Thermo Fisher Scientific - Trace Level Metabolite...

JRG 9/22/11 Thermo Users Mtg

Trace Level Metabolite Identification Using the Latest Generation of LIT-FTMS and Q-orbitrapInstruments

Jeffrey R. Gilbert*1, Jesse L. Balcer1, Yelena Adelfinskaya1, Kate Comstock2, Yingying Huang2,Suresh Annangudi1, Ayanna Jackson1, Mike J. Hastings1, Brian M. Wendelburg1, Mingming Ma1, and David G. McCaskill1

1. Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN 462682. Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95127


Mass SpectrometryLTQ-FT

LTQ

Q-Exactive

ABI 5600

LC-TOF

Chipcube-QTOF

Discovery& Regulatory

Protein ID& characterization

DevelopmentSystems biology

Bruker-TOF/TOFDiscovery

Natural ProductsActive ID

DiscoverySynthesisActive ID

RegulatoryTrace Level (14C)

Metabolite ID

Healthy OilsLipid Analysis

DiscoveryLead Optimization

Early StageMetabolite ID

FormulationsProduct Chemistry

Impurity ID

Mass Spectrometry at Dow AgroSciencesOur Mission: To solve complex identification problems encountered in the discovery, development, registration, and defense of Dow AgroSciences products using state of the art expertise and techniques in mass spectrometry.


Regulatory Metabolite Identification - Systems Studied

Metabolism:• Animal Metabolism - ruminants (goat/cow and poultry)• Plant Metabolism - groupings (root vegetables, leafy crops, fruits, cereals)

Environmental Fate:• Aerobic Soil/Sediment• Anaerobic Soil/Sediment• Hydrolysis• Photolysis

(14C) Radiolabeled Parent is Available!!Challenges

• Complex matrices – plant and soil much dirtier than urine/plasma/feces• Low dose levels/application levels – low soil dose rates ~ 0.020 ppm

must identify 5% of applied = 0.001 ppm• Slow metabolite formation – soil metabolites can take weeks/months to form

- Residue method development must incorporate required metabolites• Typical customer supplies < 200ul of an extract containing several metabolites

- We must inject 30-50 ul for on-line radiochemical detection

How can we maximize the information obtained from each injection?


MS Tools to Aid in Metabolite Identification

• Thermo LTQ-FT Ultra– Sensitivity– Pos/Neg operation– High Mass Accuracy (typically < 1ppm)– High Resolution (up to 1,000,000)– MSn

• IRMPD (Infrared Multi Photon Dissociation)– Fragmentation and Detection in the FT

• No 1/3 cut-off for fragment ions• No time-of-flight effect in product ion spectra

• Advion Nanomate– Collect fractionated sample (after RAM)– Chip-based infusion enables thorough interrogation of samples – Extended infusion allows use of slower scan functions (very high res)

• Controlled Isotopic Labeling (13C, 15N, 2H) – ‘easily’ recognize the metabolites

• Thermo Q-Exactive– Improved Sensitivity (~10-50x over LTQ-FT)– High Acquisition Speed– Pos/neg switching– High Mass Accuracy


Benefits of high resolution – M+2 Sulfur countingFlorasulam in Wheat Cell Extract

362.00 362.02 362.04 362.06m/z

0

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0

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40

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100362.03285

362.04269

362.03331

362.04305

362.03558

NL:8.23E4080326I11#508 RT: 7.92 AV: 1 SB: 1 7.94-7.98 , 8.06-8.11 T: FTMS + p ESI Full ms [120.00-1200.00]

NL:8.74E2C 12 H8 F 3 N5 O3 SH: C 12 H9 F 3 N5 O3 S 1p (gss, s /p:40) Chrg 1R: 65000 Res .Pwr . @FWHM


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Rel

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245.12832

652.40284

360.03714 674.38469

376.22293

724.38078439.29135 604.15813 1027.61789836.93613

279.17008

359 360 361 362 363m/z

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Rel

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360.03714

361.04052

362.03285

Full scan FTMS

Expanded view of isotopic cluster

[M+H]+

M+2 isotope

Measured

Theoretical(65,000 resol.)


