ABSTRACT

1. 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(732) 274 9933 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.

2. Help…my NMR Sample is a Mixture! Mixture X Pure Component

3. 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

4. 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).

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

6. SepNMR Plumbing Diagram • Bypass • Pumps A & B gradient separation • Effluent to waste • Capture • Pumps A & B gradient separation • Sample to sample loop

7. 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

8. SPSI Isolation and Reinjection of Caffeine Coffee Caffeine Sample: • Brew pot of coffee • Inject 20 onto column

9. 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

10. 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, 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)

11. MPSI OMNMR of Fractionated Model Compounds 1D 1H 500 MHz 64 scans / 4 min 13 (12ug) 17 (25ug) 20 (25ug) 21 (4ug) 22 (12ug)

12. MPSI OMNMR of Fractionated Model Compounds 13 (12ug) 17 (25ug) 2D 1H TOCSY 500 MHz 2 scans / 256 inc 2 hours 17 (25ug) 20 (25ug) 22 (12ug) 21 (4ug)

13. MPSI OMNMR of Fractionated Model Compounds 17 (25ug) 2D 1H-13C hsqca 500 MHz 32 scans / 124 inc in 4 hours

14. 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, 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)

15. MW 401 MW 385 MPSI LC/MS Fractionation of Buspirone Metabolites Parent 14

16. 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

17. 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 @ 10 %

18. Buspirone Metabolite MS-MS

19. SPSI NMR Data on Buspirone Metabolite • TOCSY • CapNMR at 600 MHz • ~ 5 ug metabolite • 24 hours

20. SPSI NMR Data on Buspirone Metabolite

21. 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 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 Fractionate