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

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. Online LC-NMR was a flail 4 sec = 60 µL 5% degradant of interest Offline LC+NMR Makes Sense: 2

Microcoil NMR Probe TypePrepared Sample Volume Relative Mass Sensitivity 1 Observed S/N (Subramanian) Conventional 5 mm550 µL20 µg 3 mm Cryoprobe 3 mm sample tube 160 µL4 µg mm Cryoprobe 1.7 mm sample tube 40 µL2 µg5021 Microcoil Direct Injection 8 µL2 µg4098 Microcoil SFA-NMR 2 µL0.5 µg 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 3

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

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

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

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

The Barnett Institute of Chemical and Biological Analysis 0.3 µg/µL beta-methyl-glucoside in D 2 O Small Samples Produce Equivalent Spectra 8

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

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

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

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

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

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

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

Total Ion Chromatogram UV NMR LC-MS-NMR cycloheximide 20 µg 1 µg.2 µg (1/15 y-scale) 1.5x y-scale 1x y-scale X The Barnett Institute of Chemical and Biological Analysis 1 hr /fraction 17

Conclusions 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 18

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