12C12H8F3N5O334S

12C1013C2H8F3N5O3

32S

NN

NN

O

F

SN

O

OF

F

Florasulam(cereal herbicide)C12H8F3N5O3S[M+H]+ = 360.03727


100 150 200 250 300 350 400 450 500m/z

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nsity

0

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800000

1000000

1200000

444.05849

464.06476

195.07512 326.08844258.02920 400.10279368.08534

195.07517

444.05888

484.07161

258.02939

326.08874

139.05033380.09699180.05177 464.06538

194.06733

164.05680 298.07371124.02683 326.08868287.08154

NL: 1.38E6DE-638_4#60-83 RT: 0.97-1.39 AV: 24 F: FTMS + c ESI Full ms2 [email protected] [130.00-1000.00]

NL: 3.25E6DE-638_4#170 RT: 3.11 AV: 1 F: FTMS + c ESI Full ms2 [email protected] [100.00-1000.00]

NL: 4.30E6DE-638_4#173-191 RT: 3.17-3.51 AV: 18 F: FTMS + c ESI Full ms2 [email protected] [100.00-1000.00]

CID vs. IRMPD – Low Mass Ion Detection

low masscut-off

NNN

NN

SF

FF

OOO

F

F

O

O

Penoxsulam (rice herbicide)C16H14F5N5O5S[M+H]+ = 484.07086

CID products of m/z 484(35% energy)

IRMPDproducts of m/z 484(20% energy)

IRMPDproducts of m/z 484(40% energy)

194258

-2HF(444.05840)

[C7H8N5O4S]+(258.02915)

[C7H9N5O2]+(195.07508)

@

80 100 120 140 160 180m/z

0

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100000

150000

200000

250000

Inte

nsity

164.05762124.02733 139.05093

180.05295

84.04478

152.0469096.03253

low mass region


CID vs. IRMPD – Energy Dependence of Fragmentation

0

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120

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 90 95 100

484.0

444.0

326.0

258.0

195.0

194.0

180.0

164.0

139.0

124.0

84.04

0

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40

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120

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

484.444.400.380.368.326.195.

Normal operation

CID

IRMPD

Rel

ativ

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n In

tens

ityR

elat

ive

Ion

Inte

nsity

NNN

NN

SF

FF

OOO

F

F

O

O

Penoxsulam (rice herbicide)C16H14F5N5O5S[M+H]+ = 484.07086

% energy

% energy

More optimization required for

IRMPD


Metabolite Identification on the LTQ-FT

Sample UPLC

2.1x100 1.9um C-18

PolyLC (HILIC)

diode-array

FinniganLTQ-FT

Gilson 205RAM

Nano-mate

IRM

PD

LTQ-FT Ultrawith IRMPD

Nanomate

RAM

General Workflow:

1) LC/MS analyses (Sample + Stds.)post column split (ESI:RAM)+/- ESI-FTMS DD-MSn

IRMPD (1-2 energies)fractionation (collect 96-well plates)

2) Retention time and mass spectral match with standards?

3) Nanomate infusion of fractionated sample from step 1.

Detailed IRMPDHigh resolution MS

TopCount

High pressure LC


Spinetoram – lettuce leaf metabolism

Radiochromatogram

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0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

RETENTION (min)

RA

DIO

AC

TIVI

TY (d

pm)

A

C

D

E

F

G HI

J/KL

M

Separation #1 Silica column

Spinetoram Lettuce LeafMetabolism DCM rinse, 3 DAT

B

Radiochromatogram

0

1000

2000

3000

4000

5000

6000

7000

0 5 10 15 20 25 30 35 40 45

RETENTION (min)

RA

DIO

AC

TIV

ITY

(dpm

)

32.2

27.828.6

Silica Fraction D

Separation #2 reverse phase column

O

OO H

H

H

H

OO

OO

O

OON

Minor factor (XDE-175-L), 759 Da

O

OO H

H

H

H

OO

OO

O

OON

Spinetoram• New natural product-derived

insect control agent

• Broad spectrum of insect activity

• Novel mode of action (resistance management)

• Low persistence in the environment

• Consists of two primary factors

Spinetoram Major Factor (XDE-175-J) C42H69NO10[M+H]+ = 748.4994


200 300 400 500 600 700 800 900 1000m/z

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100R

elat

ive

Abun

danc

e748.50016

757.53956

142.12274

NL: 5.62E6080256b#665-681 RT: 14.20-14.41 AV: 3 F: FTMS + p ESI Full ms [120.00-1200.00]

NL: 1.75E4080256c_F8_B1#255-288 RT: 4.15-5.07 AV: 34 T: FTMS + p ESI Full ms2 [email protected] [100.00-1200.00]

