Gerhard Scriba Slides

CE Enantioseparations and Application to the Determination of the Stereoisomeric Purity of Drugs

Gerhard K. E. Scriba Friedrich Schiller University Jena, Pharmaceutical Chemistry Philosophenweg 14, 07743 Jena, Germany [email protected] Outline

 Introduction  Mechanistic aspects  Examples of selector combinations  Determination of chiral purity  Levomepromazine  Dextromethorphan  Conclusions

2 Why chiral analysis of drugs?

Enantiomers: The same thing – only different?

 different pharmacological activities  different toxicological profiles  different pharmacokinetic properties  Enantiomers should be considered different entities.

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

3 Top ten best selling non-peptide drugs in 2016

# Product (company) API Form US $ billions 1 Harvoni TM (Gilead Sciences) ledipasvir enantiomer 9.081 sofosbuvir enantiomer 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 fluticasone enantiomer 4.252 (GlaxoSmithKline) salmeterol racemate 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) emtricitabine enantiomer 3.566 tenofovir enantiomer 10 Crestor TM (AstraZeneca) rosuvastatin enantiomer 3.401

4 Enantioseparation in HPLC versus CE

HPLC CE

S R S R v free v free µfree µfree

KS KR KS KR

R µ cplx

S µ cplx

 Enantioseparation  Enantioseparation

 KS ≠ KR  KS ≠ KR S R  µcplx ≠ µcplx

5 Enantiomer separation by capillary electrophoresis

S R anode detector cathode µfree µfree + + + + EOF K K + + S R +

R R S µ cplx µ + µ K [C] µ + µ K [C] ∆µ = µ − µ = f cplx R − f cplx S µ S R S 1 + K [C] 1+ K [C] cplx R S

Chromatographic principle: KS ≠ KR Electrokinetic principle: µ S ≠ µ R cplx cplx

6 Enantioseparation of Aly-Tyr by cyclodextrins

β-CD DM-β-CD TM-β-CD

pH 2.5 pH 3.5 pH 2.5 pH 3.5 pH 2.5 pH 3.5

DD DD DD DD DD DD LL LL LL LL LL LL

14 16 18 20 12 14 18 19 11 12 15 20 25 [min]

40/47 cm, 50 µm fused-silica capillary, 50 mM sodium phosphate buffer, 25 kV

7 pH-dependent enantiomer migration order

pH 2.5 pH 3.5 Ala-Tyr DD DD pH 2.5 pH 3.5 LL -1 2 -1 -1 -1 2 -1 -1 LL K [M ] µc [cm V s ] K [M ] µc [cm V s ] x 105 x 105 LL 96 7.10 35 3.12 DD 114 7.32 40 3.43 12 14 [min] 18 19

Asp-PheOMe LL LL pH 2.5 pH 3.5 DD DD -1 2 -1 -1 -1 2 -1 -1 K [M ] µc [cm V s ] K [M ] µc [cm V s ] x 105 x 105 LL 73 4.50 43 0.56 DD 84 4.65 50 0.71

14 16 18 [min] 28 30

40/47 cm, 50 µm fused-silica capillary, 50 mM sodium phosphate buffer, 20 mg/mL DM-β-CD, 25 kV

8 Complexation equilibria of chargeable analytes

µB = 0 µB·CD = 0 Kn Enantiomer 1 and 2 B B-CD ± µ CD S = eff1 µeff2

+ + pKa ± H ± H pKa/c µHB+ ≠ µHB+⋅CD and ± CD µ + ≠ µ + HB+ HB+-CD HB ⋅CD1 HB ⋅CD2 and / or K+ K ≠ K µ µ +1 +2 HB+ HB+·CD and / or

Kn1 ≠ Kn2 µ + +µ + ⋅K + ⋅[CD] 1 µ = HB HB ⋅CD ⋅ eff  1+K ⋅[CD]  1+K + ⋅[CD] pH− pK + log +   a + ⋅  1+10  1 Kn [CD] 

