Gerhard K. E. ScribaFriedrich Schiller University Jena, Pharmaceutical Chemistry

Philosophenweg 14, 07743 Jena, Germanygerhard.scriba@uni-jena.de

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