LTQ-Orbitrap An instrument dedicated to multi-fonctionnal ... · LTQ-ORBITRAP APPLICATIONSTitre du diaporama ANIMAL EXPERIMENT • Treatment of Trouts with brilliant green (bath at

LTQ-Orbitrap

An instrument dedicated to multi-fonctionnal search in molecular chemistry

AOAC-ASFILAB International Workshop, 23-24 November 2009, PARIS

Dominique Pessel, Thota Jagadeshwar Reddy & Eric VerdonAFSSA-LERMVD, Fougères, France

CRL for antibiotic/dye residues in food from animal origin

Titre du diaporamaLTQ-ORBITRAP APPLICATIONS

• Identification of metabolite of brilliant green- Dominique Hurtaud-Pessel

• Identification of antibiotic residues in

2

• Identification of antibiotic residues in biological matrices

- Thota Jagadeshwar Reddy


LC-MS instruments nowadays most frequently used in bioanalysis

• LC / triple quadrupole• LC / ion trap• LC / Q-trap

Unit Resolution

• LC / Q-trap

• LC/ToF or LC/Q-ToF• LC/Orbitrap or LC/LTQ-Orbitrap

3

High Resolution

~ 100 000 FWMH

High Mass accuracy


LTQ-Orbitrap APPLICATION :

Identification of metabolite of brilliant green

DYES

- Malachite green used in aquaculture • Against fungal and parasite infections

- High toxicity The use of Malachite green is not allowed in food producing animal

4


Metabolisation of malachite green in fish

N

C

CH3

CH3

N

H3C

H3CN

CH

CH3

CH3

N

CH3

H3C

Reduction

Malachite green Leuco Malachite green

-In treated fish, the major remaining component is the leuco compound. (> 80%)- in EU, Minimum Required Performance Limit (MRPL) = 2 µg/kg for the sum of MG + LMG.


Possible abuse of other compounds to replace Malach ite Green ?

NN+ R

R

R

R

R' • Crystal violet R=CH3, R’=N(CH3)2

- already known as possible alternative for malachite green- metabolized to leuco-Crystal violet- Not authorized

RR

Group of Triarylmethane dyes

• Brilliant green R=CH2-CH3, R’= H• Ethyl violet R=CH2-CH3 , R’=N(CH2-CH3)2

• Pararosaniline R=H, R’=NH2

Not much literature data on the metabolismNeed to know the target compounds to monitor for foodcontrol


Brilliant green, also called Malachite Green G

N

C

C2H5

C2H5

N

C2H5

C2H5

7

Is Brilliant Green metabolized in leuco brilliant green in treated fish, like MG and CV ?


ANIMAL EXPERIMENT

• Treatment of Trouts with brilliantgreen (bath at 100 ng/ml / 1 hour)

• Extraction from muscle tissue

8

• Analysis Liquid Chromatography-HRMS: LTQ-Orbitrap

Full-scan at Resolution 60 000


Research of metabolite compounds

Full High Resolution MS spectrum

Expected Analytes searchedfrom TIC

Unknowns Compounds

NON-TARGET APPROACHTARGET APPROACH

9

from TIC

Review of the Extracted Ion Chromatogram (EIC) of the exact mass of the protonatedcompounds searched.

Use of specific software allowing the comparisonof treated and untreatedsamples


1st approach : TARGET APPROACH - Extracted Ion Chromatog ramsRT: 0.00 - 24.99

0

20

40

60

80

1001.29

7.49

17.56 17.708.094.82 12.998.32 16.513.75

22.9819.566.79

NL: 2.81E8m/z= 100.00000-500.00000 F: FTMS + p ESI Full ms [100.00-500.00] MS Traitetruite03a

NL: 5.65E6

60

80

100

Rel

ativ

e A

bund

ance

06.79 NL: 5.65E6

m/z= 385.26189-385.26575 F: FTMS + p ESI Full ms [100.00-500.00] MS Brilliant green

Traitetruite03a #833 RT: 12.37 AV: 1 NL: 1.41E7F: FTMS + p ESI Full ms [100.00-500.00]

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

387.27902C 27 H35 N2

-1.17082 ppm

429.37222

Leuco Brilliant Green Theo mass =

387.27947∆ppm = -1.17C27H35N2

0 5 10 15 200

20

40

60

80

1000

12.34

7.46 12.90 15.659.545.68


0

20

40

Rel

ativ

e A

bund

ance

12.377.36 13.361.86 17.7012.34

[100.00-500.00] MS Traitetruite03a

NL: 1.52E7

LeucoBrilliant greenTheo. mass : 387.27947

Brilliant greenTheo. mass : 385.26382

Extracted window 5 ppm

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

0

10

20

30 429.37222C 29 H49 O 2

-1.12809 ppm194.14310

C 11 H18 O N2

8.92220 ppm


MS² measurement : HCD Fragmentation of m/z 387

TRAITE-truite -03

# 80 4 RT: 1 3.02

AV : 1 N L: 2.6 9E6 F: FTMS + p ES I Fu ll m s2 387 .28 @h cd 40 .00

[5 0.0 0-500 .00 ]

30

40

50

60

70

80

90

10 0

Relative A bundanc

343 .21 643 C 24 H 27 N 2 = 34 3.2 168

8 -1.2 958 2 pp m

35 8.2 3984 C 25 H 30 N 2 = 3 58.240 3

5 -1.431 59 p pm

2 81.200 62 C 19 H 2 5 N 2 = 2 81 .201 2

3 -2 .14 363 p pm

23 8.1 5854 C 17 H 20 N = 2 38 .159 0

3 -2.046 46 p pm

38 7.2 7869 C 27 H 3 5 N 2 = 3 87.279 4

8 -2.0 37 62

N N

H3C

H3C CH3

CH3

5 0 10 0 150 20 0 2 50 3 00 350 4 00 45 0 500 m/z

0

10

20 -2.0 37 62 pp m

Spectrum obtained on treated trout

• Confirmation of the identification of LBG by comparison using synthesized chemical standard of LBG (Atlanchim, Nantes)

HCD fragmentation spectrum similar,LC retention time, exact mass.

