Enhanced method development

workflow for modern LC and SFC

Davy GUILLARME

18th of September 2014

What can be done with modern LC?

1’000’000 plates1’000’000 plates

120°C 12 m column

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MONOLITHS HIGH TEMPERATURE

CORE SHELL UHPLC

What can be done with modern SFC? A

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Silica

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Ultra-fast analysis of steroids

Use of modern polar column technology (sub-2 µm fully porous or sub-3 µm core-shell)

associated with highly reliable SFC instrument (ΔPmax = 400-600 bar)

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Analysis of a mixture of 17 drugs

A. Grand-Guillaume Perrenoud et al. J. Chrom. A, 2014, 1360, 275-287

Column ChromaNik sunshell

silica 150 x 3 mm, 2.6 µm

Column Waters Acquity UPC²

BEH 100 x 3 mm, 1.7 µm

Kinetic performance of modern LC and SFC I Injection of 50 ppm of butylparaben (dissolved in water and heptane for LC and SF systems, respectively). Isocratic compositions were set

at 40% of ACN in water for LC systems and 5% and 4% MeOH in CO2 for SFC systems, respectively. T= 40°C, BPR = 150 bar.

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H (

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) SFC HPLC

UHPLC UHPSFC

pdh

L

H

LN

p

mopt

optd

Dvu

m

p

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dfC

2

SFC: Viridis 2EP - 4.6 x 150mm, 5µm

UHPSFC: UPC² BEH 2EP - 3.0 x 100mm, 1.7µm

HPLC: RP18 XTERRA - 4.6 x 150mm, 5µm

UHPLC: Acquity BEH Shield RP18 - 2.1 x 50mm, 1.7µm

A. Grand-Guillaume Perrenoud et al. J. Chrom. A, 1266 (2012) 158

Kinetic performance of modern LC and SFC II

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Isocratic compositions were set at 40% of ACN in water for LC systems and 5% and 4% MeOH in CO2 for SFC and UPC2 systems,

respectively. T = 40°C, BPR = 150 bar.

SFC: Viridis 2EP - 4.6 x 150mm, 5µm

UHPSFC: UPC² BEH 2EP - 3.0 x 100mm, 1.7µm

HPLC: RP18 XTERRA - 4.6 x 150mm, 5µm

UHPLC: Acquity BEH Shield RP18 - 2.1 x 50mm, 1.7µm

SFC

HPLC

UHPLC

UHPSFC

2

pd

uLP

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pd

uLP

HPLC vs. SFC

SFC vs. UHPSFC

+

150 bar

Phenotyping CYP450s in HLMs| our cocktail

HLMs Phase I

metabolism

acetaminophen

CYP 2A6 7-hydroxycoumarin 7-hydroxylation

1’-hydroxymidazolam CYP 3A 1’-hydroxylation

hydroxybupropion

CYP 2B6 hydroxylation

CYP 2C9 4’-hydroxyflurbiprofen 4’-hydroxylation

CYP 2C19 5-hydroxyomeprazole

5-hydroxylation

CYP 2D6 Dextrorphan O-demethylation

CYP 2E1 6-hydroxychlorzoxazone 6-hydroxylation

Buproprion 5µM

Phenacetin 50µM

Coumarin 5µM

Dextromethorphan 5µM

Midazolam 2.5µM

Flurbiprofen 5µM

Chlorzoxazone 40µM

subfamily

Omeprazole 40µM Higher activity Lower activity

Control activity

UHPLC method development workflow

1.

2.

3.

Estimation of physico-chemical properties

Screening procedure

Computer-assisted optimization procedure

Acidic / basic, polar / apolar…

Test several apolar stationary phases, mobile phase pH

and organic modifiers.

Optimization of mobile phase temperature, gradient

profile and pH.

Screening procedure in UHPLC

4 stationary phases (50 x 2.1mm, 1.7µm): C18, polar embedded C18, CSH C18, Phenyl

Most promising combination in terms of retention, selectivity and

MS sensitivity for our mixture: C18 column, pH 3, Methanol

Generic

gradient

2-90% in 4 min

3 pH values: 3, 7 and 9

2 organic modifiers: Acetonitrile and methanol

B. Debrus et al. J. Pharm. Biomed. Anal., 2014, 84, 215-223

This screening procedure is only realistic in UHPLC (rinsing steps, duplicate analysis…)

Computer-assisted optimization in UHPLC

• Computer-assisted optimization softwares

DryLab, ACDLabs, Osiris, Chromsword

Realistic approach, only if peak tracking can be efficiently performed

• Factors to be optimized in gradient mode:

Gradient steepness, temperature, pH, additive, ionic strength

• Depending on the number of investigated factors, 2 – 12 initial

experimental runs.

