Gas chromatography coupled to mass spectrometry Liquid chromatography coupled to mass spectrometry...

Gas chromatography coupled to mass spectrometry

Liquid chromatography coupled to mass spectrometry

MetMAX – Data alignment

COVAIN – Data integration, Statistics and Modelling

Data mining

Figure 1. Metabolomics platform for the characterization of medicinal plants integrating GC/MS, LC/MS, alignment tools and statistical analysis using COVAIN (Sun & Weckwerth, 2012)

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141

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Figure 2. HPLC/ESI(OrbiTrap)/MS profile (full msmode) of the crude hydroalcoholicextract of P. anserina (positive ion mode)

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Figure 3A. Sample patterns of the hydroalcoholic extracts of P.anserinaanalyzed by GC/MS.

P. anserina UniVie

P. anserina UniSa

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15

P. anserina UniVie

P. anserina UniSa

2

5

1

3

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Figure 3B.Scores plot of the hydroalcoholic extracts of P.anserinaanalyzed by LC/MS(only identified compounds used as variables).

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Figure 3C. Sample patterns of the hydroalcoholic extracts of P.anserina analyzed by LC/MS(all the compounds with RSD<25 used as variables).

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5

P. anserina UniVie

P. anserina UniSa

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Figure 4. Sample patterns of combined GC/MS and LC/MS data of the hydroalcoholic extracts of P.anserina.

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PC 1 (47.5%)PC 2 (24%)

PC

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Figure 5. Relative quantitative analysis of genistein in the commercial products (1, 2, 3, 4, 5) and in the two hydroalcoholic crude extracts of P. anserina.

Supplementary data

Supplementary Figure 1.nanoLC/ESI(Orbitrap)/MS profile (full ms mode) of the crude hydroalcoholic extract of P. anserine and its commercial products (positive ion mode)

Supplementary Figure 2.GC/EI(TSQ)/MS profile (full ms mode) of the crude hydroalcoholic extract of P. anserina and its commercial products (positive ion mode)

Supplementary Figure 3. Loadings plot of the hydroalcoholic extracts of P.anserina analyzed by GC/MS.

Supplementary Figure 4. Loadings plot of the hydroalcoholic extracts of P.anserina analyzed by LC/MS (identified compounds).

Supplementary Figure 5 .Rt, LC/MS and LC/MSMS spectra comparison between reference standards and P. anserinahydroalcoholic extract compounds.A:chlorogenic acid, [M+Na]+: 377; B: genistein, [M+H]+: 271; C: quercetin 3-O-glucoside, [M+H]+: 465; D: kaempferol 3-O-glucoside, [M+H]+: 449; E: myricetin 3-O-rhamnoside [M+H]+: 465; F: quercetin 3-O-glucuronide [M+H]+: 479

panserinaunisa120ngulposddep060412 07/04/2012 05:58:27

RT: 0.00 - 100.01 SM: 11G

0 10 20 30 40 50 60 70 80 90 100

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

46.71271.06

70.65371.10

57.64387.18

33.90465.10

76.01391.28

19.07355.10

43.32303.05

81.65371.10

4.97158.00

99.77371.10

19.02215.07

19.10377.08

23.14377.08

72.61377.33

68.570.00

78.87377.34

54.54377.10

17.09377.09

43.28377.02

37.43377.02

4.77376.95

84.67377.17

NL: 1.94E8

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

NL: 8.88E3

m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 377.08@cid40.00 [90.00-390.00] MS MixSTD1_060412

NL: 6.03E6

m/z= 376.50-377.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

RT: 0.00 - 44.91 SM: 11G

0 5 10 15 20 25 30 35 40

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.43479.08

44.86668.4437.66

493.1033.48465.106.11

294.1525.59163.04

22.49641.14

7.64371.10

12.75371.10

25.59377.0519.58

377.08

21.19377.08

6.03377.16

14.05377.08

34.80377.12

29.85377.07

43.73377.29

0.960.00

10.310.00

NL: 1.41E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 3.22E3

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 377.05@cid40.00 [90.00-390.00] MS panserinaunisa120ngulposddep060412

