: 3.0 - 5.0 Capillary temperature 15 - 25 °C Separation voltage: 15 - 25 kV
24
Fractional factorial resolution V+ design matrix
x = 25-1 = 24 = 16 + 3 center points = 19 experiments
# Run order
CD conc. (mg/mL) pH Citric acid conc.
(mM)Temperature
(°C)Voltage
(kV)1 9 1 3 25 15 252 7 60 3 25 15 153 18 1 3 200 15 154 2 60 3 200 15 255 11 1 5 25 15 156 8 60 5 25 15 257 4 1 5 200 15 258 15 60 5 200 15 159 6 1 3 25 25 1510 13 60 3 25 25 2511 12 1 3 200 25 2512 5 60 3 200 25 1513 16 1 5 25 25 2514 14 60 5 25 25 1515 3 1 5 200 25 1516 17 60 5 200 25 2517 1 30.5 4 112.5 20 2018 10 30.5 4 112.5 20 2019 19 30.5 4 112.5 20 20
25
Screening design - electropherograms
5 10 15t [min]
1
20 25 30 35t [min]
2
15 20 25 30t [min]
3
15 20 25 30t [min]
4
10 20t [min]
5
15 20t [min]
6
5 10t [min]
7
35 45 55 65t [min]
8
0 5 10 15t [min]
9
10 20t [min]
10
5 10 15t [min]
11
25 30 35 40t [min]
12
15 20 25 30t [min]
14
10 20t [min]
15
10 15 20t [min]
16
15 25 35t [min]
17 18
15 25 35t [min]
0 10t [min]
13
5
26
Defining the knowledge space – coefficient plots Fractional factorial resolution V+ design
HP-γ-CD concentration: 1 - 60 mg/mL Citric acid buffer concentration 25 - 200 mM Background electrolyte pH: 3.0 - 5.0 Capillary temperature 15 - 25 °C Separation voltage: 15 - 25 kV
Resolution drug Resolution sulfoxide
-1.0
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1.0
cCD cP p H
T U
U*U
cCD
* cP
cCD*
p H
cP*p
H
T*U
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
cCD cP pH T U
U*U
c CD
* cP
cCD
*pH
cP*p
H
T*U
27
Sweet spot plot at pH 3.0
Criteria (→ response thresholds) Resolution enantiomers 1.5 – 2.2 Resolution sulfoxide diastereomers: 0 – 0.4 Migration time levomepromazine: 6.7 – 15 min
Sweet spotCriteria met 2Criteria met 1Criteria met 0
28
Sweet spot plot at 25 kV
Criteria (→ response thresholds) Resolution enantiomers 1.5 – 2.2 Resolution sulfoxide diastereomers: 0 – 0.4 Migration time levomepromazine: 6.7 – 15 min
Sweet spotCriteria met 2Criteria met 1Criteria met 0
29
Response surface design 3-level design, -1, 0, +1 x = 2k + 2k + n
k = number of variablesn = number of replicates at center point
Central composite face centered design
# CD conc. (mg/mL) pH Citric acid
conc. (mM)1 0.5 2.5 1002 10 2.5 1003 0.5 3.5 1004 10 3.5 1005 0.5 2.5 2006 10 2.5 2007 0.5 3.5 2008 10 3.5 2009 0.5 3 15010 10 3 15011 5.25 2.5 15012 5.25 3.5 15013 5.25 3 10014 5.25 3 20015 5.25 3 15016 5.25 3 15017 5.25 3 150
Varia
ble
1
Variable 2
30
Levomepromazine electropherograms CCF design
2
5 10 15t [min]
5 10 15t [min]
1 3
10 20t [min]
4 5
6
10 20t [min]
5 10 15t [min]
7
5 10 15t [min]
10 20t [min]
8
10 20t [min]
9
0 5 10t [min]
10 20t [min]
10
5 10t [min]
11
10 20t [min]
12
5 10 15t [min]
13
5 10 15t [min]
14
5 10 15t [min]
15
31
Response surface plot
Central composite face centered design HP-γ-CD concentration, pH, buffer concentration α of enantiomers and α of diastereomers
Other conditions: 40/50.2 cm 75 µm capillary, 100 mM sodium citrate, 25 kV, 15 °C
enantiomers
diastereomers
α
CD conc.
pH
pH
CD conc.
