importance of enantiomeric profiling of chiral drugs in...

Department of Chemistry

Importance of enantiomeric

profiling of chiral drugs in

wastewater-based epidemiology

and pharmacokinetic relevance

Barbara Kasprzyk-Hordern

University of Bath, Department of Chemistry, Faculty of

Science, Bath, BA2 7AY, UK

Phenomenon of chirality


A therapeutic (+)-enantiomer of thalidomide is harmless (has

tranquilising properties) but in the human body it undergoes in vivo

inter-conversion leading to toxic (-)-enantiomer (teratogenic

properties), which leads to malformations of embryos if administered to

pregnant woman

(-)-enantiomer

Teratogen

(+)-enantiomer

Sedative

http://www.google.co.uk/url?sa=i&rct=j&q=thalidomide+enantiomers&source=images&cd=&cad=rja&docid=dcivBWb226tmkM&tbnid=g7LVHvoczRVgpM:&ved=0CAUQjRw&url=http%3A%2F%2Fwww.nature.com%2Fnrc%2Fjournal%2Fv4%2Fn4%2Fbox%2Fnrc1323_BX1.html&ei=WeyCUZakA-ml0QXa44HgBA&bvm=bv.45960087,d.ZG4&psig=AFQjCNFdhoicCd-ueFVQ2R9ufYGqyUUhCg&ust=1367621051054093



More than half of the drugs currently in use are chiral

compounds and many of these are marketed as

racemates

Worldwide sales of chiral drugs in single-enantiomer form

continuously increase

There is at the present a tendency in the pharmaceutical

industry to switch from racemates to single enantiomers


NSAIDs, analgesics and anaesthetics

CNS drugs: antipsychotic drugs, antidepressants, sedative/hypnotics, CNS stimulants and drugs used for ADHD, drugs used in neurological disorders

Respiratory drugs: bronchodilators, antihistamines, decongestants

Cardiovascular drugs: lipid regulating drugs, beta-adrenoceptor blocking drugs, antihypertensives, anticoagulants, calcium channel blockers, anti-arrhythmic drugs

Antimicrobials

Gastro-intestinal drugs: proton pump inhibitors, H2-receptor antagonists

Antineoplastics

Illicit drugs



Global market – racemic switches

Chiral drug Marketed as Prescription in England

[kg year-1]*

2004 2005 2006 2007

Ibuprofen

Dexibuprofen

Racemate

S(+)-enantiomer

129037

0

131252

54

121392

512

117281

503

Ketoprofen

Dexketoprofen

Racemate

S(+)-enantiomer

755

25

714

21

622

17

527

13

Citalopram

Escitalopram

Racemate

S(+)-enantiomer

3038

396

3270

521

4017

572

5049

223

Omeprazole

Esomeprazole

Racemate

S(-)-enantiomer

3746

1203

5494

1405

7326

1553

8864

1755

Cetrizine

Levocetrizine

Racemate

R(-)-enantiomer

807

70

839

82

924

76

1034

62

* - data obtained from National Heath Service


Chiral drugs produced in their racemic form can be divided into two groups:

drugs with only one major bioactive enantiomer (e.g. propranolol, salbutamol, verapamil, warfarin)

drugs with two bioactive enantiomers:

- equipotent (e.g. flecainide, mexiletine)

- with qualitatively different action (e.g. labetalol, ketamine)



Enantiomers of chiral drugs can be metabolised:

O O

CH3

O

OH

*

O O

CH3

OH

OH

*

O O

CH3

O

OH

OH

*

O O

CH3

O

OH

OH

*

*

S/R-warfarin Warfarin alcohol

(major metabolism pathway for (R)-warfarin)

7-hydroxywarfarin

(major metabolism pathway for (S)-warfarin)

6-hydroxywarfarin

(metabolism pathway for both (S)- and (R)-warfarin)

at different rates (quantitative differences)

by different routes (qualitative differences)

with retaining the same chiral centre

with an introduction of another chiral centre



Metabolism can also lead to:

the removal of chiral centre (e.g. omeprazole)

Clearance of

propranolol

S>R in dog

S<R in human

Clearance of

warfarin

S>R in human

S<R in rat

Interspecies differences in the stereoselectivity of metabolism and elimination are very common:

N

CH3 CH3

OCH3

S

NH

N OCH3

O

N

CH3 CH3

OCH3

S

NH

N OCH3

O

O

Omeprazole (chiral)

