Sample Preparation Strategies for Routine Trace Mixture Analysis by NMR David Detlefsen, Kenneth Ray

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Sample Preparation Strategies for Routine Trace
Mixture Analysis by NMRDavid Detlefsen, Kenneth
Ray Jeffrey WhitneyNovatia, LLC, 11 Deer Park
Drive, Suite 202, Monmouth Junction, NJ
08852www.enovatia.com [phone]

• ABSTRACT
• Mixture 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.

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Helpmy NMR Sample is a Mixture!
Mixture
X
Pure Component
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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

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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).

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Application and Equipment Matrix
Application Equipment

• 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

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SepNMR Plumbing Diagram

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

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

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SepNMR Plumbing Diagram (continued)
Peak Drying Not Shown

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

• Elute
• Pump D elutes peak
• Sample to tube

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SPSI Isolation and Reinjection of Caffeine
Coffee
Caffeine
Sample Brew pot of coffee Inject 20 onto
column
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Model Compound Mixture
proparacaine MW 294 RT 5.0
propanolol MW 259 RT 8.5
eticlopride MW 340 RT 10.4
dobutamine MW 301 RT 6.6
verapamil MW 454 RT 9.9
proadifen MW 353 RT 11.2
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MPSI LC/MS Fractionation of Model Compounds
21
22
20
13
17

• Method
• Column Waters SymmetryShield RP8, 4.6X150 mm
• Mobile Phases A 0.1 TFA in water, B0.1 TFA
  in CAN
• Method (1 mL/min)
• 0-13min at 4-63B
• 13-14 min 88B
• 14-20min equilibrate
• Collect fraction every 30 seconds (500 ul)

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MPSI OMNMR of Fractionated Model Compounds
1D 1H 500 MHz 64 scans / 4 min
13 (12ug)
17 (25ug)
20 (25ug)
21 (4ug)
22 (12ug)
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MPSI OMNMR of Fractionated Model Compounds
2D 1H TOCSY 500 MHz 2 scans / 256 inc 2 hours
13 (12ug)
17 (25ug)
17 (25ug)
20 (25ug)
22 (12ug)
21 (4ug)
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MPSI OMNMR of Fractionated Model Compounds
17 (25ug)
2D 1H-13C hsqca 500 MHz 32 scans / 124 inc in 4
hours
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Buspirone 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, B0.1 TFA
  in CAN
• Method (1 mL/min)
• 0-13min at 4-63B
• 13-14 min 88B
• 14-20min equilibrate
• Collect fraction every 30 seconds (500 ul)

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MPSI LC/MS Fractionation of Buspirone Metabolites
Parent
14
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MPSI OMNMR of Fractionated Buspirone Metabolites
1D 1H 500 MHz 64 scans / 4 min
14 (7 ug)
14 (7 ug)
9 (background)
2D 1H TOCSY 500 MHz 2 scans / 256 inc 2 hours
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SPSI Isolation and Reinjection of Buspirone
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 _at_ 10
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Buspirone Metabolite MS-MS
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SPSI NMR Data on Buspirone Metabolite

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

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SPSI NMR Data on Buspirone Metabolite
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Conclusions 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 wont
  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
Fractionate