11 Sample Preparation Strategies for Routine Trace Mixture Analysis by NMR David Detlefsen, Kenneth Ray & Jeffrey Whitney

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Sample Preparation Strategies for Routine Trace Mixture Analysis by NMR

David Detlefsen, Kenneth Ray & Jeffrey WhitneyNovatia, LLC, 11 Deer Park Drive, Suite 202, Monmouth Junction, NJ 08852

www.enovatia.com

(732) 274 9933

ABSTRACTMixture analysis continues to challenge the application of NMR in chemical and biological research. NMR spectroscopists routinely process isolates resulting from metabolism, impurity and degradation studies. Traditional preparation-scale chromatography lies outside of the realm of most NMR spectroscopy laboratories. The promise of LC-NMR, with its advantages of on-line separation and subsequent delivery to a flow probe, remains largely unrealized. It now appears that the best approach to mixture analysis is to couple a high sensitivity probe (minimizing sample requirements), with off-line sample preparation (leveraging chromatographic methods that are already available).

Novatia routinely services customers who require NMR data on trace (100 ug or less) samples. Our customers lie in two groups: those who prepare their own samples, and those who request isolation in addition to NMR analysis. To meet the needs of this second group, we routine employ two methods using UV/MS to monitor the LC separation: 1) fraction collection into a well plate, sample dry down and reconstitution or 2) a custom on-line SPE system (SepNMR) that captures and presents only chromatographic peaks of interest. The resulting isolates from either isolation method are analyzed using a CapNMR ICG 10 ul enhanced probe. Examples and advantages of each approach will be presented.



Help…my NMR Sample is a Mixture!Help…my NMR Sample is a Mixture!

X

Pure Component

Mixture



Where Does that Leave Us?Where Does that Leave Us?

• Prepare your own samples!• Strict sample requirements cause collaborators to think twice• MS rapid rise in part due to coupling with LC• Avoid customer “dilution” and “pollution” factors

• Elements of Efficient Trace Sample Preparation• Use a high sensitivity probe

• Minimize sample prep and mass requirements• Leverage chromatographic expertise

• Use methods already developed by others• Uncoupled to NMR spectrometer

• Simplify operation



What about LC-NMR?What about LC-NMR?

• The Good• Well developed vendor solutions • Growing body of expertise

• The Bad• Never on flow because of low sensitivity• Peak volume mismatch to NMR detection cell further limits sensitivity

• The Growing Consensus

• … for the occasional identification of impurities, a decoupled, preparative approach may sometimes offer the best combination of efficiency, sensitivity and flexibility (Sharman and Jones, Magn. Reson. Chem.

2003, 448-454).



Application and Equipment MatrixApplication and Equipment Matrix

• Single Peak, Single Injection (SPSI Sample)• Sample is not in limited supply

• impurities, degradants, in vitro metabolite• Use Peak Trapping

• Multiple Peak, Single Injection (MPSI Sample)• Sample is not in limited supply

• impurities, degradants, in vitro metabolite• Use Peak Trapping or Fraction Collection

• Single / Mutliple Peak, Single / Multiple Injection• Sample is in limited supply

• natural product isolate, in vivo metabolite• Use Fraction Collection

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Application Equipment



SepNMR Plumbing DiagramSepNMR Plumbing Diagram

• Bypass• Pumps A & B gradient separation• Effluent to waste

• Capture• Pumps A & B gradient separation• Sample to sample loop


are needed to see this picture. SepNMR Plumbing Diagram SepNMR Plumbing Diagram (continued)(continued)

• Trap• Pump C mix & dilute peak• Sample to Trap

• Elute• Pump D elutes peak• Sample to tube

Peak

Drying

Not

Shown


are needed to see this picture. SPSI Isolation and Reinjection of SPSI Isolation and Reinjection of CaffeineCaffeine

Sample:• Brew pot of coffee • Inject 20 onto column

Caffeine

Coffee



Model Compound MixtureModel Compound Mixture

proparacaineMW 294RT 5.0

dobutamineMW 301RT 6.6

propanololMW 259RT 8.5

verapamilMW 454RT 9.9

eticloprideMW 340RT 10.4

proadifenMW 353RT 11.2


are needed to see this picture. MPSI LC/MS Fractionation of Model MPSI LC/MS Fractionation of Model CompoundsCompounds

C:\Xcalibur\...\10x-test-mix-trap-2 8/10/2006 4:46:35 PM new test mixtrap of 10x test mix; standard SepNMR method