α
32
Design space – probability map
Critical quality attributes: αe ≥ 1.02, αd = 1.00, t ≤ 15 min Design space: ≤ 1 % risk of failure to meet critical quality attributes
prob
abili
ty o
f fai
lure
33
Levomepromazine robustness test
Method: 100 mM sodium citrate, pH 2.85, 3.6 mg/mL HP-γ-CD, 15 °C, 25 kV
Plackett-Burman design 2-level fractional factorial design 4n experiments, number of variables 4n–1
Factors: CD conc. 3.6 ± 0.2 mg/mL; pH 2.85 ± 0.15; buffer conc. 100 ± 5 mMtemp. 16 ± 1 °C; voltage 25 ± 1 kV, 2 CD batches
# CD conc. (mg/mL) pH Citric acid
conc. (mM)Temperature (°C) Voltage (kV) CD batch
1 3.8 2.7 95 17 24 two2 3.8 3 95 15 26 one3 3.8 3 105 15 24 two4 3.4 3 105 17 24 one5 3.8 2.7 105 17 26 one6 3.4 3 95 17 26 two7 3.4 2.7 105 15 26 two8 3.4 2.7 95 15 24 one9 3.6 2.85 100 16 25 one10 3.6 2.85 100 16 25 one11 3.6 2.85 100 16 25 one
34
Robustness levomepromazine
2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]
2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14
t [min]2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]2 4 6 8 10 12 14 16
t [min]
1 2 3 4 5
6 7 8 9_CD1 9_CD2LVM
DXM
LSO
IStd
35
Levomepromazine assay validation data
Parameter Level Dextromepromazine Levomepromazinesulfoxide
Range (µg/mL) 0.25 – 2.5(0.1 – 1.0 %)
0.25 – 2.5(0.1 – 1.0 %)
Coefficient of determination R2 0.9955 0.9994LOD (µg/mL) 0.08 0.05LOQ (µg/mL) 0.25 0.17Migration time (RSD)
Repeatability (n = 6) 0.25 µg/mL 0.55 1.171.0 µg/mL 2.35 0.202.5 µg/mL 2.91 0.23
Intermediate precision (n = 3) 0.25 µg/mL 3.29 2.041.0 µg/mL 2.95 1.192.5 µg/mL 2.39 0.91
Corrected peak area ratio (RSD)Repeatability (n = 6) 0.25 µg/mL 5.41 6.78
1.0 µg/mL 4.72 3.182.5 µg/mL 2.27 3.83
Intermediate precision (n = 3) 0.25 µg/mL 4.34 9.891.0 µg/mL 3.08 3.072.5 µg/mL 1.84 2.64
36
Levomepromazine method application
1 - levomepromazine, 2 - dextromepromazine, 3 - levomepromazine sulfoxide, IStd - internal standard (amitryptiline)
Experimental conditions: 40/50.2 cm, 75 µm ID fused-silica capillary; 25 kV, 15 °C100 mM sodium citrate; pH 2.85; 3.6 mg/mL HP-γ-CD
time [min]
1
2
3
1
2
1
23
IStd
Standards Ph. Eur. CRS Injection solution
IStd
5 1510 10 5 1510time [min]15time [min]
Ph. Eur. CRS Injection solutionDextromepromazine (2.84 ± 0.06 %) ~ 0.09 % (< LOQ)
Sulfoxide < LOD ~ 0.08 % (< LOQ)
37
Dextromethorphan
ent-Morphinan structure (9S,13S,14S) configuration Cough suppressant
Configuration of morphine (9R,13R,14R) configuration Opioid analgesic drug Never clinically developed
(strong respiratory depressant) Controlled substance
Dextromethorphan Levomethorphan
WHO Drug Alert 126 (2013): approx. 60 deaths caused in Pakistan due to contaminated dextromethorphan covered by test of specific rotation
