plaque hptlc/ms ? pourquoi pas - clubdeccm.com · test sur plaques tlc, hptlc et hptlc/ms après...

Plaque HPTLC/MS ? Pourquoi pas !!

Mr François Bretin, Chimie Médicinale Oncologie, Sanofi R & D Vitry-sur-Seine

Mr Francis Maquin Analytical Sciences ParisMr Francis Maquin, Analytical Sciences Paris, Sanofi R & D Vitry-sur-Seine

Test plaques HPTLC/MS

● Sur plaque TLC, HPTLC et HPTLC/MS, 5X10● Dépôt de 2, 4 et 6 µl d’une solution de 1.2 mg de VAC.FBN5.243 brut (MW=457) Soit

C=2.62 10-3 M● Distance de migration: 4 à 5 cm ● Solvant de migration: CH2Cl2/MeOH (99/1)

6/12/2012 François Bretin 2


● Analyse sur la fraction 2 µl



● Extraction sur TLC: principalement désorption du produit



● Extraction sur HPTLC: entrainement de la silice



● Extraction sur HPTLC/MS: entrainement ou soulèvement de la silice



● Analyse du spectre: rapport type100

2.2e+0052: MS ES- :TIC Smooth (Mn, 2x3)(1);100%;0.36;116333 (1);100%;0.64;116333100

9.8e+0071: MS ES+ :TIC (1)

Time10

100

%

Time10

100

%

1.2e+005ELSD-Sedex-65 (1);92%;0 39;14272

Time0

100

%

(1)34%0.32

9315620

(2)19%0.53

5190689

(3)48%0.62

13203698

Time0

100

%

-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50Time10

100

%

(1);92%;0.39;14272

(4)8%1.221229

-0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50Time10

100

%

100

%

1:MS ES+ 1.9e+007

Combine (12:15-41:43)480.4

458.44814

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0m/z0

58

419.4105.0459.4

481.4

482.4496.4



1 0 0

9 .8 e + 0 0 71 : MS E S + :T IC (1 )

3 4 %1 0 0

Comparaison des spectres TIC/ES+ des différentes plaques

%

0 .3 29 3 1 5 6 2 0

(2 )1 9 %0 .5 3

5 1 9 0 6 8 9

(3 )4 8 %0 .6 2

1 3 2 0 3 6 9 8%

TLC

-0 .1 0 0 .0 0 0 .1 0 0 .2 0 0 .3 0 0 .4 0 0 .5 0 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .1 0 1 .2 0 1 .3 0 1 .4 0 1 .5 0Tim e0

-0 .1 0 0 .0 0 0 .1 0 0 .2 0 0 .3 0 0 .4 0 0 .5 0 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .1 0 1 .2 0 1 .3 0 1 .4 0 1 .5 0Tim e0

1 00

6 .6e +0 071 : MS ES+ :TIC (1 )

25 %0.35

68 3 36 84

(2 )75 %0.5 3

2 09 35 7 64

1 00

HPTLC

Tim e0

%

(1)2 5%0 .2 6

6 83 36 8 4

Tim e0

%

HPTLC

-0.1 0 0 .0 0 0 .1 0 0 .2 0 0 .30 0 .40 0 .50 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .10 1 .20 1 .30 1 .4 0 1 .5 0-0 .1 0 0 .0 0 0 .1 0 0 .2 0 0 .30 0 .40 0 .50 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .10 1 .20 1 .30 1 .4 0 1 .5 0

1 0 0

7 .2 e + 0 0 71 : M S E S + :T IC (3 );5 1 % ;0 .8 6 ;2 5 3 1 1 3 8 0(2 )

1 8 %0 .6 8

9 0 8 8 3 4 6

(1 )1 4 %0 .3 5

6 7 6 2 1 0 7

(3 )5 1 %1 .0 1

2 5 3 1 1 3 8 0

(4 )1 7 %1 .2 6

8 3 7 3 5 1 3(4 )

1 7 %1 .2 0

8 3 7 3 5 1 3

1 0 0

HPTLC/MS

-0 1 0 0 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0T im e0

%

(1 )1 4 %0 .2 6

6 7 6 2 1 0 7

(2 )1 8 %0 .5 9

9 0 8 8 3 4 6(2 )1 8 %0 .5 3

9 0 8 8 3 4 6

-0 1 0 0 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0T im e0

%

HPTLC/MS


0 .1 0 0 .0 0 0 .1 0 0 .2 0 0 .3 0 0 .4 0 0 .5 0 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .1 0 1 .2 0 1 .3 0 1 .4 0 1 .5 00 .1 0 0 .0 0 0 .1 0 0 .2 0 0 .3 0 0 .4 0 0 .5 0 0 .6 0 0 .7 0 0 .8 0 0 .9 0 1 .0 0 1 .1 0 1 .2 0 1 .3 0 1 .4 0 1 .5 0

Test plaques HPTLC/MS100

1:MS ES+ 1.9e+007

Combine (12:15-41:43)480.4

TLC ES+

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0m/z0

%458.4

419.4105.0 420.4

459.4

460.4

481.4

482.4

496.4

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0

100

1:MS ES+ 1.9e+007

Combine (12:15-45:47)480.4

HPTLC

100 0 120 0 140 0 160 0 180 0 200 0 220 0 240 0 260 0 280 0 300 0 320 0 340 0 360 0 380 0 400 0 420 0 440 0 460 0 480 0 500 0m/z0