H3C CH3


2nd approach : NON TARGETED

Acquisition of accurate mass data

Analysis of untreated samples and treated samplesin full scan MS

Use of SIEVE softwareUse of SIEVE software

Comparison of the 2 groups to find differences

Identification of Metabolites of interest


2nd approach : NON TARGETED

• Analysis in full scan MS LTQ-Orbitrap(resolution 60,000) of a set of :

– 6 untreated samples– 6 untreated samples

– 6 treated samples

• Data processing using SIEVE


Find differences : SIEVE table of results

FrameID pValue Average Time Average m/z MZStart MZStop TimeStart TimeStop Ratio

15 8.914E-11 12.7368 387.279 387.269 387.289 11.4868 13.9868 423.471

499 7.63212E-9 12.7368 194.645 194.635 194.655 11.4868 13.9868 23.4527

166 1.11536E-8 12.7963 194.143 194.133 194.153 11.5463 14.0463 116.095

88 1.52514E-8 12.7963 388.282 388.272 388.292 11.5463 14.0463 251.448

57 2.54684E-7 8.88219 359.248 359.238 359.258 7.63218 10.1322 51.261

479 6.93342E-7 6.44377 384.31 384.3 384.32 5.19377 7.69377 10.9317

175 9.25873E-7 6.96329 386.267 386.257 386.277 5.71329 8.21329 7.2466

477 1.75513E-5 6.39916 428.337 428.327 428.347 5.14916 7.64916 12.5942

Treated trouts

Untreated trouts

477 1.75513E-5 6.39916 428.337 428.327 428.347 5.14916 7.64916 12.5942

37 2.26705E-5 6.99279 385.264 385.254 385.274 5.74279 8.24279 3.81795

204 4.90222E-5 8.88219 360.251 360.241 360.261 7.63218 10.1322 8.32198

587 6.01214E-5 12.7963 389.285 389.275 389.295 11.5463 14.0463 255153

641 6.28737E-5 6.39916 441.332 441.322 441.342 5.14916 7.64916 6.74141

97 8.79363E-5 12.0088 140.002 139.992 140.012 10.7588 13.2588 0.0827744

312 0.000146443 12.7219 181.028 181.018 181.038 11.4719 13.9719 0.109765

221 0.000147813 6.42902 402.253 402.243 402.263 5.17902 7.67902 2.4405

113 0.000162488 6.44377 356.279 356.269 356.289 5.19377 7.69377 10.3162


Extracted ion Chromatograms of potential metabolite s

RT: 0.00 - 24.99 SM: 5B

0

50

1000

50

1006.79

12.407.46 13.331.83 17.6712.37

7.49 13.109.51 15.625.658.62



NL: 2.13E6

385

387

0 5 10 15 20Time (min)

0

50

1000

50

1000

8.62

9.72 17.676.710.06 12.686.29

18.178.845.43 11.92 23.84



359

441


Identification of metabolites

RT: 0.00 - 24.99 SM: 5B

0

50

1000

50

1006.79

12.407.46 13.331.83 17.6712.37

7.49 13.109.51 15.625.658.62



NL: 2.13E6

Traitetruite03a #458 RT: 6.79 AV: 1 SB: 104 5.84-6.62 , 7.12-7.86 NL: 5.61E6F: FTMS + p ESI Full ms [100.00-500.00]

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

385.26327C 27 H33 N2

-1.43057 ppm

285.22086

Brilliant green

N

C

C2H5

C2H5

N

C2H5

C2H5

Traitetruite03a #833 RT: 12.37 AV: 1 NL: 1.41E7F: FTMS + p ESI Full ms [100.00-500.00]

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

387.27902C 27 H35 N2

-1.17082 ppm

429.37222C H O

Leuco brilliant green

N N

H3C

H3C CH3

CH3

Traitetruite03a #580 RT: 8.61 AV: 1 SB: 94 7.86-8.34 , 9.05-9.94 NL: 2.27E6F: FTMS + p ESI Full ms [100.00-500.00]

60

70

80

90

100

Rel

ativ

e A

bund

ance

359.24780C 25 H31 N2

-1.03818 ppm

Metabolite 2 :

MH – C2H4

desethylLBG

0 5 10 15 20Time (min)

0

50

1000

50

1000

8.62

9.72 17.676.710.06 12.686.29

18.178.845.43 11.92 23.84



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

0

10

20285.22086C 20 H29 O

-1.52857 ppm

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

0

10

20

C 29 H49 O 2

-1.12809 ppm194.14310

C 11 H18 O N2

8.92220 ppm

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

0

10

20

30

40

50R

elat

ive

Abu

ndan

ce

380.33664C 19 H40 N8

-1.06589 ppm

424.36267C 21 H44 O N8

-1.38707 ppm

N NH

H3C

H3C

CH3


Conclusion - Identification of metabolites

• Identification of LeucoBrilliant green in Troutstreated with BG

⇒ Accurate mass measurement⇒ Fragmentation

• Other metabolites⇒ Specific software to find differential peaks⇒ Specific software to find differential peaks

• Future : depletion studies• Control of food samples : LBG is going to beincluded in LC-MS/MS routine method.

Documents

LTQ-Orbitrap An instrument dedicated to multi-fonctionnal ... · LTQ-ORBITRAP APPLICATIONSTitre du diaporama ANIMAL EXPERIMENT • Treatment of Trouts with brilliant green (bath at