9 Complexation-induced pKa shift

µB = 0 µB·CD = 0 Kn Enantiomer 1 µf Enantiomer 2 B B-CD ± CD

µc + + pKa ± H ± H pKa/c

0 ± CD pK pK + + a a/c HB HB -CD pH →

K+ µ + µ + HB HB ·CD − µ + 10pKa / c pKa > HB + µHB⋅CD

µ + K+ > HB K > K pH ↓ pH ↑ +1 +2 + Kn µHB⋅CD Kn1 > Kn2 µf µc µc µf

10 Enantioseparation of Ala-Tyr by DM-β-CD

Parameter Ala-Tyr 9 mol/L 60 mol/L DD LL -9 2 -1 -1 µHB+ [10 m s V ] 15,88 ± 0,07 DD -9 2 -1 -1 µHB⋅CD+ [10 m s V ] 6,68 ± 0,06 6,55 ± 0,06 pH 2,2 µHB+ / µHB⋅CD+ 2,38 ± 0,02 2,42 ± 0,03 LL -1 K+ [M ] 165 ± 7 139 ± 6 -1 Kn [M ] 18,5 ± 1,5 15,0 ± 1,5

K+ / Kn 9,0 ± 0,6 9,2 ± 0,6 9.5 10.0 16 17 pK 3,12 ± 0,01 a [min] [min] pKa/c 4,07 ± 0,03 4,08 ± 0,03

9 mol/L 60 mol/L DD µf DD μ LL LL pH 3,8

µc

16 17 21 22 2.0 2.5 3.0 3.5 4.0 4.5 5.0 [min] [min] pH

11 Enantioseparation of Asp-PheOMe by DM-β-CD

Parameter Asp-PheOMe DD LL 9 mol/L 60 mol/L -9 2 -1 -1 µHB+ [10 m s V ] 15,66 ± 0,08 DD DD -9 2 -1 -1 µHB⋅CD+ [10 m s V ] 5,89 ± 0,09 5,84 ± 0,10 ± ± LL µHB+ / µHB⋅CD+ 2,66 0,04 2,68 0,05 pH 2,2 LL -1 K+ [M ] 141 ± 6 116 ± 5 -1 Kn [M ] 114 ± 7 94 ± 6

K+ / Kn 1,23 ± 0,06 1,23 ± 0,06

pKa 2,99 ± 0,01 10 11 18 19 [min] [min] pKa/c 3,08 ± 0,02 3,08 ± 0,02

9 mol/L 60 mol/L µ f DD DD LL μ pH 3,0 LL

µc

12 13 20 21 2.0 2.5 3.0 3.5 4.0 4.5 5.0 [min] [min] pH

12 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

OH OH CH3 CH3 CH3

NH2 NH2 NH2 S-amphetamine 1S,2S-(+)-norpseudoephedrine 1R,2S-(–)-norephedrine (dexamphetamine)

CH3 CH3 CH3

NH2 O N

OH R-amphetamine phenylacetone phenylacetone oxime

13 Dexamphetamine CE assays

Single CD Dual CD MEEKC-CD 100 mM sodium phosphate pH 2.5 50 mM sodium phosphate pH 3.0 0.5% ethyl acetate, 1.5% SDS, 20 °C, 25 kV 20 °C, –10 kV 3.5% 1-butanol, 2.5% 2-propanol, 10 mg/mL HDAS-β-CD 80 mg/mL SBE-β-CD 92.0% 50 mM sodium phosphate, 25 mg/mL S-β-CD pH 3.0, 20 °C, –14 kV 5.5% S-β-CD

1 1 1

2 3 4 2 3 IS 2 4 6 IS 7 3 4 7 5 6 5

5 7.5 10 12.5 15 20 25 30 10 12 14 16 18 20 22 [min] [min] [min]

1 – dexamphetamine 2 – levoamphetamine 3 – norpseudoephedrine 4 – norephedrine 5 – phenylacetone 6 – phenylacetone Z-oxime 7 – phenylacetaone E-oxime

