the impact ofenantioselectiveanalyses in clinical …..."the impact ofenantioselectiveanalyses...

1

"The impact of enantioselective analyses in clinical and forensic toxicology"

Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012

C*

A

D

C

B

C*

A

B

C

D

Isomers


IsomersChemical identities with the

same molecular formulaCxHyOz

Constitutional isomersThe same atoms attached

in different sequences

StereoisomersIsomers with the same connectivetyDiffers in their spatial arrangement

EnanatiomersStereoisomers which

are mirror images

DiasteriomersStereoisomers which not are mirror images

2

Chirality and stereoisomers

• sterogenic (chiral) centerfour valences – different substituentschiral planes / chiral axes

• 2 n, 2 2 = 4 steroisomers

• cholesterol2 8 −> one of 256 isomers

• L-amino acid, D-glucose(peptides, proteins)


C C

C

A

A

B

B

D D

B C

C

A

A

C

B

C

C

A

A

B

C

D

B C C

C

A

A

B

B D

CHCH2CH2CH2CHCH3

CH3

CH3

OH

CH3

CH3

L-alanine D-alanineNH3

+

H

C

CH3

COOH

C

COOH

CH3

NH3+

H

Stereoisomers 3-phenyl-2-butanol


-0.65° +30.9° -30.9°

Enantiomers A-B, C-D

Diasteriomers A-C, A-D, B-C, B-D

Chemical/Physical properties enantiomers equal!

Optical rotation enantiomers differ +/-

A B C D

C

C

CH3

CH3

H

HHO

Ph C

C

CH3

CH3

H

H

OH

Ph C

C

CH3

CH3

H

H

OH

Ph C

C

CH3

CH3

H

HHO

Ph

145°C 145°C 151°C 151°C+0.65°

melting pointoptical rotation

Fisher projection

3

Definitions


COOHOHH

CHO

D-Glyceraldehyde COOH

OHHCHO

OHOH

HO HHH

D-Glucose

Fisher projectionD-/L- (dextro-/levo-)glyceraldehyde (ref.)

C4

3

2

1OH

CH3

CH2CH3H

rectus

(R)-2-Butanol

Cahn-Ingold-PrelogR-/S- (rectus-/sinister-)absolute configuration

monochromatic light beam

polarized light beam

test cell

optical rotation

Optical activity(+)/(-) or d-/lplane-polarized light

D-Glyceraldehyde = R-(+)-Glyceraldehyde

Stereoselective biotransformations

subtrate/product stereoslectivity

• chiral -> chiral L-tyrosine -> L-dopa• chiral -> nonchiral L-dopa -> dopamine• nonchiral -> chiral dopamine -> norepinephrine


OH

COOH

H

OH

COOH

NH3+

H

HOOH

NH2

HO

OH

NH2

HO

HOH

4

Racemate and racemization

Many drugs are racemates!

Racemate (50:50)equimolar amount of two enantiomers (R-/S-)

Racemisation =Conversion of a “pure” enantiomer toa mixures of enantiomer

spontaneously – catalysed – by biotransformation


CY

X

Z

L X

ZY

C+

X

ZY

CNu

CY

X

Z

Nuand

Nu:- Nu:-

L:-

transition state

Chirality and drug effects

• eutomerpharmacological active isomer

• distomerinactive or less active isomer

possible differencesü potencyü desired effectü side effects and toxicityü biotransformationü kinetics


enantiomersequal chemical and physical propertiesmay differ significantly in biological action

5

Chirality in pharmacology and toxicology 1:2

• receptor interaction• enzymatic catalysis• antibody interaction

stereoselectivityprotein structures (3D, α-Helix)amino acids (L-)sugar moiety (D-)active sites (functional groups)


Pharmacodynamics-what a drug do with the human body

Chirality in pharmacology and toxicology 2:2

Absorption- active transportDistribution- protein bonding- tissue distributionMetabolism- phase I (oxidation/reduction/hydrolysis)- phase II (conjugation)Excretion

metabolism is often highlystereoselective


Pharmacokinetics-what the human body do with a drug

passive diffusion is notstereroselective

6

Enantioselective bioanalysis


more complicated than conventional analyses “a four to five time factor in cost, time and effort”

• Detection no sensitive on-line detectors are stereoselective (UV, Fluorescence, MS)

• Chromatography (or Capillary Electrophoresis)separation by differences in chemical and physically propertiesenantiomers are equal while diasteromers differs

enantiomers can not be separated by conventional chromatography (RP/NP) but diastereomers can

the rule of three point interaction (attractive or repulsive), x-y-z

Stereoselective (chiral) analysis 1:2

Prosreagents applicable for many analytesseparation on conventional chromatographyor by capillary electrophoresis

Consrequires highly (enantiomeric) pure reagents risk for racemisation during derivatisation


indirect methods• Conversion to diastereomers

ion-pairing (dynamic)derivatisation (static)

R-isomer R,R-derivative+ R-reagent

S-isomer S,R-derivative

7

Stereoselective (chiral) analysis 2:2Pros

many different stationary phases are available no chemical conversion is neededreduced risk for racemisation

Consexpensive columns (1000-3000 euro) often poor column efficiency, N (long analyses)often hard to predict the stereoselectivity


direct methods• Chiral GC

chemically modified capillary columns(less common compared to LC)

• Chiral HPLCcyclodextrins (α,β,γ-CD, derivatives)macromolecules (proteins, antibiotics)brush-type phases (Pirkle phases)cellulose/amylose-based (Okamoto phases)

Impact of stereoselectivity in toxicology 1:3

what is the true concentration of the effective drug (eutomer)?

does 50% or 100% of the doseconsist of the effective drug?

tolerance and toxicity?(e.g. d-sotalol withdrawn due to

increased mortality)


