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Our Optimized LC-MS+NMR Strategy With a CapNMR Probe

Our Optimized LC-MS+NMR Strategy With a CapNMR Probe. 1. The Barnett Institute of Chemical and Biological Analysis. Routine Manual Injection of Trace Samples. Roger Kautz, Principal Research Scientist In Collaboration with Protasis / MRM and Varian Instruments.

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Our Optimized LC-MS+NMR Strategy With a CapNMR Probe

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  1. Our Optimized LC-MS+NMR Strategy With a CapNMR Probe 1 The Barnett Institute of Chemical and Biological Analysis Routine Manual Injection of Trace Samples Roger Kautz, Principal Research Scientist In Collaboration with Protasis / MRM and Varian Instruments

  2. Online LC-NMR was a flail 2 4 sec = 60 µL 5% degradant of interest The Barnett Institute of Chemical and Biological Analysis LC-Column capacity 100 µg/peak (4 mm column) On-flow NMR sensitivity 30 µg (60 µL flow cell, 15 sec intervals) LC Peak Volume 150 µL (10 sec peak at 1 mL/min) And LC-MS-NMR had the same problems. NMR LOD 1 µg overnight (60 µL LC probe) MS LOD 1 ng 1 second Collect Fractions. Use automated NMR to analyze the fractions. Concentrate fractions and use the most sensitive NMR available µNMR LOD 0.2 µg, 1 hour (1 µL microcoil) Allocate NMR time intelligently. Offline LC+NMR Makes Sense:

  3. Microcoil NMR 3 The Barnett Institute of Chemical and Biological Analysis 1 Sensitivity radius Solenoid = 3x saddle coil Cold Probe = 4x RT probe Flow Probe is 1 mm smaller than tube probe

  4. Sample Efficiency: “ From Vial to V-observe” 4 The Barnett Institute of Chemical and Biological Analysis Percent of sample in vial that ultimately produces signal in Vobs A B C NMR Coil 30 nL 1 L observe Capillary 30,000 nL 8 L dead NMR Sensitivity is Highly Concentration-Dependent A 10-fold increase in concentration reduces the time by 100-fold, to obtain similar quality data. 1 minute to acquire spectrum 2 hours to acquire spectrum Kautz, Lacey, Wolters, Webb, Sweedler et al “capillary isotachophoresis”, JACS, 2001

  5. Ways to fill a flow cell 5 Flow Injection (solvent-filled flowcell) Direct Injection (air-filled flowcell) Direct Injection with Chaser Parabolic Flow Taylor Dispersion

  6. Direct Injection with Leader and Chaser 6 The Barnett Institute of Chemical and Biological Analysis 30% sample efficiency MRM Probe (air filled) 200 µm tubing filter 10 µL syringe,Syringe Pump 2 µL sample 2 µL solvent“leader” Chase solvent

  7. Zero-Dispersion Segmented Flow 7 The Barnett Institute of Chemical and Biological Analysis FIA (miscible carrier) Parabolic Flow Microcoil NMR flow cell (10 uL) 1 uL dye, flow injection Taylor Dispersion SFA (immiscible carrier) Sample Wets Capillary Wall Behnia & Webb “Perfluorocarbon Plugs” 1998, ; Lacey et. al “Single Bead”, 2001 Zero-Dispersion SFA Microcoil NMR flow cell (10 uL) 1 uL dye, Zero-Dispersion SFA Carrier Wets Capillary Wall Curcio & Roeraade “Continuous Flow PCR” (2003); Nord & Karlberg, 1984.