14 Analysis of dexamfetamine sulfate sample

1 2 IS

5 7 1 6

2 IS

12 14 16 18 20 22 24 26 [min]

40.2/35 cm, 50 mm ID fused-silica capillary, 50 mM sodium phosphate, pH 3.0 80 mg/mL SBE-β-CD, 25 mg/mL S-β-CD–10 kV, 20°C, 200 nm 5 mg/mL dexamphetamine sulfate , 70 mg/mL ephedrine (IS)

1 – dexamphetamine 2 – levoamphetamine 3 – norpseudoephedrine 4 – norephedrine 5 – phenylacetone 6 – phenylacetone Z-oxime 7 – phenylacetaone E-oxime

15 Dexamfetamine sulfate assay comparison

Single CD Dual CD CD-mediated MEEKC Selector 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% 0.1 – 1.0% to 0.5 – 1.0% 0.05 – 5.0% (R-AM) 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 charged and uncharged charged and uncharged impurities impurities expensive CD expensive CD inexpensive CD

HDAS-β-CD, heptakis-(2,3-di-O-acetyl-6-O-sulfo)-β-CD SBE-β-CD, sulfobutylether-β-CD S-β-CD, sulfated β-CD

16 Separation of Met(O) peptide diastereomers

NH2 NH2

O CH3 S

NO2 O O O H H H N N N H3C N N N N H H H H O O O NO2

COOH

NH2

 pH range: pH 2.5 – 9.5 no separation

 CDs: β-CD, γ-CD, CM-β-CD, (partial) separation SBE-β-CD, S-β-CD CM-β-CD, S-β-CD

 Crown ethers: 15-crown-5, no separation 18-crown-6, Kryptofix 21, Kryptofix 22

17 Met(O) peptide separation - crown ethers

 ac-KEM(O)KK-Dnp

15-crown-5 18-crown-6 Kryptofix 21 Kryptofix 22

O O O O O O NH O O O O HN

O O O HN O O NH O O O 4 3 4

] 3 3 3 2 mAU [ 2 2 2 2 1 1 Absorbance 1 1 1

0 0 0 0 0 11 12 13 11 12 13 14 12 13 14 17 18 19 20 19 20 21 Time [min] Time [min] Time [min] Time [min] Time [min]

Experimental conditions: 40/50.2 cm, 50 µm ID FS capillary, 50 mM Tris-HCl, pH 8.0 20 kV, 20 °C, 214 nm, 10 mg/mL S-β-CD, 10 mM crown ether

18 Met(O) reductase assay

Time course hMsrA Stereospecificity - Ala - S(O) K Dnp - R(O) K Dnp Fmoc - β - Ala 8 6 - R(O) K Dnp KIFM - Dnp KIFMK KIFM - S(O) K Dnp 5 Fmoc - β ] ]

6 KIFM 4 KIFM mAU mAU

- Dnp KIFMK 3 4 15 min 2

Absorbance [ Absorbance [ hMsrB2 1 2 10 min hMsrA 0 no enzyme 3 min blank 0 -1 6 8 10 12 6 8 10 12 Time [min] Time [min]

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

19 Analytical Quality by Design (AQbD)

Definition of ATP Selection of technique

Selection of experimental conditions

Method design Continuous verification Multifactor experimental Life cycle Method design Continuous management evaluation improvement Risk assessment Knowledge management Method control

Definition of Method operable method control design region strategy

20 Levomepromazine

 Levomepromazine is a chiral antipsychotic phenothiazine drug  No test for enantiomeric purity is described in pharmacopeias

CH3 CH3  CH3 CH3 Analytical target profile N N CH3  Determination of CH3 N OCH3 dextromepromazine with N OCH3 CH3 CH3 precision and accuracy of S N CH3 S ≤ 15 % at the 0.1 % level O N OCH Levomepromazine and ≤ 10 % at > 0.1 % levels Levomepromazine 3 sulfoxide  The diastereomers of S levomepromazine sulfoxide Dextromepromazine should not be separated allowing the determination with precision and accuracy of ≤ 15 % at the 0.1 % level and ≤ 10 % at > 0.1 % levels

