Use of Supercritical Fluid

Chromatography (SFC) for

Quantification and Bioanalysis

Utilizing MS/MS and HRMS

Detection

Chester Bowen

GlaxoSmithKline Pharmaceuticals,

Bioanalytical Science and Toxicokinetics,

Upper Merion PA

Outline

A combination of SFC and chemical derivatization to separate four

enantiomers of GSK compound A utilizing SFC-MS/MS

SFC application to separate a complex mixture of multiple enantiomeric

metabolites of GSK compound B

Separation of different classes of diastereomers on reverse ULPC-

MS/MS (M2; M3; M4; M5; M6; and M13)

SFC application to separate diastereomers within classes and in the

complex mixture utilizing SFC-MS/MS

A combination of SFC high resolution mass spectrometry to separate isobaric

analytes utilizing SFC-HRMS

Isomers

FDA Policy states1:

– “To evaluate the PK of a single enantiomer or mixture of enantiomers, manufacturers should develop quantitative assays for individual enantiomers in in vivo samples early in drug development.”

1. FDA’s Policy Statement for the Development of New Stereoisomeric Drugs

Supercritical Fluid Chromatography

Image: Larry Taylor, Modern Supercritical Fluid Chromatography

Image: www.jasco.de

SFC Column Screening

Image: John Langley, Supercritical Fluid Chromatography

Image: www.lamondlab.com

Complex mixture of parent and three stereoisomers Combining SFC and Triple Quadrupole Mass Spectrometry

Example #1

Method Development In

tensity

0.0

5000.0

10000.0

15000.0

20000.0

25000.0

30000.0

35000.0

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Parent

Diastereomer

Diastereomer

Enantiomer

Chiral Column A, IPA with 0.4% DEA as modifier

Method Transfer to GSK –

Chiral Column A, Different Lot #

20 minute run time

13 minute run time

Method Development – Different Lots of Chiral Column A

20 minute run time

20 minute run time 20 minute run time

Column Transfer

Waters shipped the chiral column they performed method development with

Adjust the BPR to achieve separation shown below.

Issue was peak at 4.10 min had significant tailing

20 minute run time

Chemical Derivatization with Camphanic Chloride

Camphanic Chloride Optimization

Parameters:

Concentration – 1 mg/mL

Solvent - ACN

Derivatization Time – 15 min

Derivatization Temperature – 37ºC

Analytical Method Conditions

Assay Range: 5 – 5000 ng/mL

100 µL plasma, protein crash with ACN, derivatize with camphanic chloride

SFC Conditions

ABPR: 2750 psi

Mobile Phase B: IPA with 0.4% DEA

Washes: MeOH

Flow: 3 mL/min, isocratic at 20% MPB

Column Temp: 40°C

Column: Chiral PAK AD-H 4.6 x 150 mm

Run time of 10 minutes

Mass Spec Conditions

ESI temp at 150ºC

Dessolvation temp at 200ºC

Dessolvation gas (L/Hr) at 800

Cone gas (L/Hr) at 150

Capillary (Kv) at 4

Cone (Kv) at 25

Collision at 30

Chromatography Comparison

Time (minutes)

0 20 40

Peak In

tensity

0

2e6

4e6

6e6

8e6

1e7

1.2e7

HPLC-UV Chromatography –

Normal Phase

Underivatized SFC-MS/MS

Chromatography

Derivatized SFC-MS/MS

Chromatography

STD5000

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

GSK13223322HUPLVALE01_007 MRM of 2 Channels ES+ 660 > 462.2 (Diasteromers derivatized)

1.12e7

5.07

7.597.546.83

6.78

5.96

45 minute run time

20 minute run time

10 minute run time

Chromatogram of Derivatized Compounds with UPLC Conditions

5000 ng/mL of all 4 derivatized compounds. No separation using BEH C-18 column.

Co-elution of all

derivatized peaks

Validation Statistics

Parent Molecule

Mean (n=18) 4.72 14.46 247.76 3956.5 4859.67

Precision (%CV) 5.6 4.7 2.4 1.3 1.2

Bias % -5.6 -3.6 -0.9 -1.1 -2.8

Between-run Precision (%) 2.1 4.0 0.5 0.3 0.8

Diastereomer 1

Mean (n=18) 4.63 14.98 247.17 3936.67 4843.79

Precision (%CV) 5.1 2.8 2.1 1.9 1.7

Bias % -7.4 -0.1 -1.1 -1.6 -3.1

Between-run Precision (%) 3.9 Negligible 1.1 1.2 1.8

Diastereomer 2

Mean (n=18) 4.86 15.27 246.72 3830.84 4697.48

Precision (%CV) 5.6 4.0 2.3 2.6 2.0

Bias % -2.8 1.8 -1.3 -4.2 -6.1

Between-run Precision (%) 4.2 2.3 1.0 1.7 1.9

Enantiomer

Mean (n=18) 4.77 15.02 244.72 3883.63 4771.53

Precision (%CV) 3.6 2.9 2 2.0 0.9

Bias % -4.7 0.2 -2.1 -2.9 -4.6

Between-run Precision (%) Negligible 1.2 0.9 Negligible Negligible

Complex mixture of parent and enantiomeric metabolites Utilizing UPLC-MS/MS and SFC-MS/MS

Example #2

GSK compound B has multiple enantiomeric metabolites

Separation of different class of diastereomers on reverse UPLC (M2; M3;

M4; M5; M6; and M13)

The Project Team needed a method mainly for M3 and M13.

