SMASH 2008 Recap:Introducing Citation, Your Hard-

Working Lab Assistant

Dean Olson, Ph.D.Director of Customer Support

Protasis Corporation

Citation:Push-Button Sample Loader

• Chromatography-grade Fluidics• Easy-to-use, Web-based Software• Built-in Instrument Controller• Integral Degassing• Field-serviceable Valves• Small-footprint benchtop packaging• Enjoy the reassurance of feedback

control, auto-calibration, and system self-maintenance in applications where the robot is not required

• Completely upgradeable to full robotic automation

Chromatography grade pumps

Field-serviceable valves

Integrated feedback control

sensors

Degas, blanket, sparge

On-board instrument controller

Protasis One-Minute™ NMR Lite Software

The size of a plant metabolome is unknown it is estimated at a few thousand constituents

Plant metabolomics

LC-NMR 10-6 mol

LC-UV 10-9 mol

GC-MS 10-12 mol

LC-MS 10-15 mol

LC-LIF 10-19 mol

CE-LIF 10-22 mol

Sumner et al. Phytochemistry 2003, 62,

817-836.

Rapid, Reproducible and Non-Destructive

Low Sensitivity

NMR Metabolomics

High Sensitivity

Instrumentation variability

LC-MS: problem in RT repeatability

MS Metabolomics

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

0

100

%

291.72

277.71

265.66

113.43

159.46133.48 175.45 223.57

305.74

309.70339.60

577.63353.73

397.77

381.76 531.80431.71 441.78623.61593.62

823.81807.83

739.60

291.71

277.70

265.66

113.43

223.60

305.73

319.76339.63

353.73397.75

381.75 441.76 577.69531.77 609.68 805.80647.65

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

0

100

%

291.72

277.71

265.66

113.43

159.46133.48 175.45 223.57

305.74

309.70339.60

577.63353.73

397.77

381.76 531.80431.71 441.78623.61593.62

823.81807.83

739.60

291.71

277.70

265.66

113.43

223.60

305.73

319.76339.63

353.73397.75

381.75 441.76 577.69531.77 609.68 805.80647.65

CONTROL

WOUNDED

PCA of total crude extract MS spectra

WA

CA

WB

CB

WA

CA

WB

CB

spiked

spiked

Total crude extract TOF MS spectra

RANKED LIST OF INDUCED IONS

MATLABm/z coord t1209 0.092353338 0.088507308 0.087165226 0.086949352 0.086062237 0.079287446 0.078662307 0.077829256 0.076834263 0.076811194 0.076683272 0.075681250 0.075639238 0.07556

Total crude extract TOF MS spectra

CONTROL

WOUNDED

C12

H17

O3

209.1256

209.1256

Data Courtesty of Data Courtesty of Jean-Luc Wolfender, Jean-Luc Wolfender, University of Geneva, University of Geneva,

SwitzerlandSwitzerland

UPLC-TOF-ES-MS of A. thaliana

11:06:54wt wound conc NEG new col 150 mmm aper 1 30 v

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- 209.12 10PPM

5.41e326.37209

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- 209

5.44e326.37209

1.15210 22.63

20910.76371

1.30;436 12.76338

31.46309

28.31501

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- BPI

3.84e419.14577

14.3673911.24

492

8.72285

1.26404

1.85436

6.263385.07

500

9.66447

13.68431

17.26593

16.47431

26.88545

26.51517

23.74579

19.69498

28.00689

33.8129130.83

26329.26527

37.1881935.94

809 38.75311

11:06:54wt wound conc NEG new col 150 mmm aper 1 30 v

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- 209.12 10PPM

5.41e326.37209

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- 209

5.44e326.37209

1.15210 22.63

20910.76371

1.30;436 12.76338

31.46309

28.31501

060316_wt_w_conc_neg_1_ap30 1: TOF MS ES- BPI

3.84e419.14577

14.3673911.24

492

8.72285

1.26404

1.85436

6.263385.07

500

9.66447

13.68431

17.26593

16.47431

26.88545

26.51517

23.74579

19.69498

28.00689

33.8129130.83

26329.26527

37.1881935.94

809 38.75311

Induced by wounding

m/z 209.12 (+/- 10 ppm)

m/z 209.12 (+/- 0.5 Da)

CAP-NMRCAP-NMR

Multiple collection Multiple collection triggered by MStriggered by MS

Data Courtesty of Jean-Luc Wolfender, Data Courtesty of Jean-Luc Wolfender, University of Geneva, SwitzerlandUniversity of Geneva, Switzerland

CAP-NMR of ion m/z 209

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5Chemical Shift (ppm)

CAP-NMRca 1 gNT=5000 12

10 9 7 3,2,8 11,5 4 12

Standard 50 g

COOH

O 1187

5

43

2

19 10

Data Courtesty of Jean-Luc Wolfender, Data Courtesty of Jean-Luc Wolfender, University of Geneva, SwitzerlandUniversity of Geneva, Switzerland

Further example details can be found in…

A LC-MS approach is presented for the isolation of minor key plant biomarkers, in view of their characterization by NMR at the microgram scale …using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS) … then transferred to semi-preparative LC conditions with MS detection … and subsequent characterization by capillary NMR (CapNMR).

Hot Off the Press: Natural Products Workflow

Herein we present a method of extract evaluation using HPLC profiling with biological evaluation16 followed by capillary probe NMR spectroscopy/ESMS/UV combined with NMR database (AntiMarin) evaluation, which reduces the crude extract requirement for dereplication to submilligram quantities.17

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