Post on 12-Aug-2020
transcript
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