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LCLC--NMR in Drug DiscoveryNMR in Drug Discovery
Brian L Marquez, Ph.D.
Principal Scientist Tel: (860) 686-1360Structure Elucidation Group Fax: (860) 686-6227Pharmaceutical Sciences NMR Laboratory Email: [email protected] Inc.445 Eastern Point Road, MS 8118A-2011Groton, CT 06340
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Personal BackgroundPersonal Background
• Undergraduate in Biochemistry and Biophysics in 1997• Ph.D. in Medicinal Chemistry in 2001
– Pharmacognosy -- Marine Natural Products
• Wyeth Pharmaceuticals (PA) -- NMR Spectroscopist in Discovery• Amgen Inc. (CA) -- NMR Spectroscopist in Discovery/PKDM/Process
Chemistry• Pfizer Inc. (CT) -- NMR Group Leader in Pharmaceutical Sciences
(Development)
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What we do in the Pharm. Sci. NMR groupWhat we do in the Pharm. Sci. NMR group
• NMR need throughout all of drug development. This includes but is not limited to:– Filing characterizations– Lot confirmations– Solution confirmations
– New technology development and integration– STRUCTURE ELUCIDATION
• Impurities (e.g. process related)• Degradants (e.g. process related or forced degradation)
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Intent of this LectureIntent of this Lecture
– What is LC-NMR – No NMR theory, just application oriented.
– Limitations – Configurations/Options– Practical Considerations – Overlying Theme
• When/When not to use it
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LCLC--NMR: Simplistic ConceptNMR: Simplistic Concept
• HPLC is plumbed in line with a “flow” NMR system• Sample components are physically separated by HPLC• Each component flows, in turn, from the LC column and
UV detector to the NMR sample cell– Multiple different configurations for this
• NMR is performed on each desired fraction / peak– Always the rate limiting step
• Continuous or stopped flow mode– Additional methods are also available – Peak “parking” and
“trapping”.
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Modes of OperationModes of Operation
• Continuous Flow– Eluent sampled in “real-time” as flowing through NMR Detection
Coil
• Time Slices– Regions, or “time-slices” of interest are analyzed
• Stopped Flow– Pump is stopped at desired location and data acquired
• Peak Parking– Peaks of interest are “parked” in off-line sample loops
• Peak Trapping– Solid Phase Extraction cartridges are used to “re-concentrate”
samples.
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General Schematic for an LCGeneral Schematic for an LC--NMRNMR
LC Pump
Injector
Column/ColumnHeater
UV
X
NMRSpectrometer
LC Pump
X
Waste OrFractionCollector
LC ControlSystem
NMR
Control System
36 LoopCassette
OR
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General Cartoon of Loop CollectionGeneral Cartoon of Loop Collection
DADDetector
XLC Pump
NMRSpectrometer
….
….
36 LoopCassette
Loop Collection
Direct Stop Flow
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LCLC--NMR Hardware ConfigurationNMR Hardware Configuration
DegasserBinary or Quaternary LC Pump
UV Detector Manual Injection RF Gradients
TemperatureControlUnit
LC Column Temperature Control Loop Collection600 MHz NMR Console
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LCLC--NMR Probe SchematicNMR Probe Schematic
IN OUT
240µL
NMR detection coil built directly onto flow cell (4mm OD)
120µL
From LC To Waste
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Traditional Probe ConfigurationsTraditional Probe Configurations
• Most common configuration – inverse probes – best proton sensitivity
• Flow Cells – Active Volumes– 3mm – 60µL– 4mm – 120µL– 5mm – 240 µL– Others “non-traditional” will be covered later
• Typically probes are outfitted with z-gradients– For gradient experiments and shimming
Active Volume
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General Application StrategyGeneral Application Strategy
4.6 mm or 6mm Reverse-phase HPLC
Mobile phase: CH3CN/D2O+(0.1%TFA or DCOOD), solvent gradient < 2%/min
~50 µg of metabolites
From microsomal
incubations
PM2
M1M3
1. Structural determination of metabolites:
4.6 mm or 6 mm reverse-phase HPLC
Mobile phase: CH3CN/D2O+(0.1%TFA or DCOOD), solvent gradient < 2%/min
Large volume
injections in high % of
aqueous phase
More then 1 mg
i2i1
P
Min~1 µg
2. Structural determination of impurities:
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Structure Elucidation Using NMRStructure Elucidation Using NMR
• 1D 1H and homonuclear NMR experiments are the most sensitive and accessible experiments for LC-NMR
• 1D 13C and heteronuclear NMR experiments are very insensitive and are typically inaccessible to LC-NMR applications (in most cases).