15 20 25 30 time [min]

Experimental conditions: 40/50.2 cm, 50 µm ID fused-silica capillary; 20 °C; 20 kV 110 mM sodium citrate, pH 4.0; 30 mg/mL HP-γ-CD

21 Selector screening

CD conc. Polarity of Migration CD t t α R (mg/mL) voltage 1 2 S order sulfated α-CD 2 + 12.06 13.10 1.086 4.03 Levo > Dex sulfobutyl-α-CD 30 - 7.15 7.28 1.018 0.91 Levo > Dex sulfopropyl-α-CD 30 - 15.44 16.40 1.062 2.64 Levo > Dex (2-hydroxy-3-N,N,N- 30 + 16.56 16.77 1.013 0.64 Levo > Dex triethylamino)propyl-β-CD carboxymethyl-β-CD 30 + 21.41 21.58 1.008 0.31 Dex > Levo succinyl-β-CD 2 + 12.89 13.49 1.047 1.30 Levo > Dex sulfated β-CD 2 - 5.40 5.59 1.035 0.45 Levo > Dex sulfobutyl-β-CD 30 - 6.63 6.71 1.012 1.22 Levo > Dex sulfopropyl-β-CD 10 - 21.91 23.20 1.059 1.89 Levo > Dex γ-CD 2 + 7.90 8.07 1.022 1.03 Levo > Dex carboxymethyl-γ-CD 2 + 11.24 11.98 1.066 2.25 Dex > Levo hydroxypropyl-γ-CD 2 + 7.63 8.09 1.060 2.83 Dex > Levo succinyl-γ-CD 2 + 7.48 7.65 1.023 0.96 Levo > Dex sulfated 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

22 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

 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 Variable 1 Variable  Separation voltage: 15 - 25 kV

Variable 2

23 Fractional factorial resolution V+ design matrix

x = 25-1 = 24 = 16 + 3 center points = 19 experiments

Run CD conc. Citric acid conc. Temperature Voltage # pH order (mg/mL) (mM) (°C) (kV) 1 9 1 3 25 15 25 2 7 60 3 25 15 15 3 18 1 3 200 15 15 4 2 60 3 200 15 25 5 11 1 5 25 15 15 6 8 60 5 25 15 25 7 4 1 5 200 15 25 8 15 60 5 200 15 15 9 6 1 3 25 25 15 10 13 60 3 25 25 25 11 12 1 3 200 25 25 12 5 60 3 200 25 15 13 16 1 5 25 25 25 14 14 60 5 25 25 15 15 3 1 5 200 25 15 16 17 60 5 200 25 25 17 1 30.5 4 112.5 20 20 18 10 30.5 4 112.5 20 20 19 19 30.5 4 112.5 20 20

24 Screening design - electropherograms

1 2 3 4 5 6

5 10 15 20 25 30 35 15 20 25 30 15 20 25 30 10 20 15 20 t [min] t [min] t [min] t [min] t [min] t [min]

7 8 9 10 11 12

5 10 35 45 55 65 0 5 10 15 10 20 5 10 15 25 30 35 40 t [min] t [min] t [min] t [min] t [min] t [min]

13 14 15 16 17 18

0 5 10 15 20 25 30 10 2010 15 20 15 25 35 15 25 35 t [min] t [min] t [min] t [min] t [min] t [min]

25 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 0.3 1.0

0.2 0.8 0.6 0.1 0.4 0 0.2

-0.1 0

-0.2 -0.2 -0.4 -0.3 -0.6 -0.4 -0.8

-0.5 -1.0 T P P D U U U H H H P P D T H U H U H U c * * c p p * c c p * * C p p p C * * * * U T * c T U c D P D D P D c C C c C C c c c c

26 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 spot Criteria met 2 Criteria met 1 Criteria met 0

