1. 11 www.enovatia.com www.enovatia.com Sample Preparation Strategies for Routine Trace Mixture Analysis by NMR David Detlefsen, Kenneth Ray & Jeffrey Whitney Novatia, LLC, 11 Deer Park Drive, Suite 202, Monmouth Junction, NJ 08852 www.enovatia.com (732) 274 9933 www.enovatia.com 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. 22 Help…my NMR Sample is a Mixture! X Pure Component Mixture

3. 33 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. 44 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. 55 Application 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 ApplicationEquipment

6. 66 SepNMR Plumbing Diagram Bypass Pumps A & B gradient separation Effluent to waste Capture Pumps A & B gradient separation Sample to sample loop

7. 77 SepNMR Plumbing Diagram (continued) Trap Pump C mix & dilute peak Sample to Trap Elute Pump D elutes peak Sample to tube Peak Drying Not Shown

8. 88 SPSI Isolation and Reinjection of Caffeine SPSI Isolation and Reinjection of Caffeine Sample: Brew pot of coffee Inject 20 onto column Caffeine Coffee

9. 99 Model Compound Mixture proparacaine MW 294 RT 5.0 dobutamine MW 301 RT 6.6 propanolol MW 259 RT 8.5 verapamil MW 454 RT 9.9 eticlopride MW 340 RT 10.4 proadifen MW 353 RT 11.2

10. 1010 MPSI LC/MS Fractionation of Model Compounds 1317 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)

11. 1111 MPSI OMNMR of Fractionated Model Compounds 13 (12ug) 17 (25ug) 20 (25ug) 21 (4ug) 22 (12ug) 1D 1 H 500 MHz 64 scans / 4 min

12. 1212 MPSI OMNMR of Fractionated Model Compounds 21 (4ug) 2D 1 H TOCSY 500 MHz 2 scans / 256 inc 2 hours 13 (12ug) 17 (25ug) 20 (25ug)22 (12ug)

13. 1313 MPSI OMNMR of Fractionated Model Compounds 2D 1 H- 13 C hsqca 500 MHz 32 scans / 124 inc in 4 hours 17 (25ug)

14. 1414 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. 1515 MPSI LC/MS Fractionation of Buspirone Metabolites MW 385 MW 401 14 Parent

16. 1616 MPSI OMNMR of Fractionated Buspirone Metabolites 9 (background) 2D 1 H TOCSY 500 MHz 2 scans / 256 inc 2 hours 1D 1 H 500 MHz 64 scans / 4 min 14 (7 ug)

17. 1717 SPSI Isolation and Reinjection of Buspirone Metabolite 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. 1818 Buspirone Metabolite MS-MS

19. 1919 SPSI NMR Data on Buspirone Metabolite TOCSY CapNMR at 600 MHz ~ 5 ug metabolite 24 hours

20. 2020 SPSI NMR Data on Buspirone Metabolite

21. 2121 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 Fractionate SPE