Separation of diastereomers within classes and in the mixture

– M2; one enantiomer

– M3; two enantiomers

– M4; two enantiomers

– M5; three enantiomers

– M6; four enantiomers

– M13; two enantiomers

SFC Application to a Complex Mixture of

Enantiomeric Metabolites – Case #2

14 Possible

Distinct Peaks

Chiral Separation on SFC-MS/MS System

Column: Chiralpak AD-H 4.6 mmx150 mm; 5 µm

Flow rate: 1.5 ml/min

Mobile phase B: 0.5% Formic Acid in Isopropanol

Retention Time (min)

0 2 4 6 8 10 12 14 16

Inte

ns

ity

(c

ps

)

0

2e5

4e5

M13 SFC-MS/MS Separation

Retention Time (min)

0 2 4 6 8 10 12 14 16

Inte

ns

ity

(c

ps

)

0

20000

40000

60000

80000

1e5M3 SFC-MS/MS Separation

Retention Time (min)

0 2 4 6 8 10 12 14 16

Inte

ns

ity

(c

ps

)

0

20000

40000

60000

80000

1e5

1.2e5

M2

M3

M4

M2, M3 and M4 SFC-MS/MS Separation

Chiral Separation on SFC-MS/MS System

Column: Chiralpak AD-H 4.6 mmX150 mm; 5 µm

Flow rate: 1.5 ml/min

Mobile phase B: 0.5% Formic Acid in Isopropanol

Retention Time (min)

0 2 4 6 8 10 12 14 16

Inte

ns

ity

(c

ps

)

0

10000

20000

30000

M5

M6

M5 and M6 SFC-MS/MS Separation

Chiral Separation on SFC-MS/MS System

Column: Chiralpak AD-H 4.6 mmX150 mm; 5 µm

Flow rate: 1.5 ml/min

Mobile phase B: 0.5% Formic Acid in Isopropanol

Retention Time (min)

4 6 8 10 12 14

Inte

ns

ity

(c

ps

)

0

20000

40000

60000

80000

1e5

1.2e5M2

M3

M4

M5

M6

M2 through M13 SFC-MS/MS Separation

M13

Complex mixture of isobaric analytes Can technology overcome poor ionization and non-selective transitions?…….. Utilizing SFC-HRMS

Example #3

Initial Separation Attempts with UPLC-MS/MS Isobaric analytes; long gradient required……

m/z = 299 255 m/z = 313 269

–Method required quantitation of 6

analytes – Transitions not particularly selective

– Poor Ionization (negative mode)

Secondary Separation Attempts with SFC-MS/MS

Neat solutions look great, issues noted in human plasma……

Neat solutions (100 ng/mL)

Control Plasma (200 uL)

100 ng/mL in Plasma (200 uL)

Study requires a LLQ of 0.3 ng/mL

Initial Attempts Using a New Platform (SFC-HRMS)

Major Improvement in Sensitivity and Selectivity; work in progress……..

Neat solutions (10 ng/mL)

10 ng/mL in Plasma (200 uL)

–SFC-TOF-MRM acquisitions – Baseline separation

– Notable improvement in selectivity

and sensitivity

– Work in progress; experiments ongoing…..

m/z 299255

m/z 313269

m/z 313269

m/z 299255

Conclusion

Example 1 – SFC-MS/MS

application with chemical

derivatization. Full validation

and clinical study support

Example 2 – SFC-MS/MS

application without chemical

derivatization.

Example 3 – SFC-HRMS

application of isobaric

analytes.

STD5000

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

GSK13223322HUPLVALE01_007 MRM of 2 Channels ES+ 660 > 462.2 (Diasteromers derivatized)

1.12e7

5.07

7.597.546.83

6.78

5.96

Retention Time (min)

4 6 8 10 12 14

Inte

ns

ity

(c

ps

)

0

20000

40000

60000

80000

1e5

1.2e5M2

M3

M4

M5

M6

M2 through M13 SFC-MS/MS Separation

M13

Customer Value

The characterization of the drug metabolism and pharmacokinetic

(DMPK) profiles of stereoisomers is a fundamental aspect of the drug

discovery and development processes

Fully validated rapid and robust assay for parent and 3 stereoisomer

metabolites was developed and In vivo chiral inversion was investigated

in pooled clinical and preclinical samples to determine stereoselective

metabolism

Upon request, stored clinical or preclinical samples could be assayed to

answer questions about stereoselective metabolism for the various

projects

Achiral work currently being investigated on SFC

SFC in DMPKs Future

2015

–Possible study support for two “high profile” GSK assets

–Unique selectivity may be useful for difficult to separate achiral

compounds

–Less solvent use

2015 and beyond

–With a single detector independent chromatography

platform, assays could be easily transferred between various

departments within GSK

–Minimize method development time

Acknowledgements

GSK

–Hermes Licea-Perez

–Christopher Evans

–Dana Knecht

–Molly Karlinsey

–Jonathan Kehler

Waters

–Thomas DePhillipo

–Denise Heyburn

–Paul Rainville

–Rob Plumb

–Contact email Chester.L.Bowen@gsk.com