27 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 spot Criteria met 2 Criteria met 1 Criteria met 0

28 Central composite face centered design

 Response surface design CD conc. Citric acid # pH  3-level design, -1, 0, +1 (mg/mL) conc. (mM) 1 0.5 2.5 100 k  x = 2 + 2k + n 2 10 2.5 100 k = number of variables 3 0.5 3.5 100 n = number of replicates at 4 10 3.5 100 center point 5 0.5 2.5 200 6 10 2.5 200 7 0.5 3.5 200 8 10 3.5 200 9 0.5 3 150 10 10 3 150 11 5.25 2.5 150 12 5.25 3.5 150

Variable 1 Variable 13 5.25 3 100 14 5.25 3 200 15 5.25 3 150 Variable 2 16 5.25 3 150 17 5.25 3 150

29 Levomepromazine electropherograms CCF design

1 2 3 4 5

5 10 15 5 10 15 5 10 15 10 20 0 5 10 t [min] t [min] t [min] t [min] t [min]

6 7 8 9 10

10 20 5 10 15 10 20 10 20 10 20 t [min] t [min] t [min] t [min] t [min]

11 12 13 14 15

5 10 10 20 5 10 15 5 10 15 5 10 15 t [min] t [min] t [min] t [min] t [min]

30 Response surface plot

 Central composite face centered design  HP-γ-CD concentration, pH, buffer concentration  α of enantiomers and α of diastereomers

pH CD conc.

α α

enantiomers CD conc. diastereomers pH

Other conditions: 40/50.2 cm 75 µm capillary, 100 mM sodium citrate, 25 kV, 15 °C

31 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 probability failure of probability

32 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 mM temp. 16 ± 1 °C; voltage 25 ± 1 kV, 2 CD batches

CD conc. Citric acid Temperature (°C) Voltage (kV) CD batch # pH (mg/mL) conc. (mM) 1 3.8 2.7 95 17 24 two 2 3.8 3 95 15 26 one 3 3.8 3 105 15 24 two 4 3.4 3 105 17 24 one 5 3.8 2.7 105 17 26 one 6 3.4 3 95 17 26 two 7 3.4 2.7 105 15 26 two 8 3.4 2.7 95 15 24 one 9 3.6 2.85 100 16 25 one 10 3.6 2.85 100 16 25 one 11 3.6 2.85 100 16 25 one

33 Robustness levomepromazine

1 2 3 4 5

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 t [min] t [min] t [min] t [min] t [min]

6 7 8 9_CD1 LVM 9_CD2 LSO IStd DXM

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 t [min] t [min] t [min] t [min] t [min]

34 Levomepromazine assay validation data

Parameter Level Dextromepromazine Levomepromazine sulfoxide

Range (µg/mL) 0.25 – 2.5 0.25 – 2.5 (0.1 – 1.0 %) (0.1 – 1.0 %) Coefficient of determination R2 0.9955 0.9994 LOD (µg/mL) 0.08 0.05 LOQ (µg/mL) 0.25 0.17 Migration time (RSD) Repeatability (n = 6) 0.25 µg/mL 0.55 1.17 1.0 µg/mL 2.35 0.20 2.5 µg/mL 2.91 0.23 Intermediate precision (n = 3) 0.25 µg/mL 3.29 2.04 1.0 µg/mL 2.95 1.19 2.5 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 3.18 2.5 µg/mL 2.27 3.83 Intermediate precision (n = 3) 0.25 µg/mL 4.34 9.89 1.0 µg/mL 3.08 3.07 2.5 µg/mL 1.84 2.64

35 Levomepromazine method application

Standards 2 Ph. Eur. CRS 1 Injection solution 1 1 2 IStd

3 IStd 3 2

10 15 5 10 15 5 10 15 time [min] time [min] time [min] 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 °C 100 mM sodium citrate; pH 2.85; 3.6 mg/mL HP-γ-CD