*

Sulfone (achiral)

enzymatic chiral inversion (e.g. NSAIDs)


PRINCIPLES OF CHIRALITY

Enantiomers of the same chiral molecule have similar physico-

chemical properties but may differ in their biological properties

Enantiomers of chiral drugs reveal different potency:

• S(+)-enantiomers of AMPH-derived drugs have much higher stimulant activity

than R(-)-enantiomers

• S(+)-MDMA is more amphetamine-like stimulant and R(–)-MDMA is more

hallucinogenic

• S-ibuprofen is 100x more potent than R-ibuprofen

• S(-)-enantiomers of beta-blockers reveal much higher potency in humans

• Cardiovascular effects of verapamil are mediated by S(-)-enantiomer

Chiral drugs have stereoselective disposition in the body: • S(+)-enantiomers of amphetamine-derived drugs metabolise faster in human

than R(-)-enantiomers



B. Kasprzyk-Hordern, D. Baker, STOTEN, 423 (2012) 142

RP (C18)-UPLC-QqQ

RP-chiral (CBH) LC-QqQ

S (+) - MDA

R ( - ) - MDA

R ( - ) - MDMA

S (+) - MDMA

R ( - ) - AMPH

S (+) - AMPH

Analysis of chiral biomarkers



SPE (HLB, 60mg)

UPLC

Column 1:

CBH

(100x2.1mm; 5um);

90% H2O, 10% IPA, 1mM

NH4OAc;

25oC, 0.075 mL/min; isocratic

Column 2:

Chirobiotic V

(250x2.1mm; 5um);

100% MeOH, 4mM NH4OAc,

0.005% HCOOH; 25oC, 0.1

mL/min; isocratic

Mass Spectrometry

MS1:

(ESI+) QqQ (Waters TQD)

MS2:

(ESI+) QTOF (micrOTOFQ, Bruker)

Condition: 4mL MeOH, 4mL H2O; Sample load: 100mL wastewater; 500mL river water; Elution: 4mL

MeOH; Evaporation and reconstitution to 0.5mL with mobile phase

ANALYSIS OF CHIRAL DRUGS IN LIQUID MATRICES

S(+)-MDA

R(-)-MDA

R(-)-Atenolol

S(+)-Atenolol

R(-)-MDMA

S(+)-MDMA

S(+)-METH

R(-)-METH

E1-Venlafaxine

E2-Venlafaxine

R(-)-AMPH

S(+)-AMPH

1R,2S(-)-Ephedrine

1S,2S(+)-Pseudoephedrine

S(+)-MDA

R(-)-MDA

R(-)-Atenolol

S(+)-Atenolol

R(-)-MDMA

S(+)-MDMA

S(+)-METH

R(-)-METH

E1-Venlafaxine

E2-Venlafaxine

R(-)-AMPH

S(+)-AMPH

1R,2S(-)-Ephedrine

1S,2S(+)-Pseudoephedrine

LC-MS/MS chromatograms

of chiral drugs in

wastewater influent

Column:

CBH (100x2.1mm; 5um);

90% H2O, 10% IPA, 1mM

NH4OAc;


MS:

TQD

B.Kasprzyk-Hordern, V.V.R. Kondakal, D.R. Baker, J. Chromatogr 1217 (2010) 4575



LC-MS/MS chromatograms of

chiral drugs in river water spiked

with chiral drugs and extracted

by SPE (concentration, 100 ng/L)

Column:

Chirobiotic V (100 x 2.1mm; 5um);

100% MeOH, 4mM NH4OAc; 0.005%FA


MS:

QTOF

J. Bagnall, S.E. Evans, M.T. Wort, A.T. Lubben, B. Kasprzyk-

Hordern, J. Chromatogr 1249(2012)115

Importance of chirality Wastewater-based epidemiology

• Distinction between consumption and direct

disposal

• Identification of whether drug residue results

from consumption of illicit drug or metabolism

of other (illicit) drug

• Distinction between legal and illicit use of drugs

• Verification of the method of synthesis of illicit

drugs

• Verification of potency of abused drugs


NH

O

O

H

mirro

r im

age

NH

O

O

H

Enantiomers of MDMA

Importance:

Potency and metabolism Synthesis: Non-stereoselective method

S(+)-MDMA

• More AMPH-like

stimulant

• Metabolises

faster in humans

R(-)-MDMA

• More

hallucinogenic

• Dominant in urine


No medical use

Produced as racemate

Metabolism favours S(+)-enantiomer

Preferential metabolism of S(+)-MDA

leads to enrichment of MDA with R(-)-

enantiomer

CH3

NH2

O

O

*CH3

NH

O

O CH3

*

MDA MDMA

No medical use

Produced as racemate

Metabolism favours S(+)-enantiomer

Preferential metabolism of S(+)-

MDMA leads to enrichment of MDMA

with R(-)-enantiomer and formation of

S(+)-MDA (twice as much S(+)-MDA

is excreted in urine as compared to

R(-)-MDA)


MDMA IN 7 WWTPs IN ENGLAND

𝐸𝐹 =𝑅 − 𝑀𝐷𝑀𝐴

𝑅 − 𝑀𝐷𝑀𝐴 + 𝑆 + 𝑀𝐷𝑀𝐴


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7

Load

[g/

day

]

JANFEBMARAPRAUG

0

100

200

300

400

500

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7

Co

nce

ntr

atio

n [

ng/

L] JAN

FEB

MAR

APR

AUG

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7

Enan

tio

me

ric

frac

tio

n

JAN FEBMAR APR

Excess of R(-)-MDMA

CH3

NH

O

O CH3

*

MDMA


MDA in 7 WWTPs in England

𝐸𝐹 =𝑅 − 𝑀𝐷𝐴

𝑅 − 𝑀𝐷𝐴 + 𝑆 + 𝑀𝐷𝐴


CH3

NH2

O

O

*

MDA

0

10

20

30

40

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7Co

nce

ntr

atio

n [

ng/

L]

JANFEBMARAPRAUG

0

0.02

0.04

0.06

0.08

0.1

0.12

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7

Load

[g/

day

]

JAN

FEB

MAR

APR

AUG

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

WWTP1

WWTP2

WWTP3

WWTP4

WWTP5

WWTP6

WWTP7

Enan

tio

me

ric

frac

tio

n

JANFEBMARAPRAUG

Excess of S(+)-MDA


2011 averaged weekly loads

in mg/d/1000 i.e. MDMA

Bel

gium

, Ant

wer

p

Bel

gium

, Bru

ssel

s

Cro

atia

, Zag

reb

Cze

ch R

ep.,

Bud

wei

s

Finla

nd, H

elsi

nki

Finla

nd, T

urku

France

, Par

is

Italy

, Mila

n

NL, A

mst

erdam

NL, E

indho

ven

NL, U

trec

ht

Norw

ay, O

slo

Spai

n, Bar

celo

na

Spai

n, Cas

tello

n

Spai

n, San

tiago

Spai

n, Val

enci

a

Swed

en, S

tock

holm

Swed

en, U

mea

UK, L

ondon

0

50

100

150250500750

1000

< < <

mg

/da

y/1

00

0 in

h.

MDMA – Utrecht 2011

0

50

100

150

200

250

300

350

400

0 2 4 6 8

Lo

ad

[g

/da

y]

Days

STP Utrecht2011

2010

Thu Fri Sat Sun Mon Tue Wed Fri

10/03/2011-16/03/2011

17/02/2010-23/02/2010

SAMPLING:

Emke et al., STOTEN 487 (2014) 666–672


9 March 2011

two people were

arrested for having

a facility to

manufacture XTC

pills

-Typical flow 1m/s

-1.8 km distance

-Travel time 30 min

MDMA – Utrecht 2011

Emke et al., STOTEN 487 (2014) 666–672


MDMA –

Utrecht 2011

Thu Fri Sat Sun Mon Tue Wed Fri

Excess of R(-)-MDMA

0

50

100

150

200

250

300

350

400

0 2 4 6 8L

oa

d [

g/d

ay]

Days

STP Utrecht2011

2010

2011 – racemate

DIRECT DISPOSAL

2010 – excess of R(-)-MDMA

CONSUMPTION

Emke et al., STOTEN 487 (2014) 666–672


Conclusions Enantiomeric profiling of chiral drugs in environmental matrices provides yet another dimension to wastewater-based epidemiology as it helps with:

• Distinction between legal and illicit use of drugs • Verification of the method of synthesis of illicit drugs • Identification of whether drug residue results from consumption of illicit

drug or metabolism of other (illicit) drug • Verification of potency of abused drugs


importance of enantiomeric profiling of chiral drugs in...

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