HPLC test of levomethorphan developed for the United States Pharmacopeia and the International Pharmacopoeia (limit 0.1 %)
N
H3CO
CH3 NH3C
OCH3
38
Dextromethorphan
ent-Morphinan structure (9S,13S,14S) configuration Cough suppressant
Configuration of morphine (9R,13R,14R) configuration Opioid analgesic drug Never clinically developed
(strong respiratory depressant) Controlled substance
Dextromethorphan Levomethorphan
N
H3CO
CH3 NH3C
OCH3
Analytical target profile Determination of levomethorphan with precision and accuracy
of ≤ 15 % at the 0.1 % level and ≤ 10 % at > 0.1 % level
39
Dextromethorphan method scouting
Phosphate buffer, pH 2.5 Baseline resolution: CM-γ-CD, HP-α-CD, CM-α-CD, SBE-α-CD (-),
S-β-CD (-) Partial resolution: α-CD, M-α-CD, HP-γ-CD, CM-β-CD Baseline noise high, relatively low RS values (< 3)
Phosphate buffer, pH 7.0 Baseline resolution: S-β-CD (RS ~ 21) Stable baseline, tailing peaks
Borate buffer, pH 8.5 Baseline resolution: S-β-CD (RS ~ 24) Relatively noisy baseline, tailing peaks
Other conditions: 40/50.2 cm, 50 µm id fused-silica capillary, 20 °C, 20 kV
40
Separation of methorphan enantiomers
30/40.2 cm, 50 µm ID fused-silica capillary; 50 mM sodium phosphate, pH 7.0; 20 °C, 16 kV
[min]5 10 15
20 mg/mL S-β-CD / 10 mg/mL M-α-CD20 mg/mL S-β-CD
Sulfated β-CD Sulfated-β-CD / Methyl-α-CD
2.5 5 7.5[min]
DXM
LVM
DXM
LVM
41
Sulfated β-cyclodextrin
Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 30 mM sodium phosphate, pH 6.50; 20 kV, 20 °C
DXM LVM
K (M-1) 259(211 / 319)
606(516/ 716)
µcplx
(10-9m2V-1s-1)– 38.0
(-34.3 / -42.3)– 43.3
(-41.2 / 45.5)
2 4 6 8 10 12 14 16 18 20Time (min)
[S-β-CD] = 2 mM[S-β-CD] = 4 mM[S-β-CD] = 8 mM[S-β-CD] = 12 mM
[S-β-CD] = 0 mM
DXM
LVM
Numbers in brackets represent 95 % confidence intervalData calculated with CEval
42
Methyl-α-cyclodextrin
Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 100 mM sodium phosphate, pH 2.12; 20 kV, 20 °C
DXM LVM
K (M-1) 354(292 / 429)
399(329 / 585)
µcplx
(10-9m2V-1s-1)9.97
(9.42 / 10.48)9.90
(9.38 / 10.40)
Numbers in brackets represent 95 % confidence intervalData calculated with CEval
4 6 8 10 12Time (min)
[M-α-CD] = 0 mM[M-α-CD] = 2 mM[M-α-CD] = 5 mM[M-α-CD] = 10 mM[M-α-CD] = 20 mM
DXMLVM
43
Defining the knowledge space – initial screening Fractional factorial resolution IV design (x = 26-2 + 3)
S-β-CD conc.: 10 - 24 mg/mL; M-α-CD conc.: 6 - 20 mg/mL Sodium phosphate buffer concentration 30 - 100 mM; pH 6.4 - 8.0 Capillary temperature 15 - 25 °C; Voltage: 10 - 20 kV
# S-β-CD (mg/mL)
M-α-CD (mg/mL) Buffer (mM) pH Temp. (°C) Voltage (kV) RS RT (min)