Spinetoram labeling scheme aids in metabolite detection

Spinetoram Major factor C42H69NO10[M+H]+ = 748.4994

O

OO H

H

H

H

OO

OO

O

OON

DD

D DD

[C8H16NO]+

142.12263IRMPD(40% energy)

Full Scan FTMS p [ ]

748 750 752 754 756 758m/z

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748.50016

749.50478 753.53170

750.51004

754.53528

751.51446

755.53765752.51773

756.54080

EnhancedM+5

low masscut-off


Spinetoram – Turnip Leaf ExtractFTMS TIC and Radiochemical Traces

0

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400

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800

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1400

0.25 2.25 4.25 6.25 8.25 10.25 12.25 14.25 16.25 18.25 20.25 22.25

Time (min.)

Parent

RT: 0.07 - 23.16

2 4 6 8 10 12 14 16 18 20 22Time (min)

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19.08

20.31

21.1814.3021.4016.50 18.2212.20 21.7610.22 14.778.988.614.63 5.74

14.4714.54

12.2410.33 14.89 16.65 18.11 20.2014.10 23.108.784.65 7.767.171.67 2.170.57 5.533.84

NL: 1.45E8m/z= 50.00000-1200.00000 F: FTMS + p ESI Full ms [120.00-1200.00] MS 080256b

NL: 1.35E3A/D Card Ch. 1 A/D card 080256b

+FTMS

On-lineRAM

Topcountfractions Parent

metabolitesImproved radiochemicalS/N using TopCount

A BC


RT: 0.15 - 25.10 SM: 3B

2 4 6 8 10 12 14 16 18 20 22 24Time (min)

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milli

Volts

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50

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0

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100

19.20338.3415514.30

749.50519 21.30763.67609

16.50464.357798.98

329.2473810.22

739.515567.74

313.321174.63

232.1330714.41

748.50000

14.20753.53162

10.22753.51959

12.20606.40027

13.95606.39063

21.060.00000

10.34734.48444

12.20734.48413

14.470.00000

12.240.0000010.33

0.0000014.89

0.0000016.65

0.0000020.20

0.0000023.10

0.000008.78

0.000004.65

0.000007.17

0.000001.67

0.00000

NL: 1.31E7Base Peak F: FTMS + p ESI Full ms [120.00-1200.00] MS 080256b





NL: 1.35E3A/D Card Ch. 1 A/D card 080256b

Spinetoram – Turnip Leaf Extract FTMS TIC, selected ion, and RAM traces

Parent

RAM

m/z 734

m/z 753

m/z 606

m/z 748

TIC

D5-Parent

Metabolite B

Metabolite A


Spinetoram – Turnip Leaf Extractfull scan FTMS spectra from LC run

200 300 400 500 600 700 800 900 1000 1100 1200m/z

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100748.50015

770.48249198.18521 332.33117 439.31871 571.39772 639.28154 853.99453 1171.72861

732.03984

1103.01760606.40027546.39998

502.37378

634.45343441.32106

203.12769 678.47905271.15388

722.50548397.29490766.53147

359.20638

854.58528922.61262 1199.712981077.65232

734.48446

796.54232383.25784198.18511 299.11003 884.59476708.48939457.27128 620.43693259.11769 972.65231 1154.70024




m/z 606 (metabolite B)(RT = 12.2 min)

m/z 734 (metabolite A)(RT = 10.34 min)

m/z 748, Parent(RT = 14.4 min)

Spinetoram Major factor C42H69NO10, [M+H]+ = 748.4994

O

OO H

H

H

H

OO

OO

O

OON

DD

D DD

750 755m/z

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

Inte

nsity

749.50518748.50029

750.51044

753.53195

751.51474

754.53667

755.53993745.78710

606 608 610 612 614m/z

0

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300000

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Inte

nsity

606.40027

607.40522

611.43213608.41105

612.43535609.41492613.36714

614.37004

735 740 745m/z

0

100000

200000

300000

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Inte

nsity

734.48454

735.48933739.51569

736.49437

740.51979

741.52241

742.52470• 2 major metabolites detected in LC run• Isotopic label key to finding metabolites


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Rel

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609.27090

734.48479

454.29283 884.59508581.36612

625.39259

757.47314

972.64566385.14688299.11006191.09131

128.10705

735.49443

NL: 4.34E5080260c#479-489 RT: 10.31-10.41 AV: 2 F: FTMS + p ESI Full ms [120.00-1200.00]