2-3 runs 4-6 runs 6-12 runs

Efficient peak tracking with QDa detector

• When developing chromatographic methods, it is important to track

peaks when changing analytical conditions.

• For this task, UV-DAD can be employed, but often lacks specificity.

• MS would be the best solution but remains expensive and difficult to use,

particularly for beginners.

In this study, a compact, user-friendly single

quadrupole MS detector (Waters Acquity

QDa) was employed to efficiently develop

chromatographic methods and track peaks.

D. Spaggiari et al. J. Chromatogr. A, 2014, Submitted

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UHPLC method screening / optimization

Simulated chromatogram

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T°C

Final optimized conditions

An HPLC modeling software (Drylab) was employed to optimize the gradient profile, temperature

and pH, based on 12 initial experiments. Peak tracking was performed with QDa detector.

Selected working point

T° 35°C

pH 3.7

plate number ~ 7000

Critical resolution 2.89

Gradient Table

Time (min) %MeOH

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Time (minutes) 0.0 2.5 5.0 7.5

3D

-mo

del

2D

-mo

del

Final UHPLC-MS separation 1. acetaminophen, 2. 6-hydroxychlorzoxazone, 3. 7-hydroxycoumarin, 4. dextrorphan, 5. coumarin, 6. hydroxybupropion, 7. phenacetin, 8. bupropion, 9. 5-hydroxyomeprazole,

10. chlorzoxazone, 11. dextromethorphan, 12. omeprazole, 13. midazolam, 14. 4’-hydroxyflurbiprofen, 15. 1’-hydroxymidazolam, 16. flurbiprofen.

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ESI - overlaid SIR

10

ESI+/ESI- overlaid SIR

The differences between predicted and experimental retention times

were comprised between 0 and 5.4%. Analysis time of 7 minutes.

UHPSFC method development workflow

1.

2.

3.

Estimation of physico-chemical properties

Screening procedure

Manual optimization procedure

Acidic / basic, H-bond donor groups, polar / apolar…

Test several polar stationary phases, organic modifiers

and mobile phase additives.

Optimization of temperature, backpressure, gradient

profile and additives concentration.

Screening procedure in UHPSFC

4 stationary phases (100 x 3mm, 1.7µm): Hybrid silica, C18 with no endcapping, 2-ethylpyridine, CSH PFP

2 additives: No water, 2% water

2 organic modifiers: Methanol and isopropanol

Generic

gradient

2-30% in 4 min

Most promising combination in terms of retention,

selectivity and MS sensitivity: 2-EP, Methanol, 2% water

10 mM ammonium formate was systematically added to the mobile phase

Why adding ammonium formate with bases?

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III I

IV II

V VI

VII

I. Benzocaine V. Alprazolam

Low range bases

pKa < 6

II. Noscapine III. Midazolam IV. Papaverine VI. Nortriptyline VII. Duloxetine

Middle range bases

6 < pKa < 8 High-range bases

pKa > 8

Without additive With 10 mM ammonium formate

Need to add 10 mM ammonium formate in the mobile phase

Interfacing SFC with MS

There are various options for SFC-MS hyphenation. Some of them are more universal or user-

friendly and others are more sensitive. The goal is always to avoid precipitation and improve

ionization yield.

Affect sensitivity of mass-dependent ionization source (APCI).

Additional extra-column volume prior to MS.

Flexible operating conditions thanks to the active backpressure regulator

(BPR).

No analytes precipitation due to the addition of sheath liquid (Ethanol).

Ionization enhancers could be added post-column. A. Grand-Guillaume Perrenoud et al. J. Chromatogr. A, 1339 (2014) 174

Sheath

pump BPR

MS UV

Final UHPSFC-MS separation

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1. acetaminophen, 2. 6-hydroxychlorzoxazone, 3. 7-hydroxycoumarin, 4. dextrorphan, 5. coumarin, 6. hydroxybupropion, 7. phenacetin, 8. bupropion, 9. 5-hydroxyomeprazole,

10. chlorzoxazone, 11. dextromethorphan, 12. omeprazole, 13. midazolam, 14. 4’-hydroxyflurbiprofen, 15. 1’-hydroxymidazolam, 16. flurbiprofen.

