Roger Kautz, Principal Research ScientistIn 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

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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:

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Microcoil NMR

Probe Type PreparedSample Volume

Relative Mass Sensitivity1

Observed S/N(Subramanian)

Conventional 5 mm 550 µL 20 µg

3 mm Cryoprobe 3 mm sample tube

160 µL 4 µg 3204

3 mm Cryoprobe 1.7 mm sample tube

40 µL 2 µg 5021

Microcoil Direct Injection

8 µL 2 µg 4098

Microcoil SFA-NMR

2 µL 0.5 µg

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1 Sensitivity radius Solenoid = 3x saddle coil Cold Probe = 4x RT probe Flow Probe is 1 mm smaller than tube probe

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NMR Coil 30 nL

Capillary 30,000 nL

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2 hoursto acquire spectrum

1 minuteto acquire spectrum

NMR Sensitivity is Highly Concentration-DependentA 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

A B C

Kautz, Lacey, Wolters, Webb, Sweedler et al “capillary isotachophoresis”, JACS, 2001

Percent of sample in vial that ultimately produces signal in Vobs 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

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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

Dispers ion of 2 uL sam ple injected into MRM ICG Probe

0.00

20.00

40.00

60.00

80.00

100.00

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Injection Volum e (uL)

Pea

k In

tens

ity

lead_integral

lead_height

sam ple_integral

sam ple_height

chase_integral

chase_height

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filter

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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

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Behnia & Webb “Perfluorocarbon Plugs” 1998, ; Lacey et. al “Single Bead”, 2001

Curcio & Roeraade “Continuous Flow PCR” (2003); Nord & Karlberg, 1984.

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0.3 µg/µL beta-methyl-glucoside in D2OSmall Samples Produce Equivalent Spectra 8

NMR Quality of D2O Plugs in FC 43 Carrier FluidPassing Through MRM ICG Microcoil Probe

0

1

2

3

4

5

6

7

8

-3 -2 -1 0 1 2 3

3 uL lock

3 uL linewidth

1 uL lock

1 uL linewidth

2 uL lock

2 uL linewidth

2 uL lock

2 uL linewidth

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Preparing Trace Sampleswith keeper no keeper no keeperwith 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

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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)

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1.5 µg Erythromycin gives COSY and TOCSY in 10 hr

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High Throughput Segmented Flow Microcoil NMR

The Barnett Institute of Chemical and Biological AnalysisKautz et al., J. Combi Chem 7: 14-20. (2005)

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TMSP peak, 2 sec intervals

Start flow,(Well 74 in probe)

Wash PlugDetected Flow Stopped,

Well 75 centered in probe

Detect and Position Sample Plugs

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flow

WashplugSample

plug

Washplug

Sampleplug

Previous Sample

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UV-DAD

LC-MS

Nanosplitter ESI-MS

Culture

Bioactivity

Segmented Flow Loading

Sample Recovery

LC-MS-microNMRNatural Product Identification

Bioactive Fraction

LC Separation

Fraction Collection200 µL Fractions

NMR

Microcoil

Evaporate LC Solvent

Resuspend in 2 uL NMR solvent

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The NanoSplitter LC-MS Interface

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

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Total Ion Chromatogram

min2 4 6 8 10 12 14

mAU

0

250

500

750

1000

1250

1500

1750

2000

DAD1 C, Sig =210,8 Ref=360,100 (YIQING \ YIQING_092706_5.D)

1.61

8

4.61

1

10.4

37

13.0

49

5.09

1

7.33

2

9.65

7

11.8

78

RT: 0.06 -16.05 SM: 7B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Time (min)

5101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

4.42

12.8910.26

9.49

4.93 8.597.185.251.18 7.504.09 6.162.33 2.52 10.65 12.24 13.300.84 14.13 14.55

UV

NMR

LC-MS-NMR

cycloheximide

20 µg

1 µg

.2 µg

(1/15 y-scale)

1.5x y-scale

1x y-scale

X

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1 hr /fraction

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ConclusionsImproving 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. DirectionsData Dependent AcquisitionSample Recovery Better Software; LEAP autosampler The Barnett Institute

of Chemical and Biological Analysis

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Northeastern Yiqing LinCarmelina Freni

Paul Vouros

Barry KargerFrantisec ForetTomas Rejtar

James Waters

Illinois

Jonathan SweedlerAndrew WebbMichael LaceyAndrew Wolters

Protasis / MRM

David Strand

Tim PeckDean OlsonJim Norcross

Varian

Daina AvizonasSteve Smallcombe

Paul Keifer

With Gratitude To:

Flow InjectionCharles Patton

MGHJ. Manuel Perez

ArquleWolfgang GoetzingerJun Zhao

Univ. Illinois ChicagoJimmy Orjala

NIH R01 GM075856-01

The Barnett Institute of Chemical and Biological Analysis

Yiqing Lin

Jimmy Orjala

Paul Vouros

Roger Kautz