1 10 6 30 6.4 15 10 9.12 7.662 24 6 30 6.4 25 10 13.86 10.283 24 20 30 6.4 15 20 12.06 4.344 10 20 30 6.4 25 20 4.99 4.455 24 20 100 6.4 15 10 3.96 8.46 10 20 100 6.4 25 10 5.28 5.647 10 6 100 6.4 15 20 8.43 3.978 24 6 100 6.4 25 20 14.02 10.169 10 20 30 8.2 15 10 3.68 6.0710 24 20 30 8.2 25 10 8.8 6.3511 24 6 30 8.2 15 20 15.86 5.4312 10 6 30 8.2 25 20 6.86 2.7113 24 6 100 8.2 15 10 11.41 11.5114 10 6 100 8.2 25 10 8.03 6.2815 10 20 100 8.2 15 20 0.01 2.7816 24 20 100 8.2 25 20 3.79 3.1617 17 13 65 7.2 20 15 5.97 4.4018 17 13 65 7.2 20 15 6.19 4.5319 17 13 65 7.2 20 15 6.45 4.58
44
Defining the knowledge space – initial screening Fractional factorial resolution IV design
S-β-CD concentration: 10 - 24 mg/mL M-α-CD concentration: 6 - 20 mg/mL Sodium phosphate buffer concentration 30 - 100 mM Background electrolyte pH: 6.4 - 8.0 Capillary temperature 15 - 25 °C Separation voltage: 10 - 20 kV
SB-β
-CD
M-α
-CD
SB-β
-CD
M-α
-CD
45
# S-β-CD (mg/mL)
M-α-CD (mg/mL)
Voltage(kV)
MT (min) SDXM
1 10 5 10 10.12 8.302 10 5 20 5.23 4.893 30 5 10 22.98 8.094 30 5 20 10.50 5.485 10 15 10 7.82 0.696 10 15 20 3.45 0.917 30 15 10 12.00 0.638 30 15 20 5.63 0.859 20 10 10 18.61 3.9910 20 10 20 5.25 2.7911 10 10 15 5.33 2.4812 30 10 15 9.67 2.6613 20 5 15 14.86 7.9714 20 15 15 6.27 0.9515 20 10 15 7.26 3.2316 20 10 15 8.22 3.1717 20 10 15 7.23 3.13
Variables S-β-CD: 10 - 30 mg/mL M-α-CD: 5 - 15 mg/mL Voltage: 10 - 20 kV
2k + 2k + n = 17 experiments
Responses Migration time (MT)
levomethorphan (≤ 8 min) Peak symmetry (S)
dextromethorphan (0.5 – 3, target value 1)
Central composite face centered design
46
Dextromethorphan CCC design
2 6 10 14t [min]
1
0 2 4 6t [min]
2
2 6 10 14t [min]
4
0 2 4 6 8t [min]
5
0 2 4 6 8t [min]
6
4 8 12t [min]
7
2 4 6t [min]
8
2 6 10t [min]
9
4 10 16 22t [min]
3
2 4 6t [min]
10
2 4 6t [min]
11
2 6 10t [min]
12
2 6 10 14t [min]
13
2 4 6t [min]
14
2 4 6 8t [min]
15
2 4 6 8t [min]
16
2 4 6 8t [min]
17 LVM
47
Design space – probability map
Critical quality attributes: t ≤ 8 min, NLVM ≥ 3000, 3 > SDXM > 0.5, peak height (LVM) ≥ 3000 µAU
Design space: 1 % risk of failure to meet critical quality attributes
%
0.5
1
2
5
10
50
0.5
1
2
5
10
50
Voltage 10 kV Voltage 15 kV Voltage 20 kV
12
15
18
21
24
27
30
6 9 12M-α-CD (mg/mL)
6 9 12M-α-CD (mg/mL)
6 9 12 15M-α-CD (mg/mL)
1515
S-β
-CD
(mg/
mL) 0.5
1
2
5
10
50
1050
48
Design space – probability map
Critical quality attributes: t ≤ 8 min, NLVM ≥ 3000, 3 > SDXM > 0.5, peak height (LVM) ≥ 3000 µAU
Design space: 1 % risk of failure to meet critical quality attributes
%
0.5
1
2
5
10
50
10
14
18
22
26
30
10 12 14 16 18 20Voltage (kV)
S-β
-CD
(mg/
mL)
10
502
51 0.5
M-α-CD 14 mg/mL