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In UHPSFC, a baseline separation was achieved in about 7 minutes

Complementarity UHPLC vs. UHPSFC

In UHPLC, the retention of substrates and metabolites is driven by hydrophobic

interactions with the stationary phase.

In UHPSFC, the retention of these compounds is driven by H-bond interactions. The

stationary phase acts mostly as a H-bond acceptor group. The presence of H-bond donor

groups on analyzed compounds generally increases retention.

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UHPLC-MS retention time (min)

UH

PS

FC

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rete

nti

on

tim

e (

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)

High LC retention

Moderate SFC retention

Poor LC retention

High SFC retention

Moderate LC retention

Poor SFC retention

1: Acetaminophen

5: Coumarin

16: Flurbiprofen

http://commons.wikimedia.org/wiki/File:(%C2%B1)-Flurbiprofen_Structural_Formulae_V.1.svg

Final LOD and LOQ values

LOD (ng/mL) LOQ (ng/mL) CYP450 isoform

Substrate / metabolite UHPLC-MS UHPSFC-MS UHPLC-MS UHPSFC-MS

1A2 phenacetin 2 5 5 10

acetaminophen 5 10 20 20

2A6 coumarin 2 10 5 40

7-hydroxycoumarin 20 3 50 10

2B6 bupropion 1 5 3 20

hydroxybupropion 1 3 2 10

2C9 flurbiprofen 20 10 50 50

4’-hydroxyflurbiprofen 50 75 100 200

2C19 omeprazole 1 3 3 10

5-hydroxyomeprazole 2 4 5 20

2D6 dextromethorphan 1 1 2 2

dextrorphan 1 2 3 10

2E1 chlorzoxazone 2 1 5 5

6-hydroxychlorzoxazone 10 40 30 100

3A midazolam 1 1 5 4

1’-hydroxymidazolam 1 5 5 20

Despite the fact that the QDa detector was extremely compact, the

achieved sensitivities were comparable to the ones obtained with other

commercially available single quadrupole detectors.

In average, sensitivity was 3-fold lower in UHPSFC-MS vs. UHPLC-MS.

S

N

x 5

÷ 8

Application of the methods to in vitro incubation

For in vitro metabolism study, the reaction medium is relatively complex and contains the

mixture of 8 CYP probe substrates, 25 mM HEPES buffer at pH 7.4, 0.25 mg/mL of proteins

(HLMs), an excess of NADPH as co-factor, acetonitrile as stopping agent for the microsomal

reaction. A precipitation of proteins and centrifugation is finally performed.

This incubation medium is perfectly compatible with UHPLC-MS conditions, but has never

been tested in UHPSFC-MS.

Because of a possible adsorption of HEPES, NADPH and residual proteins at the surface of

the polar UHPSFC stationary phase, the retention times stability was checked.

During all this study, the RSD values on retention times of the 16 compounds were in average

equal to 0.14% in UHPLC-MS and 0.15% in UHPSFC-MS.

HEPES

NADPH PROTEINS

http://commons.wikimedia.org/wiki/File:HEPES.svg

Inhibition study of two phytochemicals

UHPSFC-MS

UHPLC-MS

Yohimbine Resveratrol

Yohimbine is a strong inhibitor of CYP2D6, while resveratrol moderately inhibits

CYP2E1 activity and weakly inhibits CYP1A2 and CYP3A subfamily activities.

The conclusions drawn in UHPLC-MS and UHPSFC-MS were reliable and identical

SFC vs. LC?

More expensive instrument than LC (20 - 30%)

Less possibility to tune mobile phase, need several stationary phases

2.1 mm I.D. columns hardly compatible with UPC² instrument

Compatibility with MS less straightforward than RPLC

Alternative selectivity compared to RPLC

Better retention of polar compounds in SFC (polar stationary phase)

Possibility to analyze very apolar compounds (triglycerides, carotenoids…)

Green technology (limited consumption of organic solvents)

High throughput chiral and achiral separation on one unique system

Acknowledgments

Hélène BOITEUX

Marleen VAN WINGERDEN

Joel FRICKER

Frédéric FORINI

Dany SPAGGIARI

Florence MEHL

Vincent DESFONTAINE

Alexandre GRAND-GUILLAUME PERRENOUD

Szabolcs FEKETE

Serge RUDAZ

Jean-Luc VEUTHEY