NL: 1.75E5080260c#483 RT: 10.34 AV: 1 F: FTMS + p ESI Full ms2 [email protected] [80.00-800.00]

IRMPD of Spinetoram Metabolite A at 10.3 min

N-demethyl Spinetoram Metabolite C41H67NO10[M+H]+ = 734.48377

O

OO H

H

H

H

OO

OO

O

OON

DD

D DD

C7H14NO128.10699

Predicted Metabolite

734 736 738 740 742m/z

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Rel

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bund

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734.48479

735.48948

736.49437

739.51562

737.49814

740.51938

741.52452


Nanomate infusion of Spinetoram Turnip Leaf FractionFragmentation of m/z 762 (metabolite C) at varying IRMPD energies

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1200000

1400000

1600000370.20140

762.48058532.44023328.32127432.23850 780.51163300.28995

462.14702 722.37002652.45832212.20100

156.10198

562.37166

407.27709295.18761 764.49147

156.10199

276.17060

NL: 1.68E6080257c_H8_b1#875-889 RT: 25.51-25.64 AV: 15 T: FTMS + p ESI Full ms [205.00-1000.00]

NL: 2.52E5080257c_H8_b1#994-1021 RT: 27.99-28.62 AV: 28 T: FTMS + p ESI Full ms2 [email protected] [100.00-1000.00]

NL: 3.37E5080257c_H8_b1#1074-1084 RT: 29.84-30.07 AV: 11 T: FTMS + p ESI Full ms2 [email protected] [100.00-1000.00]

150 200 250 300m/z

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0

50000

100000

150000

200000

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Inte

nsity

156.10198

125.05977

295.18761

156.10199

125.05978

276.17060

IRPMD ofm/z 762at 5% energy

IRPMD ofm/z 762at 25% energy

Full ScanFTMS

Expansion oflow mass region

O

OO H

H

H

H

OO

OO

O

OON

O

N-Formyl Metabolite of XDE-175-JC42H67NO11, [M+H]+ = 762.47869

[C8H14NO2]+

156.10191

762 764 766 768 770 772m/z

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100

Rel

ativ

e A

bund

ance

762.48058

763.48513

764.49103767.51230

768.51532765.49476

769.51713

-CH3O31.0183931.04221

sugarloss

sugarloss

-CH5N31.04220

C10H19O4

203.12833

N-formyl Spinetoram J represented a novel metabolite. Use of Nanomate allowed extended data acquisition from initial sample injection


Nanomate infusion of spinetoram fractionIRPMD product of m/z 762 at increasing resolution

560 561 562 563m/z

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0

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40

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100562.37195

561.36412

560.35873

561.36452

560.35847

562.37164

562.37137

561.36516560.35887

562.37143

561.36519

560.35893

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nsity

0

20

40

60

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100156.10201

562.37195

407.27684764.49284

295.18765

156.10194

561.36452

406.27009763.48427

294.18106

562.37137156.10196

407.27642

764.4928295.18744

562.37143156.10196

407.27644764.49278

654.39804294.18121

562.32 562.34 562.36 562.38 562.40 562.42m/z

0

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0

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100562.37195

562.37164

562.36030

562.38521

562.37137

562.36549562.37640

562.35658

562.37143

562.36561

562.37639562.35682

full spectrum m/z 560 - 562 m/z 562.3 – 562.4

50,0001.4 sec/scan

100,0001.9 sec/scan

400,0004.26 sec/scan

750,0007.4 sec/scan

[C32H48D2O7N]+562.36853

sugar loss[C32H50NO7]+560.35818

[13C2C30H50O7N]+

[C32H49DO7N]+

[14CC31H50O7N]+

Delta = < 3mDa


Spinetoram (XDE-175) Plant Metabolic Pathway

H

O

O

O

O H

CH3

H HH

O

O

O

O

O

O

N

H

O

O

O

O H

CH3

H HH

O

O

O

O

O

O

N

H

H

O

O

O

O H

CH3

H HH

O

O

O

O

O

O

N

O

H

H

O

O

O

O H

CH3

H HH

O

O

O

O

OH

O

N

H

O

O

O

O H

CH3

H HH

OH

O

N

XDE-175-J

N-demethyl-175-J

N-formyl-175-J

1

1

3'-O-deethyl-175-J(5,6-dihydro-spinosyn J)

