Coupling of spectroscopic, thermoanalytic and chemometric techniques for the resolution of

enantiomeric mixtures

Federico Marini, Remo Bucci, Ilaria Ginevro & Antonio MagrìDipartimento di Chimica, Università di Roma “La Sapienza”

ENANTIOMERS

PAIN DRUG

ENANTIOMERIC MIXTURE

DIFFERENT PHARMACOKINETIC

BEHAVIOUR: study of the absorption process,

distribution, metabolism and excretion

DIFFERENT PHARMACODYNAMICAL BEHAVIOUR: study of the

mechanisms of action between the active principle

and the receptor

ADVANTAGES IN THE USE OF THE ACTIVE ENANTIOMER ONLY

Half quantity of active principle

Elimination of the undesirable effects of the inactive form

Reduction of the metabolic burden on the liver

Reduction of the metabolic burden on the kidneys

Elimination of the intermediate products of the bio-inversion R→S

SINGLE ENANTIOMER DRUGS

d-ibuprofen (S)-citalopram

KETOPROFEN

O

COOH

3HC H

(R)- 2- (3- benzoylphenyl)propanoic acid

O

COOH

H CH3

(S)- 2- (3- benzoylphenyl)propanoic acid

Analgesic

Non-steroidic anti-inflammatory

Antipyretic

RACEMIC KETOPROFEN

d-KETOPROFEN

KETOPROFEN (2)

• (S) is the active enantiomer (cyclooxigenase, which is the target enzyme is stereo-selective)

• (S) is not gastrolesive while (R) is• (S) doesn’t lead to the formation of ulcerae, while

(R) is ulcerogenic at the level of both intestines.

More pharmaceutical industries are moving towards the use of single enantiomers and therefore it is necessary to device a suitable way of determining enantiomeric excess

SCOPE OF THE WORK

Study of the possibility of devicing a rapid and cheap method to determine enantiomeric excess in

pharmaceutical preparation

DSC

Tf (racemate) = 95°C

Tf (enantiomer) = 78°C

IR

3500-2500 cm-1

1700-1300 cm-1

DSC Analysis

• Particularly suited for the determination of purity of compounds (Van’t Hoff law)

• There can be deviations from linearity that need to be corrected.

• Some enantiomeric mixtures melt with decomposition so it’s difficult to estimate the melting temperatures (that are needed for the computation)

EXPERIMENTAL DESIGN

• Check repeatability, reproducibility and effect of some experimental factors:– Way of preparing lab mixtures (directly in mortar

or by dissolution and riprecipitation)– DSC scan rate– Open/sealed capsula– Identity of mixtures prepared starting from the

single enantiomer plus the racemate and of those prepared mixing the two enantiomers

Preliminary study using mandelic acid

OH

O

OH

(s)-mandelic acidOH

O

OH

(R)-mandelic acid

MANDELIC ACID

DSC

Tf (racemate) = 118°C

Tf (enantiomer) = 132°C

IR

3500-2500 cm-1

1700-1300 cm-1

PHASE DIAGRAM

105

110

115

120

125

130

135

0 20 40 60 80 100

% (S)-mandelic acid

T (°C)

fmix

fen

en

T

1

T

1

R

H =ln x

f

fmix

frac

rac

T

1

T

1

R

H2 = x-14xln

f

Prigogine-Dufay Schroeder-Van Laar

SOME CONSIDERATIONS

• No difference in preparing the mixtures by directly weighing and mixing in mortar or by dissolution and riprecipitation.

• No detectable effect of scan rate• No difference in mixing R+S or Rac+S• Sealed capsula provided better reproducibility• Presence of one eutectic

• It’s difficult to measure Tf close to the composition of the eutectic

IR SPECTROSCOPY

500 1000 1500 2000 2500 3000 3500 4000

2

3

4

5

6

7

8

9

x 10-4

ni

A

racemate

(S)-mandelic

PLS CALIBRATION

• 2 significant components (about 94% Y-variance explained)

MCR

Better results with three component, probably due to background effects

MCR (2)

KETOPROFEN - PLS

KETOPROFEN – MCR

0 10 20 30 40 50 60 70 80 90 1000

10

20

30

40

50

60

70

80

90

100

pred

icte

d en

antio

mer

ic e

xces

s

measured enantiomeric excess

MCR 3 components

MCR(2)

500 1000 1500 2000 2500 3000 3500 40000

0.02

0.04

0.06

0.08

0.1

ni

A

MCR component

ketoprofen racemate

500 1000 1500 2000 2500 3000 3500 4000

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

ni

A

MCR component

(s)-ketoprofen

CONCLUSIONS

• Coupling of IR spectroscopy and chemometrics seems to provide a rapid and cheap tool to determine enantiomeric excess to an acceptable accuracy

• Possibility of improving these results by variable selection (GA, iPLS_GA)

• Next step is the validation on real formulates

СПАСИБО!