Ph. Eur. CRS Injection solution Dextromepromazine (2.84 ± 0.06 %) ~ 0.09 % (< LOQ) Sulfoxide < LOD ~ 0.08 % (< LOQ)

36 Dextromethorphan

Dextromethorphan Levomethorphan

H3CO OCH3

CH H C N 3 3 N

 ent-Morphinan structure  Configuration of morphine  (9S,13S,14S) configuration  (9R,13R,14R) configuration  Cough suppressant  Opioid analgesic drug  Never clinically developed (strong respiratory depressant)  Controlled substance

 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 %)

37 Dextromethorphan

Dextromethorphan Levomethorphan

H3CO OCH3

CH H C N 3 3 N

 Analytical target profile  Determination of levomethorphan with precision and accuracy of ≤ 15 % at the 0.1 % level and ≤ 10 % at > 0.1 % level

38 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

39 Separation of methorphan enantiomers

Sulfated β-CD Sulfated-β-CD / Methyl-α-CD

DXM DXM

LVM LVM

5 10 15 2.5 5 7.5 [min] [min]

20 mg/mL S-β-CD 20 mg/mL S-β-CD / 10 mg/mL M-α-CD

30/40.2 cm, 50 µm ID fused-silica capillary; 50 mM sodium phosphate, pH 7.0; 20 °C, 16 kV

40 Sulfated β-cyclodextrin

[S-β-CD] = 0 mM DXM LVM β [S- -CD] = 2 mM -1 [S-β-CD] = 4 mM K (M ) 259 606 [S-β-CD] = 8 mM (211 / 319) (516/ 716) [S-β-CD] = 12 mM µcplx – 38.0 – 43.3 (10-9m2V-1s-1) (-34.3 / -42.3) (-41.2 / 45.5)

DXM Numbers in brackets represent 95 % confidence interval Data calculated with CEval

LVM

2 4 6 8 10 12 14 16 18 20 Time (min)

Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 30 mM sodium phosphate, pH 6.50; 20 kV, 20 °C

41 Methyl-α-cyclodextrin

[M-α-CD] = 0 mM DXM LVM [M-α-CD] = 2 mM -1 [M-α-CD] = 5 mM K (M ) 354 399 [M-α-CD] = 10 mM [M-α-CD] = 20 mM (292 / 429) (329 / 585)

µcplx 9.97 9.90 (10-9m2V-1s-1) (9.42 / 10.48) (9.38 / 10.40)

Numbers in brackets represent 95 % confidence interval DXM Data calculated with CEval LVM

4 6 8 10 12 Time (min)

Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 100 mM sodium phosphate, pH 2.12; 20 kV, 20 °C

42 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 M-α-CD # Buffer (mM) pH Temp. (°C) Voltage (kV) RS RT (min) (mg/mL) (mg/mL) 1 10 6 30 6.4 15 10 9.12 7.66 2 24 6 30 6.4 25 10 13.86 10.28 3 24 20 30 6.4 15 20 12.06 4.34 4 10 20 30 6.4 25 20 4.99 4.45 5 24 20 100 6.4 15 10 3.96 8.4 6 10 20 100 6.4 25 10 5.28 5.64 7 10 6 100 6.4 15 20 8.43 3.97 8 24 6 100 6.4 25 20 14.02 10.16 9 10 20 30 8.2 15 10 3.68 6.07 10 24 20 30 8.2 25 10 8.8 6.35 11 24 6 30 8.2 15 20 15.86 5.43 12 10 6 30 8.2 25 20 6.86 2.71 13 24 6 100 8.2 15 10 11.41 11.51 14 10 6 100 8.2 25 10 8.03 6.28 15 10 20 100 8.2 15 20 0.01 2.78 16 24 20 100 8.2 25 20 3.79 3.16 17 17 13 65 7.2 20 15 5.97 4.40 18 17 13 65 7.2 20 15 6.19 4.53 19 17 13 65 7.2 20 15 6.45 4.58

43 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 CD CD CD CD - - - - β β α α - - - - M M SB SB