49
Assay robustness – coefficient plots Method: 30 mM sodium phosphate, pH 6.5; 16 mg/mL S-β-CD, 14 mg/mL M-α-CD;
20 °C; 20 kV
Plackett-Burman design S-β-CD conc. 16 ± 1 mg/mL; M-α-CD conc. 14 ± 1 mg/mL; pH 6.5 ± 0.1;
buffer conc. 30 ± 1 mM; temp. 20 ± 1 °C; voltage 20 ± 1 kV, 2 batches of each CD
Run Time (MT LVM) Peak Symmetry DXM
Temp, capillary temperatureV, voltageBuf, buffer concentrationpH, buffer pHSB-C, S-β-CD concentrationma-C, M-α-CD concentrationSB-B, S-β-CD batchma-B, M-α-CD batch
50
Assay robustness test - run time scatter
51
Dextromethorphan assay validation data
Parameter Level Levomethorphan
Range (µg/mL) 1.0 – 15(0.07 – 1.0 %)
Coefficient of determination R2 0.9989LOD (µg/mL) 0.3 (0.02 %)LOQ (µg/mL) 1.0 (0.07 %)Accuracy1) 1.5 µg(mL (0.10 %) 88.9 ± 3.6 %
7.5 µg/mL (0.50 %) 96.1 ± 2.0 %13.5 µg/mL (0.90 %) 99.2 ± 0.9 %
Content repeatability2) 5.3 %Content intermediate precision3) 5.7 %Migration time
Repeatability 1.2 %Intermediate precision 5.8 %
1) expressed as recovery in percent ± 95 % confidence interval2) 3 concentrations analyzed 3 times on one day3) 3 concentrations analyzed 3 times on 3 consecutive days
30/40.2 cm, 50 µm ID fused-silica capillary; 20 °C; 20 kV, 30 mM sodium phosphate buffer, pH 6.5; 16 mg/mL S-β-CD, 14 mg/mL M-α-CD
52
Dextromethorphan method application
Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 20 °C; 20 kV; HDI 0.7 psi x 5 sec30 mM sodium phosphate buffer, pH 6.5; 16 mg/mL S-β-CD, 14 mg/mL M-α-CDIStd: 30 µg/mL procainamide hydrochloride; DXM concentration: 1.5 mg/mL
4
Standards1.0 % LVM
DXMIS
tdLV
M
3 4t (min)
LOQ 0.067 % LVM
DXM
IStd
LVM
X
3 4t (min)
DXM capsule
DXM
IStd
LVM
X
3 4t (min)
53
Conclusions
CE is a powerful technique for enantioseparations including the determination of the enantiomeric purity of compounds.
CE often allows the simultaneous separation of stereoisomeric impurities and (achiral) related substances.
Robust CE methods can be developed for the analysis of stereoisomeric as well as achiral impurities at LOQ levels comparable to HPLC methods.
Analytical Quality by Design (AQbD) strategies including predefined method characteristics and chemometric Design of Experiments (DoE) for rational method development result in robust methods with known risk of failure.
CE is a suitable technique to study mechanistic aspects of selector-selectand interactions and mechanistic aspects of stereoisomer separations.
54
Acknowledgements
FSU Jena Manuela Hammitzsch Qingfu Zhu Sudaporn Wongwan Stephan Niedermeier Sulaiman Krait Henrik Harnisch
Charles University Prague Pavel Dubský Michal Malý
World Health Organization Dr. Herbert Schmidt
Funding