O H
H CH3
H
1H-1H COSY
1H-1H NOESY
1H-13C HMBC
1H-13C HSQC
Homonuclear Homonuclear CouplingCoupling
Heteronuclear Heteronuclear CouplingCoupling
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When to use LCWhen to use LC--NMRNMR
• Fairly resolved peaks.• Relative abundance of entities similar.• Known stability issues.• A significant amount of information is known
about the compound
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20µL injected
t %B0 1048 10050 10050.5 1051 10
1.5mL/min4.6x150mm Luna 5u C8(2)A: 0.1% TFA/D20B: 0.1% TFA/MeCN
Impurity Analysis: Impurity Analysis: Low Volume (20Low Volume (20µµL) Injection of SampleL) Injection of Sample
0 5 10 15 20 25 30 35 40 45Time [min]
200
400
600
800
Intens.[mAU]
4 5 6 8
7
~0.3-0.5% Impurities (215nm)
In order to achieve sufficient NMR sensitivity it was necessary to overload the HPLC column without sacrificing peak resolution. This goal was achieved by maximizing sample concentration in the highest content of aqueous phase followed by large volume column injections.
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500µL injected
t %B0 1048 10050 10050.5 1051 10
1mL/min4.6x150mm Luna 5u C8(2)A: 0.1% TFA/D20B: 0.1% TFA/MeCN
UV (215.0nm)
Pressure
0 5 10 15 20 25 30 35 40 45 Time [min]
0
200
400
600
800
Intens.[mAU]
4
56
8
7
Impurity Analysis: Large Volume (500µL) Injection of Sample
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1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
7,83-5
9
6,6’1
*
3-5 7 8 9 6,6’
10
1
11-13,3-58 7 9 2 6,6’
10
1
11,123-5
8 7 9 2 6,6’
10
1
11, 128 7 9 6
10
3’-5’
1
3-5
#4
#5
#6
#7
#8
3
45
B78
AD
3
45
B78
A
3
45
B78
AC11
12
3
45
B78
AC11
12
3
45
B78
A 1112
13
Impurity Analysis: Solved StructuresImpurity Analysis: Solved Structures
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100µL of ~4mg/mL injected
t %B0 355 3530 9537 9538 3540 35
1.0mL/min4.6x250mm YMC-AQ 5u 120AA: 0.1% d-FA/D20B: 0.1% d-FA/MeCN
Metabolite Analysis:Metabolite Analysis:
DAD (254)
Analog only
0 5 10 15 20 25 30 35Time [min]
0
500
1000
1500
2000
Intens.[mAU]
12
3
5
4
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3.54.04.55.05.56.06.57.07.58.08.59.0 ppm
N N
F
F
F
O
O
O N1
2
345
6
8
79
10
11
12
13
1 8 3 4 9 6 275
1110
12
13
N N
F
F
F
O
O
O N1
345
6
8
79
10
11
12
13
OH
189 5 6 3
7 11
10,4
1213
N N
F
F
F
O
O
O N1
345
6
8
79
10
11
12
13
2
O8 1 6 4
3
9 75 10
1213
8
N N
F
F
F
O
O
O N1
45
6
8
79
10
11
12
13OH
OH
4 9 71110
6
5
12
13
8 9 4710
11N N
F
F
F
O
O
O N
45
6
8
79
10
11
12
13
O
23
5 6 23
M_678351_1, LC/MS run 396 MS(H)/MS(D)
428/429
444/446
444/445
460/462
1
2
3
4
5
478/482
12
13
2
1 1
1
Metabolite Analysis: Solved StructuresMetabolite Analysis: Solved Structures
NA
3
61
24
5
NA
OH3
61
45
NAO
3
61
24
5
NA
OH
OH
61
45
NA
3
61
2
45
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Metabolite Analysis:Metabolite Analysis:
500µL injected
t %B0 202 2040 9045 9046 2050 20
1.0mL/min6x150mm YMC-AQ 3u 12nA: 0.1% d-AcOH/D20B: 0.1% d-AcOH/MeCN
DAD (254)
0 5 10 15 20 25 30 35 40 Time [min]
0
1000
2000
3000
4000
5000
Intens.[mAU]
1 2
3
5
4
6
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Metabolite Analysis: Solved StructuresMetabolite Analysis: Solved Structures
1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
6
6
6
6
6
1
1
1
1
2, 5 3, 4
2, 3, 4, 5
3, 4
45
5
52
23
7, 8
9
7, 8
23
1
S
1
23
4
5A
O
S
1
23
4
5A
O
S
1
5A
HO OH23
4
S
1
23
5A
HO
S
1
23
4
5A
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When not to use LCWhen not to use LC--NMRNMR
min0 10 20 30 40 50 60 70 80
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=254 nm (DC110403\1EAR4633.D)
11.