44 Central composite face centered design

S-β-CD M-α-CD Voltage MT # SDXM  Variables (mg/mL) (mg/mL) (kV) (min)  S-β-CD: 10 - 30 mg/mL 1 10 5 10 10.12 8.30 2 10 5 20 5.23 4.89  α M- -CD: 5 - 15 mg/mL 3 30 5 10 22.98 8.09  Voltage: 10 - 20 kV 4 30 5 20 10.50 5.48 5 10 15 10 7.82 0.69 6  2k + 2k + n = 17 experiments 10 15 20 3.45 0.91 7 30 15 10 12.00 0.63 8 30 15 20 5.63 0.85  Responses 9 20 10 10 18.61 3.99  Migration time (MT) 10 20 10 20 5.25 2.79 levomethorphan (≤ 8 min) 11 10 10 15 5.33 2.48  Peak symmetry (S) 12 30 10 15 9.67 2.66 dextromethorphan (0.5 – 3, 13 20 5 15 14.86 7.97 target value 1) 14 20 15 15 6.27 0.95 15 20 10 15 7.26 3.23 16 20 10 15 8.22 3.17 17 20 10 15 7.23 3.13

45 Dextromethorphan CCC design

1 2 3 4 5 6

0 2 4 6 8 0 2 4 6 8 2 6 10 14 0 2 4 6 4 10 16 22 2 6 10 14 t [min] t [min] t [min] t [min] t [min] t [min]

7 8 9 10 11 12

4 8 12 2 4 6 2 6 10 2 4 6 2 4 6 2 6 10 t [min] t [min] t [min] t [min] t [min] t [min]

16 13 14 15 17 LVM

2 6 10 14 2 4 6 2 4 6 8 2 4 6 8 2 4 6 8 t [min] t [min] t [min] t [min] t [min]

46 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

Voltage 10 kV Voltage 15 kV Voltage 20 kV 30 %

27 50 0.5 24 0.5 1 10 1 21 2

CD (mg/mL) 2 - 5 β

- 5 18 S 5 10 2 15 10 50 50 50 12 10 1

6 9 12 15 6 9 12 15 6 9 12 15 M-α-CD (mg/mL) M-α-CD (mg/mL) M-α-CD (mg/mL) 0.5

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

M-α-CD 14 mg/mL % 30

10 50 26 1 0.5 5 50 10 22 2

CD (mg/mL)- CD 18

S - β 2 14 1 10 10 12 14 16 18 20 0.5 Voltage (kV)

48 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 temperature V, voltage Buf, buffer concentration pH, buffer pH SB-C, S-β-CD concentration ma-C, M-α-CD concentration SB-B, S-β-CD batch ma-B, M-α-CD batch

49 Assay robustness test - run time scatter

50 Dextromethorphan assay validation data

Parameter Level Levomethorphan

Range (µg/mL) 1.0 – 15 (0.07 – 1.0 %) Coefficient of determination R2 0.9989 LOD (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 interval 2) 3 concentrations analyzed 3 times on one day 3) 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

51 Dextromethorphan method application

Standards LOQ DXM capsule 1.0 % LVM 0.067 % LVM

DXM DXM DXM IStd IStd 4 LVM

X LVM X LVM IStd

3 4 3 4 3 4 t (min) t (min) t (min)

Experimental conditions: 30/40.2 cm, 50 µm ID fused-silica capillary; 20 °C; 20 kV; HDI 0.7 psi x 5 sec 30 mM sodium phosphate buffer, pH 6.5; 16 mg/mL S-β-CD, 14 mg/mL M-α-CD IStd: 30 µg/mL procainamide hydrochloride; DXM concentration: 1.5 mg/mL

52 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.

53 Acknowledgements

 FSU Jena  Charles University Prague  Manuela Hammitzsch  Pavel Dubský  Qingfu Zhu  Michal Malý  Sudaporn Wongwan  Stephan Niedermeier  World Health Organization  Sulaiman Krait  Dr. Herbert Schmidt  Henrik Harnisch

 Funding