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: brian_marquez@groton.pfizer.comPfizer Inc.445 Eastern Point Road, MS 8118A-2011Groton, CT 06340

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)

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)

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

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”.

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.

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

General Cartoon of Loop CollectionGeneral Cartoon of Loop Collection

DADDetector

XLC Pump

NMRSpectrometer

….

….

36 LoopCassette

Loop Collection

Direct Stop Flow

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

LCLC--NMR Probe SchematicNMR Probe Schematic

IN OUT

240µL

NMR detection coil built directly onto flow cell (4mm OD)

120µL

From LC To Waste

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

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:

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

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

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.

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

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

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

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

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

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

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

Regiochemistry Regiochemistry

• Sample submitted for determination of the regiochemistry of the primary amine moiety

N ONH2

N ONH2

Structure A Structure B

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

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

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

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.

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

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

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

Other OptionsOther Options

LCLC--SPESPE--MSMS--NMRNMR

Peak TrappingPeak Trapping

Chromatographic System

Spark Prospekt 2

NMR Spectrometer

SparkProspekt 2

Drying

Closer Look at the SPECloser Look at the SPE

Spark HollandProspekt SPE

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

Major New DevelopmentsMajor New Developments

• Miniaturization – Microcoil Probes• Integration of new (to commercial NMR) MS• Cryogenic NMR Flow Probes

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

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

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

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

CryoProbesCryoProbes

Installation of a 600MHz triple

resonance 1H{13C,15N}

CryoProbeTM system.

Picture taken from presentation made by Dr. Kim Colson at Bruker Biospin

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

AcknowledgementsAcknowledgements

• David Chow• Kim Colson• Linda Lohr and Andy Jensen