3

3

C9-(Reverse)-Pseudoaglycone-175-J

Theorized Macrolide RingCleavage Products

2

22 2

2

Systems studied as required for registration:

Soil biodegradation - aerobic conditionsbiodegradation - anaerobic conditionsabiotic degradationaerobic photodegradation

Sedimentbiodegradation - aerobic conditionsbiodegradation - anaerobic conditions

Water Hydrolysisphotodegradation in buffered waterphotodegradation in natural water

MetabolismLettuce (foliar applications)Turnips (foliar applications)Apples (foliar applications)Rice (after treatment of seedling box soil)

MetabolismPoultryLactating goats.Rats

SpinetoramPlant

MetabolicPathway


Schematic of the Q Exactive

Capabilities that may aid in metabolite ID:SensitivityMass accuracy < 5ppm (ext. cal.), <1ppm (int. cal.)Pos/Neg switchingFast MS, and MS/MS acquisition rates (up to 12Hz) High resolution up to 140,000 (FWHM @ m/z 200)


General Q Exactive Experiments Used to Study theSoil Metabolism of pyroxsulam (herbicide)

Experiments:

1) FTMS - p ESI Full ms [100.00-1000.00] @ 70K resolutionFTMS - p ESI Full ms2 top-5@HCD35 FTMS + p ESI Full ms [100.00-1000.00] @ 70K resolutionFTMS + p ESI Full ms2 top-5@HCD35

Goal: General screen for metabolites at medium resolution using pos/neg switching, acquire MS/MS spectra for anticipated metabolites

2) FTMS + p ESI Full ms [100.00-1000.00] @ 140K resolutionFTMS + p ESI Full ms2 [100.00-1000.00] @ 140K resolutionFTMS + p ESI mSIM top-5@HCD35 @ 140K resolution

Goal: Target metabolites detected in experiment #1 (+ESI) for high resolution +ESI MS, MS/MS, and mSIM spectra – optimal mass measurement and characterization of isotopic cluster

3) FTMS - p ESI Full ms [100.00-1000.00] @ 140K resolutionFTMS - p ESI Full ms2 [100.00-1000.00] @ 140K resolutionFTMS - p ESI mSIM top-5@HCD35 @ 140K resolution

Goal: Target metabolites detected in experiment #1 (-ESI) for high resolution -ESI MS, MS/MS, and mSIM spectra – optimal mass measurement and characterization of isotopic cluster

S

NO

NO

O

N

N

N

N

O

O

F FF

PyroxsulamC14H13F3N6O5SM = 434.0620[M+H]+ = 435.0693[M-H]- = 433.05475


Benefits of High Resolution – M+2 Sulfur CountingFlorasulam in Wheat Cell Extract

362.00 362.02 362.04 362.06m/z

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Re

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bu

nd

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ce

0

20

40

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100362.03285

362.04269

362.03331

362.04305

362.03558

NL:8.23E4080326I11#508 RT: 7.92 AV: 1 SB: 1 7.94-7.98 , 8.06-8.11 T: FTMS + p ESI Full ms [120.00-1200.00]



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Rel

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245.12832

652.40284

360.03714 674.38469

376.22293

724.38078439.29135 604.15813 1027.61789836.93613

279.17008

359 360 361 362 363m/z

0

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360.03714

361.04052

362.03285

Full scan FTMS

Expanded view of isotopic cluster

[M+H]+

M+2 isotope

Measured



12C12H8F3N5O334S

12C1013C2H8F3N5O3

32S

NN

NN

O

F

SN

O

OF

F

Florasulam(cereal herbicide)C12H8F3N5O3S[M+H]+ = 360.03727

Typically requires fairly concentrated sample because of low M+2 intensity


200 400 600 800m/z

0

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1000000

0

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0

20

40

60

80

100 435.0681

154.9898 457.0500381.2237 568.2950 684.2010

195.0746

435.0682

276.0391 460.9586433.0538

174.9547 501.0417541.2648242.9429

394.8899

705.0040179.0560

323.0504

433.0537

454.2881

1 2 3 4 5 6 7 8 9Time (min)

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Rel

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400000000

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1000000000

Rel

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0.8557275.11

7.9062183.08

2.9929565.15

8.2023409.255.8347

435.070.9841275.11 3.3753

609.187.8213183.08

5.8347435.07

2.9825563.14

0.8661297.12

2.8546371.10 9.3416

329.236.9384426.97

3.3648607.17 5.5634

505.232.6828352.10 4.5099

541.27

Soil Extract Containing Pyroxsulam Metabolites (0.5 ppm, 0.5 day), Q Exactive Experiment #1 (70K, +/-, top-5 DD MS/MS)