  8. Small Samples Produce Equivalent Spectra 8 The Barnett Institute of Chemical and Biological Analysis 0.3 µg/µL beta-methyl-glucoside in D2O

  9. 9

  10. Preparing Trace Samples 10 The Barnett Institute of Chemical and Biological Analysis with keeper no keeper with keeper no keeper Recovery from FAS-coated glass vial insert. Note DMSO contact angle even as drawn down. (360 µm o.d. capillary)

  11. Microcoil NMR Sensitivity Enhanced by SFA 11 Peaks labeled “D” are seen in similar acquisition of clean solvent (DMSO) Indirect Carbon NMR Data from 50 ug taxol (HSQC, HMBC)

  12. 12 The Barnett Institute of Chemical and Biological Analysis 1.5 µg Erythromycin gives COSY and TOCSY in 10 hr

  13. High Throughput Segmented Flow Microcoil NMR 13 The Barnett Institute of Chemical and Biological Analysis Kautz et al., J. Combi Chem 7: 14-20. (2005)

  14. Detect and Position Sample Plugs 14 The Barnett Institute of Chemical and Biological Analysis flow Washplug Sampleplug Washplug TMSP peak, 2 sec intervals Previous Sample Sampleplug Start flow, (Well 74 in probe) Wash PlugDetected Flow Stopped, Well 75 centered in probe

  15. LC-MS-microNMRNatural Product Identification 15 NMR Segmented Flow Loading Culture Resuspend in 2 uL NMR solvent Bioactive Fraction Microcoil LC Separation Sample Recovery UV-DAD Nanosplitter ESI-MS LC-MS Evaporate LC Solvent Bioactivity The Barnett Institute of Chemical and Biological Analysis Fraction Collection 200 µL Fractions

  16. The NanoSplitter LC-MS Interface 16 The Barnett Institute of Chemical and Biological Analysis Sampling Flat Region of Parabolic FlowPreserves Chromatographic Resolution. Sampling 0.1% of LC Flow Makes MS A Non-destructive Method 100-fold Better S/N. Nano-electropray Avoids Ion Suppression

  17. LC-MS-NMR 17 RT: 0.06 - 16.05 SM: 7B 4.42 100 95 90 85 80 75 12.89 10.26 70 65 60 55 50 45 Relative Abundance 40 35 9.49 30 25 20 4.93 8.59 15 7.18 10 5.25 1.18 7.50 4.09 6.16 5 2.33 2.52 10.65 12.24 13.30 0.84 14.13 14.55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (min) DAD1 C, Sig =210,8 Ref=360,100 (YIQING \ YIQING_092706_5.D) mAU 2000 10.437 4.611 1750 1500 13.049 1250 1.618 5.091 1000 750 9.657 7.332 500 11.878 250 0 2 4 6 8 10 12 14 min Total Ion Chromatogram NMR UV X .2 µg 1.5x y-scale 1 µg 1x y-scale 1 hr /fraction 20 µg (1/15 y-scale) The Barnett Institute of Chemical and Biological Analysis cycloheximide

  18. Conclusions 18 Improving sample efficiency can give several-fold gains in sensitivity. Dry with keeper in low-retention vial; recover in 1-3 uL Minimize dead volume CapNMR LOD’s (with segmented-flow sample loading) : 50 ng overnight for dereplication (200 ng, 1 hr) 1-2 µg for COSY, TOCSY overnight 20 µg for HSQC 50 µg for HMBC Offline LC-NMR (or LS-MS+NMR) can be recommended Can acquire comprehensive LC-NMR data overnight; Can review LC or MS data to select samples of interest. Could be performed using any automated loading method. Lets chromatographer use his own, validated equipment. Can be done retrospectively. Directions Data Dependent Acquisition Sample Recovery Better Software; LEAP autosampler The Barnett Institute of Chemical and Biological Analysis

  19. With Gratitude To: The Barnett Institute of Chemical and Biological Analysis NIH R01 GM075856-01 Paul Vouros Yiqing Lin Varian Daina Avizonas Steve Smallcombe Paul Keifer Northeastern Yiqing LinCarmelina Freni Paul Vouros Barry Karger Frantisec Foret Tomas Rejtar James Waters Jimmy Orjala Roger Kautz Illinois Jonathan Sweedler Andrew Webb Michael Lacey Andrew Wolters Univ. Illinois Chicago Jimmy Orjala Protasis / MRM David Strand Tim Peck Dean Olson Jim Norcross Flow Injection Charles Patton Arqule Wolfgang GoetzingerJun Zhao MGH J. Manuel Perez

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