740
12.
429
15.
382
18.
085
21.
642
23.
354
29.
261 3
3.19
0 3
4.34
0
42.
550
43.
497
44.
583
44.
950
45.
288
45.
589
46.
437
46.
903
47.
221
47.
831
48.
372
48.
828
49.
137
49.
509
50.
444
51.
044
52.
481
52.
958
54.
584
57.
740
58.
518
62.
646
63.
083
64.
261
65.
099
65.
991
71.
301
72.
122 7
3.74
3 7
4.22
7
76.
648
78.
252
78.
883
79.
723
80.
553
82.
475
86.
873
87.
267
89.
427
17
24 45
2728
23
582
525
582
580
598
582
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Regiochemistry Regiochemistry
• Sample submitted for determination of the regiochemistry of the primary amine moiety
N ONH2
N ONH2
Structure A Structure B
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5.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.9 ppm
1.93
1.06
1.00
1.05
1.00
A BC D
E
11H AssignmentH AssignmentVery little help using Chemical Shift ArgumentsVery little help using Chemical Shift Arguments
N ONH2
A
B
C
D
E
N ONH2
A
B
C
D
E
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11HH--1515N HMBC DataN HMBC DataBackground Background ---- SimplifiedSimplified
• Pulse sequence allows one to detect 1H’s long-range coupled (~8 Hz) to 15N– Depending on the experiment used one can choose to
omit or retain the 1JNH
NNH
OnJNH (n = 2,3) ? Will result in a correlation centered at
the 1H and 15N chemical shift
1JNH ? Will result in a correlation centered at the 15N chemical shift and a split signal centered on 1H
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ppm
6.06.26.46.66.87.07.27.47.67.8 ppm
80
90
100
110
120
130
140
150
160
170
180
190
1H-15N HMBC OF CP-325,366 LOT52203-059-01~ 22, MG IN 0.8 ML DMSO, BY AJJTEMP=25C, Liquids 600 MHz NMR Instrument, GNMR06
A BC D
E
11HH--1515N HMBC Data Allows Assignment N HMBC Data Allows Assignment
5.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.9 ppm
1.93
1.06
1.00
1.05
1.00
A BC D
E
N O
NH2
nJNH (n = 2,3)
1JNH
A
B
C
D
E
Primary Amine
Tertiary Amide
70 Hz
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Other Options AvailableOther Options Available
• LC-NMR/MS– Allows on-line MS and NMR evaluation of samples
• Peak Trapping (Column Trapping)– Potentially allows multiple LC peaks to be “trapped” and
concentrated prior to NMR data acquisition.• Microcoil Probes
– Has potential to allow microscale separation mechanisms (e.g. CapLC).
• CryoProbe Technology– Significantly lowers “noise floor” through cryo-cooling RF
electronics in the probe.