• Chiral swithesoriginal drug as a racemic mixuturenew drug as a pure enantiomer

• Racemization and toxic enantiomersa toxic isomer (distomer) conversions can not always be avoided

8

Chiral switches- increased efficiency of drug therapy- unacceptable adverse effects- lowering doses (no isomeric ballast)- commercial aspects (prolonged patent)


Drugs marketed as single enantiomers

Ecitalopram/Lexapro Citalopram/Celexa (SSRI)(S)-citalopram rac-citalopram

Focalin Ritalin/Concerta (ADHD)(R,R)-methylphenidate R,R-/S,S-methylphenidated-threo-isomer d-/l-threo-isomer

Levosalbutamol/Xopenex Salbutamol/Ventoline (Astma)(R)-Salbutamol rac.-salbutamol

1992 - FDAinitial guidance on Chiral drugs

not availablein all countries

chiral switches cont…

Citalopram - Escitalopram


Chiral Chromatography by HPLC

B-cyclodextrin (Cyclobond I, Astec)250 x 4.6 mm, isocratic within 30 minMeOH/100mM citric acid TEA (55:45, pH 6.3)Fluorescence detection (240/300 nm)

Carlsson B et. al, Linköping Sweden

9


do we know what drug thatwas taken? (prohibited or legal)


• Illicit drugslegal drug prescribed as a pure enantiomer“home made” copy drugs as racemic mixtures

the true identity of a new designer drug (RC)

Illicit drugs – amphetamines

ü S-amphetamine,Metamina – ADHDü R-amphetamine, metabolite for Seliginine – Parkinson’s diseaseü rac-amphetamine (or other ratios) from illicit drugs - Abuse


H

N

CH 3

CH 3

3

H

N

CH3

CHH

H

N

CH3

HH

H CH3NH

Selegilin

Dm-selegilin

R-amfetamin

R-metamfetamin

H

N

CH 3

CH 3

3

H

N

CH3

CHH

H

N

CH3

HH

H CH3NH

Selegilin

Dm-selegilin

R-amfetamin

R-metamfetamin

10

amphetamines cont…

Samples R/S-ratio

Case 1,25

Ref. (abuse case) 1,34

Metamina patients 0.10 (S-)

Selegilin patients 11.2 (R-)


Chiral derivatisation and GC-MS

N-trifluoroacetyl-l-prolylkloridHP5-MS 30 m 0.25mm/0.25µm

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 237.00 (236.70 to 237.70): 8401005.D

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 241.00 (240.70 to 241.70): 8401005.D

S(+)-amphetamineMetamina

suspected abuse

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 237.00 (236.70 to 237.70): 8401005.D

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 241.00 (240.70 to 241.70): 8401005.D

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 237.00 (236.70 to 237.70): 8401005.D

7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80

Ion 241.00 (240.70 to 241.70): 8401005.D

S(+)-amphetamineMetamina

suspected abuse


are there adverse effects due tosteroselective biotransformation?


• Enantioselective metabolismInterindividual differences in metabolism Cytochrom P450 system/genetic variationpoor or extensive metabolisers, PM/EMdrug efflux transporter (Pgp), BBB

Citalopram (SSRI)Venlafaxine (SNRI)Tramadol (opiate, painkiller)Methylphenidate (ADHD)

11

metabolism cont… Venlafaxine (VEN)O-desmethyl (ODV), N-desmethyl (NDV)N,O-didesmethyl (DDV)


Chirobiotic V, 5 µm particle, 250 x 2.1 mmcolumn temperature was kept at 10°C

tetrahydrofuran: ammonium acetate 10 mM pH 6.0 (10:90; v/v), 0.2 ml/min

0.05% formic acid in acetonitrile was addedpost column, 0.2 ml/min

ESI (pos) 3Q LC-MS

2 4 6 8 10 12 14 16 18 22 24 26 28 30 32

Time, min0.0

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

Intensity, cps

20

S-D

DV

R

-DD

V S-

OD

V

R-O

DV

S-V

EN

R-V

EN

S-N

DV

R-N

DV

Compound Transition (Q1/Q3) Collision energy (V)

S-enantiomer R-enantiomer S-enantiomer R-enantiomer

VEN 278/58 45 100 102 19.6 23.4278/260 20

ODV 264/58 40 98.1 104 14.4 16.2264/246 20

NDV 264/121 35 49.2 54.2 19.1 21.6264/246 20

DDV 250/107 40 52.7 57.8 13.9 15.2250/232 18

Mexiletine 180/58 22 N.C. N.C. N.C. N.C.180/105 27 N.C. N.C. N.C. N.C.

Relative area intensity (%) Retention time (min)

Challenges in enantioselective analysis by LC


• Reference materialmany drugs are not commercially available as pure enantiomershigh costs

• Chromatographic selectivitycomplete baseline separation could be hard to achievesmall changes in mobile phase composition could give drastic change in chromatography

• Requires sensitive detection the “pure” enantiomer is 50% of the racemate (often other ratio 75:25, 90:10)detection under non-optimized conditions (e.g. pH, type and content of organic solvent)MS-detection not always applicable (e.g. normal phase conditions)

• Complex bioanalytical methods chiral parent drug and chiral metabolites must be separated under the same conditionschiral phases often more sensitive for long term matrix effects

12

Thank you for your attention!


Further reading

Chiral Toxicology: It’s the Same Thing…Only Different (Review)Silas W. Smith (2009) Tocixol Sci 110(1)4-30

Chiral recognition by enantioselective liquid chromatography: Mechanisms and modern chiral stationary phases (Review)Michael Lämmerhofer (2010) J Chrom A 1217:814-856

the impact ofenantioselectiveanalyses in clinical …..."the impact ofenantioselectiveanalyses...

Documents