1.266 sec between scans ~0.8 Hz

S

NO

NO

O

N

N

N

N

O

O

F FF

PyroxsulamC14H13F3N6O5S M = 434.0620[M+H]+ = 435.0693[M-H]- = 433.05475

+ESI TIC

-ESI TIC

+ESI EIC m/z 435

+ESI MS

+ESI MS/MSprod of m/z 435

-ESI MS

-ESI MS/MSprod of m/z 433

Error = 1.2 mDa

Error = 1.1 mDa

Error = 0.9 mDa

Error = 1.1 mDa Average Error = 1.1 mDa (2.5 ppm)


433 434 435 436 437 438 439 440m/z

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

0

20

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60

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100R

elat

ive

Abu

ndan

ce435.0681

436.0710

437.0618 438.0666 439.0679433.0538

434.0563

435.0478 436.0519 439.1949 440.0826437.0559

Soil Extract Containing Pyroxsulam Metabolites (0.5 ppm, 0.5 day), Q Exactive Experiment #1 (70K, +/-, top-5 DD MS/MS)

435.0 435.1m/z

0

500000

1000000

1500000

Rel

ativ

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unda

nce

435.0475

437.0 437.1m/z

0

1000000

2000000

3000000

4000000

Rel

ativ

e Ab

unda

nce

437.0618

Insufficient Resolution for sulfur counting experiment

+ESI MS

-ESI MS

Insufficient Resolution for sulfur counting experiment


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0

200000

400000

600000

0

50000000

100000000

150000000

200000000

0

5000000

10000000

15000000

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30000000

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0

20

40

60

80

100 435.0693

457.0510381.2246167.0128

195.0753

435.0695

166.0725 276.0398 351.1013435.0695

435.0693

RT: 0.72 - 10.30

1 2 3 4 5 6 7 8 9 10Time (min)

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0

20

40

60

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100 5.80

8.17

7.37

8.05 10.236.054.732.91 4.492.68 9.826.70 8.870.81 2.00 5.64

5.80

5.84

5.79

7.97 9.057.87 8.16

7.36 9.608.486.695.342.922.35 4.741.950.82 4.26

Soil Extract Containing Pyroxsulam Metabolites Q Exactive Experiment #2 (+ESI 140K MS, 140K MS/MS and 140k mSIM)

Full scan@ 140K res

EIC m/z 435@ 140K res

DD HR MS/MS

DD HR mSIM

Full scan@ 140K res

DD HR MS/MS

DD HR mSIM

Theoretical[C14H13F3N6O5S+H]+

1.128 sec between scans ~0.9 Hz

Error = 0.0 mDa

Error = 0.2 mDa

Error = 0.3 mD

Average Error = 0.2 mDa (0.4 ppm)


437.04 437.06 437.08 437.10 437.12m/z

0

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0

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0

20

40

60

80

100 437.0652

437.0743

437.0652

437.0756

437.0648

437.0732

437.0652

437.0745

434 435 436 437 438m/z

0

5000

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15000

0

200000000

400000000

600000000

800000000

0

2000000

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6000000

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0

50000000

100000000

150000000

200000000435.0695

436.0722437.0652

435.0695

436.0730437.0652

435.0693

436.0709437.0648

435.0693

436.0726437.0652 438.0686

Soil Extract Containing Pyroxsulam Metabolites Q Exactive Experiment #2 (+ESI 140K MS, 140K MS/MS and 140k mSIM) – expanded view

Full scan@ 140K res

DD HR MS/MS

DD HR mSIM

Theoretical[C14H13F3N6O5S+H]+

Full scan@ 140K res

DD HR MS/MS

DD HR mSIM

TheoreticalM+2 region

[C1213C2H13F3N6O5

32S+H]+

[C12H13F3N6O534S+H]+

M+2Error = 0.4 mDa

Error = 0.0 mDa

Error = 0.2 mDa

Error = 1.4 mDa

Error = 1.0 mDa

Error = 0.2 mDa


Soil Extract Containing Pyroxsulam Metabolites Q Exactive MS/MS Spectrum from Experiment #2 (140K MS/MS )