RT: 0.00 - 18.98 SM: 3B

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (min)

0

50000

100000

150000

200000

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uAU

4.77 8.13 10.134.45 9.606.32

10.976.47

2.37

5.30

15.6215.436.05 7.80 12.202.55

9.43 14.487.68 12.52 14.051.87 18.3216.781.00

NL:6.79E5nm=239.5-240.5 PDA 10x-test-mix-trap-2

13 17

20

21

22

•Method•Column: Waters SymmetryShield RP8, 4.6X150 mm•Mobile Phases: A: 0.1% TFA in water, B:0.1% TFA in CAN•Method (1 mL/min)

•0-13min at 4-63%B•13-14 min 88%B•14-20min equilibrate

•Collect fraction every 30 seconds (500 ul)


are needed to see this picture. MPSI OMNMR of Fractionated MPSI OMNMR of Fractionated Model CompoundsModel Compounds

13 (12ug)

17 (25ug)

20 (25ug)

21 (4ug)

22 (12ug)

1D 1H 500 MHz64 scans / 4 min



21 (4ug)

2D 1H TOCSY 500 MHz2 scans / 256 inc2 hours

13 (12ug)

17 (25ug)

17 (25ug)

20 (25ug) 22 (12ug)



2D 1H-13C hsqca 500 MHz 32 scans / 124 inc in 4 hours

17 (25ug)



Buspirone MetabolitesBuspirone Metabolites

• Metabolite Production• 1.4 mg of Buspirone in 3ml with HLM• Dry down to 400 ul

• Method• Column: Waters SymmetryShield RP8, 4.6X150 mm• Mobile Phases: A: 0.1% TFA in water, B:0.1% TFA in CAN• Method (1 mL/min)

• 0-13min at 4-63%B• 13-14 min 88%B• 14-20min equilibrate

• Collect fraction every 30 seconds (500 ul)


are needed to see this picture. MPSI LC/MS Fractionation of MPSI LC/MS Fractionation of Buspirone MetabolitesBuspirone Metabolites

C:\Xcalibur\...\081006\busp-mets-FC2 8/10/2006 2:55:51 PM buspirone incubationStandard SepNMR method, 15 s fractions

RT: 0.00 - 13.98 SM: 3B

0 1 2 3 4 5 6 7 8 9 10 11 12 13Time (min)

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

uAU

2.051.92 2.45 7.723.25

3.48

6.857.62

5.52

5.97

6.52

4.78

3.759.30

11.779.934.15 11.278.97 11.9210.908.27 12.53 13.721.13

0.67

NL:6.78E5nm=239.5-240.5 PDA busp-mets-FC2

MW

385

MW

401

14

Paren

t


are needed to see this picture. MPSI OMNMR of Fractionated MPSI OMNMR of Fractionated Buspirone MetabolitesBuspirone Metabolites

9 (background)

2D 1H TOCSY 500 MHz2 scans / 256 inc2 hours

1D 1H 500 MHz64 scans / 4 min

14 (7 ug)

14 (7 ug)


are needed to see this picture. SPSI Isolation and Reinjection of SPSI Isolation and Reinjection of Buspirone MetaboliteBuspirone Metabolite

Buspirone HLM Incubation

Purified Buspirone Metabolite

Sample:• Incubate 700 ug buspirone with HLM• Dry down to 400 ul• Inject 80 ul on column• Estimate metabolite @ 10 %



Buspirone Metabolite MS-MSBuspirone Metabolite MS-MS



SPSI NMR Data on Buspirone MetaboliteSPSI NMR Data on Buspirone Metabolite

• TOCSY• CapNMR at 600 MHz• ~ 5 ug metabolite• 24 hours



SPSI NMR Data on Buspirone MetaboliteSPSI NMR Data on Buspirone Metabolite



Conclusions & SummaryConclusions & Summary

• Prepare your own samples

• Use a CapNMR Probe

• Leverage chromatography experience

• Use offline approach

• Fractionate when peaks are many or sample is precious• Pro: all column effluent in the well (you won’t lose anything)• Con: requires more equipment, may have buffer interference

• SPE when peaks are few or sample is copious• Pro: less equipment, get samples more quickly• Con: risk losing sample if trapping is not effective

SPE vs Fractionation

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Sample Volume (ul) Fractionate

SPE

Documents

11 Sample Preparation Strategies for Routine Trace Mixture Analysis by NMR David Detlefsen, Kenneth Ray & Jeffrey Whitney