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LC-MS Plus NMR

LC-MS Plus NMR. Building And Confirming Modern Molecular Libraries. Bob Albrecht Protasis Corporation. LC / Mass Spectrometry Alone. First choice for compound identification Very sensitive and fast, but… Not enough information to elucidate novel compounds Often ambiguous verification

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LC-MS Plus NMR

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  1. LC-MS Plus NMR Building And Confirming Modern Molecular Libraries Bob AlbrechtProtasis Corporation

  2. LC / Mass Spectrometry Alone • First choice for compound identification • Very sensitive and fast, but… • Not enough information to elucidate novel compounds • Often ambiguous verification • Ionization problems complicate identification

  3. CapNMR From Purified Fractions • Simple • No tricky SPE method or LC solvents • No stop-flow • No HPLC solvent suppression • No re-shimming • Inexpensive • No interferences = easier elucidation • Best NMR Sensitivity vs. online LC-NMR or LC-SPE-NMR • Largest fraction capacity vs. LC-SPE-NMR (384+)

  4. LC-MS+NMR HPLC Column MS HPLC CapNMR H11D, HMBC, 13C. 15N, … FractionCollector Auto-sampler Splitter UV ACD/LabsStructure Elucidator

  5. Micro-plateFraction Collection CapNMR is designed foranalytical-scale separations.

  6. MS isn’t enough for novel structure determination, but…CapNMR Has The Power • MS + 1D Proton NMR in deuterated solvent is often enough for verification, but when it isn’t… • CapNMR enables full structure elucidation of unknowns • 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY, ROESY, NOESY without changing probes! • One-Minute NMR keeps the system running non-stop

  7. Chromatographic Purification Strategies • Time / Volume • UV • MS • MS + UV

  8. Time / Volume Directed Purification • Fractions can contain 2+ compounds • Less mass of purified material • Many fractions • Collects everything

  9. UV-Directed Purification • More pure compound • Few impure fractions • Too many worthless fractions • No chromaphore, no fraction

  10. MS-Directed Purification • Pure fractions • MS picks valuable fractions • “Worthless” fractions may actually have value • Poor ionization can waste valuable fractions • Unknown mass range a problem

  11. MS + UV Purification • Poor ionization, fractions still collected • No chromaphore, fractions still colleted • Mass range can screen-out unlikely hits • NMR on fractions with interesting MS spectra

  12. C18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection Bioactivity profile Workflow Example: Natural Products DereplicationActive Peak(s) John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND 12

  13. John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND 500 MHz 8 mg in 6 mL CD3OD PRESAT1.5 min Recognisable features: 5 CH3 groups, of which1 methoxyl, 3 singlet CH3-C,1 doublet CH3-C; 1 aldehyde 13

  14. Purification Scale • UPLC™ (2.1x150mm 1.7µ) • < ~2 mg sample, plenty for MS (.1µg) + CapNMR (50µg) • Amazing chromatographic resolution • highest purity fractions • CapNMR on 2.5% of total fraction mass • Analytical – (4.6x150mm 3-5µ) • < ~7mg sample • Good chromatographic resolution • CapNMR on 0.7% of total fraction mass • Semi-Prep • 50mg to grams of sample • Longer runs • Less resolution, more impure fractions • Typical for compound libraries • CapNMR on 0.1% of total fraction mass

  15. Purification Scale-Down Saves Time and Money t ≈ $ ∆t + ∆$ = ∆productivity

  16. Analytical ScaleChromatographic Resolution Matters There are three compounds under this peak.The MS sees them, but you will not getthe purity you need for NMR elucidation.

  17. But… Trade-Off With Sensitivity You still need to scale-down NMR to Elucidate the 0.1% fraction. A second analytical run resolves a poorly resolvedsemi-prep fraction.

  18. One-Minute NMR Software • Web / E-Mail enabled • Import Fraction Lists • Run NMR Experiments • Maintain NMR Quality • Archive and Process Data • Customizable

  19. One-Minute NMR • Interfaces with: • People (Web, e-mail) • NMR • LC-MS • ACD/Labs • OneMoon • CustomSoftware Manages Samples fromPlates and Vials

  20. CapNMRSpecifically Designed for NMR of LC Peaks • Excellent NMR Results from: • 20 – 200µg samples • 0.1% fraction for semi-prep • 0.7% fraction for analytical scale • 2.5% fraction for UPLC CapNMR TXI Runs all of your NMR experiments for structure elucidationwithout changing the probe! 1H, 13C, 15N, 1D, 2D: APT, INEPT, DEPT, NOE, COSY, RELAY, DQFCOSY, TQCOSY, TOCSY, HOMJ, NOESY, ROESY, INADEQUATE, PFG, gCOSY, gDQFCOS, HETCOR, HMQC, HSQC, HMBC, gHMBC, gHMQC, gHSQC… Dual flow cell now available!

  21. Isolated Model Compound NMR • 5 Model Compound Mixture • CapNMR at 600 MHz • 64 Scans (4 minutes) • 5 ug of 5 components • Isolated proparacain • CapNMR at 600 MHz • 16 Scans (1 minute) • 5 ug of 1 component on column Data Courtesy Novatia, LLC

  22. MW 740 -> agl 286 On flow NMR NT=512 CAP-NMR of fraction C47 NT=16 Online LC-NMR vs. Fractionation + CapNMR Data Courtesy of Jean-Luc Wolfender MetabolomicsUniversity of Geneva, Switzerland

  23. exfl10mg # 676-690 RT: 22,87-23,34 AV: 15 SB: 77 21,55-22,66, 23,61-25,02 NL: 5,92E5 F: - c ESI Full ms [ 140,01-1000,02] 100 95 90 85 80 75 70 65 60 55 50 Relative Abundance 45 40 35 30 25 20 15 10 5 0 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z LC/UV/MSof the chromatogram used for micro-fractionation [M-H]- 739,3 m/z 739 477,5 445,6 740,8 421,6 216,2 741,8 506,6 446,7 TIC 921,7 313,3 609,8 775,8 281,3 593,7 863,8 922,9 738,8 253,3 567,7 387,6 404,9 776,8 468,5 364,9 742,9 957,7 670,4 515,2 650,8 832,2 902,0 956,1 997,5 Data Courtesy of Jean-Luc Wolfender MetabolomicsUniversity of Geneva, Switzerland UV (254nm)

  24. Cap-NMR gCOSY spectrum of fraction C47 NT=1 NI=512 Time=12 min Data Courtesy of Jean-Luc Wolfender MetabolomicsUniversity of Geneva, Switzerland

  25. Thanks • John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND • Jean-Luc Wolfender, University of Geneva • David Detlefsen, Novatia Protasis Corporationhttp://protasis.com508-481-4163

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