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LCLC--NMR/NMR/MSMS
LC Pump
Injector
Column/ColumnHeater
MassSpectrometerUV
5%95%
X
600MHzNMR
Spectrometer
LC Pump
Waste OrFractionCollector
LC & MS ControlSystem
NMRControl System
LoopCassette
OR
MS Component
Splitter
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Actual SystemActual System
Hardware Setup for LC-NMR-MS ( without magnet )
HPLC
LC-NMR-MS Interface, BNMI
UV detectorBPSU-36/BSFU-O
Esquire Ion Trap Mass-Spectrometer
NMR Spectrometer electronics
MS-Rough pump
Graphic Borrowed from Bruker-Biospin
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Actual SystemActual System
36 Loop Cassette
Column Heater/Injector
3 Port Switching ValvesMS Rough
PumpNMR
Electronics
BNMI Esquire 3000+ Ion Trap MSAgilent 1100
DAD Detector
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Other OptionsOther Options
LCLC--SPESPE--MSMS--NMRNMR
Peak TrappingPeak Trapping
Chromatographic System
Spark Prospekt 2
NMR Spectrometer
SparkProspekt 2
Drying
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Closer Look at the SPECloser Look at the SPE
Spark HollandProspekt SPE
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Robot gripper for SPE cartridges
2 flow lineswhere trapcartridges areinserted
SPARK HOLLAND SPE-UNIT
Trap cartridge size10mm * 2mm (ID)10mm * 1mm (ID)10mm * 3mm (ID)
~ 2 $ per cartridge
Various commercialpackings available
Bruker provides a set of 4 different solid phase types to start
Graphic Borrowed from Bruker-Biospin
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Major New DevelopmentsMajor New Developments
• Miniaturization – Microcoil Probes• Integration of new (to commercial NMR) MS• Cryogenic NMR Flow Probes
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MicroCoilMicroCoil ProbeProbe
• Horizontal copper RF solenoid Coil• Vertical (Z) pulse field gradient (PFG) coil• Flow cell is surrounded by CF-43 fluorocarbon for
susceptibility matched to copper coil• 1.5 µL active volume with a 5 µL total volume• 7 µL total volume from inlet to outlet (3 µL transfer
from injection assembly)• Lock power > 45 db to prevent saturation• π/2 pulse width of 8.4 µs at 18 db power level• Low power needed for 90% H2O/ 10% D2O (75 db)
saturation
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Advantages vs. DisadvantagesAdvantages vs. Disadvantages
• Advantages• Extremely mass sensitive• Capillary-scale fluidics allow transport of µL volume samples over
distances of 5-10 meters with virtually no degradation in analyte peak volume.
• Diffusion and mixing effects at the capillary scale are very limited so that peaks can be parked overnight with negligible loss of S/N.
• Residual protonated solvents are significantly reduced – need for multiple solvent suppression avoided in most cases.
• Acquisition of data in fully protonated solvents is reasonable.
• Disadvantages• Manipulation of 5µL aliquots can tedious.• Availability of HT platform poor• Samples of poor solubility
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Capillary Probe Data Capillary Probe Data
2.02.53.03.54.04.55.05.56.06.57.0 ppm
Sample in 5µL cell (conc. mg/mL) S/N
100µg (20 mg/mL)
50µg (10 mg/mL)
25µg (5 mg/mL)
12.5µg (2.5 mg/mL)
6.3µg (1.3 mg/mL)
3.1µg (0.63 mg/mL)
1.5µg (0.31 mg/mL)
780ng (0.16 mg/mL)
390ng (0.08 mg/mL)
845
440
229
116
62
36
25
18
16
HOD Saturation
One Hour acquisition time per sample
O
N
NH
NN
F
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Bruker’s Bruker’s New New MicroTOF MicroTOF ---- MetabolitesMetabolites
• The microTOF-LC can provide the exact mass of the analyzed sample and therefore access to the sum formula.
• microTOF-LC allows HyStar™ to trigger collection of chromatographic peaks into loops (LC-NMR), or SPE cartridges (LC-SPE™NMR) based on mass chromatograms
• However, no additional structural information is provided (e.g. fragmentation). This information is sometimes more relevant than the molecular formula.
Picture taken from www.bruker-biospin.con
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CryoProbesCryoProbes
Installation of a 600MHz triple
resonance 1H{13C,15N}
CryoProbeTM system.
Picture taken from presentation made by Dr. Kim Colson at Bruker Biospin
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Overall ConclusionsOverall Conclusions
• LC-NMR is an extremely useful tool in very specific instances.
• Additional “hyphenation”, in some cases, provides an enormous amount of pertinent structural information.
• Decreasing the noise floor (cryoprobes) is allowing NMR to routinely analyze samples that were previously impossible by NMR
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AcknowledgementsAcknowledgements
• David Chow• Kim Colson• Linda Lohr and Andy Jensen