%

458.4419.4

105.0 420.4459.4

481.4

482.4496.4

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0

100

1:MS ES+ 2.0e+007

Combine (10:13)480.4

HPTLC/MS

415.0 420.0 425.0 430.0 435.0 440.0 445.0 450.0 455.0 460.0 465.0 470.0 475.0 480.0 485.0 490.0 495.0 500.0m/z0

%

419.4 458.4

481.4

496.4482.4497.4


Test plaques HPTLC/MS100

2:MS ES- 1.3e+005

Combine (10:14)353.4

ES-TLC

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0m/z0

%

119.1

143.2 261.1

354.4456.5

355.4403.2

490.5457.5472.6

100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0

100

2:MS ES- 1.3e+004

Combine (12:15-44:46)456.5

180.3

353.4490.4

HPTLC

80 0 100 0 120 0 140 0 160 0 180 0 200 0 220 0 240 0 260 0 280 0 300 0 320 0 340 0 360 0 380 0 400 0 420 0 440 0 460 0 480 0 500 0 520 0 540 0 560 0 580 0 600 0 620 0m/z0

%

138.2121.2

117.1

101.1

130.2

147.2

174.3149.5

166.1

241.5181.3199.3

239.2216.2 261.2243.2 268.3 283.6313.5 332.2 347.3

439.5429.5407.6402.9391.6357.2

446.4

490.4

472.5

457.5

458.5 473.5 541.5501.4 525.2 610.5549.4586.6

576.5

80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0 520.0 540.0 560.0 580.0 600.0 620.0

100

2:MS ES- 2.8e+004

Combine (9:13)353.4

490.4

HPTLC/MS

80 0 100 0 120 0 140 0 160 0 180 0 200 0 220 0 240 0 260 0 280 0 300 0 320 0 340 0 360 0 380 0 400 0 420 0 440 0 460 0 480 0 500 0 520 0 540 0 560 0 580 0 600 0m/z0

%

113.2

103.1151.3119.1

138.3 219.5157.3 255.4227.3 248.9

456.5

439.5354.4

429.5 440.5

472.5

457.5

458.3473.4

522.4

491.5501.5 523.5

554.7537.6 576.4


80.0 100.0 120.0 140.0 160.0 180.0 200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 420.0 440.0 460.0 480.0 500.0 520.0 540.0 560.0 580.0 600.0

Test plaques HPTLC/MS● Test sur plaques TLC, HPTLC et HPTLC/MS après migration sans

dépôt d’échantillon.1 MS ES

0

100

%

1:MS ES+ 2.1e+006

Combine (14:17-47:48)165.1

159.3135.1

307.1193.4167.2 297.2239.3219.2 395.5367.5347.2 434.4414.6 ES+ et ES- sur TLC/blanc

100

%

2:MS ES- 8.6e+004

Combine (13:17-46:48)119.2

261.2

121 1 255 5

m/z0 ES et ES sur TLC/blanc

150.0 200.0 250.0 300.0 350.0 400.0 450.0m/z0

159.1121.1 180.3255.5 283.5 403.1313.4

100

1:MS ES+ 8.1e+005

Combine (11:15-48:50)165.2

100

2:MS ES- 2.2e+004

Combine (11:14-48:49)119.1

m/z0

%

136.2121.1

297.2

185.4193.3

288.4

219.2395.6

367.5434.4 487.6 633.7605.7507.6

776.8661.9 748.2 ES+ et ES- sur

HPTLC/MS/blanc

100.0 200.0 300.0 400.0 500.0 600.0 700.0 800.0m/z0

% 116.1

103.1 261.2187.2179.1 249.1

283.6407.3318.7

397.1 679.0657.3443.3 688.3 781.7


Test plaques HPTLC/MS● Test sur plaques TLC, HPTLC et HPTLC/MS après migration sans

dépôt d’échantillon.

ES+ et ES- sur /0

100

%

1:MS ES+ 6.6e+006

Combine (17:20)122.2

115.2

108.1

171.3157.3

129.2 185.3 215.3 267.4225.3 257.2

297.4 395.5367.5 413.5

HPTLC/blanc

0

100

%

2:MS ES- 1.1e+005

Combine (16:20)121.2

113.2

101.2180.4147.2

143.3 151.3 196.4 241.4206.3 255.4

m/z0

100

1:MS ES+ 8.1e+005

Combine (11:15-48:50)165.2

100.0 150.0 200.0 250.0 300.0 350.0 400.0m/z0

100

2:MS ES- 2.2e+004

Combine (11:14-48:49)119.1

m/z0

%

136.2121.1

297.2

185.4193.3

288.4

219.2395.6

367.5434.4 487.6 633.7605.7507.6

776.8661.9 748.2 ES+ et ES- sur

HPTLC/MS/blanc

100.0 200.0 300.0 400.0 500.0 600.0 700.0 800.0m/z0

% 116.1

103.1 261.2187.2179.1 249.1

283.6407.3318.7

397.1 679.0657.3443.3 688.3 781.7



●ConclusionConclusion Dans nos conditions d’utilisation nous ne voyons pas d’amélioration au niveau du spectre de masse. Peutd amélioration au niveau du spectre de masse. Peut être plus adapté sur de l’analyse quantitative ?


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