100 150 200 250 300 350 400 450m/z

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195.0746R=160202

435.0687R=107102

166.0722R=170302 276.0393

R=133802

MS/MS(products of m/z 435at ~ 130,000 resolution)

Proposed:[C7H10N5O2]+= 195.0751Error = 0.5 mDa

Proposed: (would require rearrangement)[C7H10O5N5S]+ = 276.0397 ,Error = 0.4 mDa

Proposed:[C6H8N5O]+= 166.0723Error = 0.1 mDa

S

NO

NO

O

N

N

N

N

O

O

F FF


C8H10N5O2[-H]+= 195.0751

Error = 0.2 mDa


Pyroxsulam Soil Metabolism – Q Exactive Results

S

NO

NO

O

N

N

N

N

O

O

F FF


O-demethylationC13H11F3N6O5S M = 420.0464[M+H]+ = 421.0536[M-H]- = 419.0391

Sulfonic AcidC7H6F3NO4S M = 256.9970[M+H]+ = 258.0042[M-H]- = 255.9897

S

NO

NO

O

N

N

N

N

O

O

F FF H

S

NO

NO

O

N

N

N

N

O

O

F FF

H

S

NO

O

O

F FF

OH

All with external calibration

Only detectedIn -ESI

Experiment 1 Experiment 2 Experiment 3pyroxsulam pos neg pos neg

theo 435.0693 433.0548 435.0693 433.0548 +/- MS 435.0681 433.0538 140k MS 435.0693 140k MS 433.0550MS/MS 435.0682 433.0537 140k MS/MS 435.0695 140k MS/MS 433.0551

Ave = 435.0682 433.0538 mSIM 435.0696 mSIM 433.0552 ave. error (Da) = 0.0011 0.001 Ave = 435.0695 Ave = 433.0551

error (ppm) = 2.53 2.31 StDev = 0.0002 StDev = 0.0001 ave. error (Da) = 0.0002 ave. error (Da) = 0.0004

ave error (ppm) = 2.42 0.23 0.69

Overall for all metabolites Detectedave error (ppm) = 2.47 0.15 1.18


484.0 484.5 485.0 485.5 486.0m/z

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485.8121484.0711

485.1128

486.1118484.3483

485.8762

484.8283 485.2867 486.3064484.2239

485.0344

484.0709

485.0742

486.0667

NL:1.28E4DOW_STD_140K_mSIM_Top1_#A_10pg_OC#450 RT: 7.37 AV: 1 T: FTMS + ESI SIM msx ms [465.56-474.56, 479.57-488.57, 727.97-736.97, 741.98-750.98, 804.00-813.00]

NL:1.82E4C 16 H14 O5 N5 S 1 F 5 +H: C 16 H15 O5 N5 S 1 F 5p (gss, s /p:40) Chrg 1R: 140000 Res .Pwr . @FWHM

At 10 pg on column in mSIM mode - A+2 splitting is still observed

486.055 486.060 486.065 486.070 486.075 486.080 486.085m/z

0

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486.0666

486.0753

486.0667

486.0762

~20-30x lower than we can achieve on our LTQ-FT Ultra

theoretical

observed


Conclusions• Metabolite identification remains a challenging component of agrochemical discovery and

development. • Constantly lowering application rates = lower levels of resulting metabolites.• Sample complexity is high, but timelines often require ID with limited sample clean-up.

•New instruments, including the LTQ-FT, have significantly improved our ability to meet these challenges by providing:

• Improved Sensitivity• High Mass Accuracy (typically better than 1ppm)• High Resolution (up to 1,000,000)• MSn

•Additional methods to interrogate an unknown, including IRMPD and high resolution MS combined chip-based nanospray infusion allows the acquisition of a wide array of data with minimal sample consumption.

•Addition of the Thermo Q Exactive, a new benchtop quadrupole-orbitrap, may further expand our capabilities by providing:

• Improved MS and MS/MS sensitivity• Pos/Neg switching• Maintaining Fast MS, and MS/MS acquisition rates • High resolution (~140,000 FWHM) – allowing high resolution experiments, including sulfur

counting experiments at levels 20-30x better than our LTQ-FT Ultra.


Dow AgroSciencesGerry DeboerJeff GodbeyDebbie Schwedler

ThermoMary BlackburnTim StrattonDavid PeakeBjoern RoseMarkus KellmannEsther LewisButler John

Acknowledgements

Thermo Fisher Scientific - Trace Level Metabolite...

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