NL: 1.18E6

m/z= 376.50-377.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

MixSTD1_060412 #1754 RT: 19.02 AV: 1 NL: 1.34E4F: ITMS + c NSI d Full ms2 377.08@cid40.00 [90.00-390.00]

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

m/z

0

10

20

30

40

50

60

70

80

90

100Relativ

e A

bundance

215.07

359.07

185.07197.09 331.14

179.08

163.09151.13 341.10315.14271.14 299.02 360.02216.91117.05 258.17

panserinaunisa120ngulposddep060412 #1694 RT: 19.17 AV: 1 NL: 1.45E2F: ITMS + c NSI d Full ms2 377.05@cid40.00 [90.00-390.00]

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

m/z

0

10

20

30

40

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60

70

80

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Relativ

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359.11

215.11

361.10

331.07197.21 297.07

179.04 315.12 341.10279.01216.77 368.14260.99137.17 247.06177.72115.11E:\DATA\ORBI\ORBISTDMIX\MixSTD1_060412 06/04/2012 14:40:58

RT: 0.00 - 100.01 SM: 11G

0 10 20 30 40 50 60 70 80 90 100

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

46.71271.06

70.65371.10

57.64387.18

33.90465.10

76.01391.28

19.07355.10

43.32303.05

81.65371.10

4.97158.00

99.77371.10

46.88153.00

69.34215.06

46.71271.06

47.96271.06

67.54271.06

43.94271.15

73.53271.06

63.29271.06

82.32271.06

32.45271.08

1.95271.19

27.23271.19

11.92271.19

97.28271.19

NL: 1.94E8

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

NL: 8.71E4

m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 271.06@cid40.00 [60.00-285.00] MS MixSTD1_060412

NL: 2.05E8

m/z= 270.50-271.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

RT: 39.48 - 50.00 SM: 11G

40 41 42 43 44 45 46 47 48 49 50

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

44.86668.44 45.71

668.4441.77593.19 43.57

595.1441.36797.25

47.54266.17

49.25527.33

47.16371.10

43.38137.03

48.03271.19

46.95271.06

43.36271.1039.90

271.0649.93271.23

41.09271.11

46.22271.06

44.56271.10

42.04270.98

NL: 1.26E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 7.71E2

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 271.06@cid40.00 [60.00-285.00] MS panserinaunisa120ngulposddep060412

NL: 4.55E5

m/z= 270.50-271.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

MixSTD1_060412 #4130 RT: 45.86 AV: 1 NL: 3.39E2F: ITMS + c NSI d Full ms2 271.06@cid40.00 [60.00-285.00]

60 80 100 120 140 160 180 200 220 240 260 280

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

253.05153.08

215.06

282.72

243.16

149.05163.19

224.98173.13107.14 145.11

121.1781.04 187.12 229.29197.0593.11254.26 272.95

80.26

panserinaunisa120ngulposddep060412 #4112 RT: 46.91 AV: 1 NL: 2.69E2F: ITMS + c NSI d Full ms2 271.06@cid40.00 [60.00-285.00]

60 80 100 120 140 160 180 200 220 240 260 280

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

282.76153.05

253.08

215.05

243.12

254.70149.11 229.08163.22145.04 211.20 272.94121.07 197.21185.04

93.17 107.0285.11 271.12

panserinaunisa120ngulposddep060412 07/04/2012 05:58:27

RT: 0.00 - 50.31 SM: 11G

0 5 10 15 20 25 30 35 40 45 50

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

46.71271.06

33.90465.10

19.07355.10

43.32303.05

36.59449.11

25.01686.32

30.18832.43

4.97158.00

9.73371.10

12.02371.10

34.35303.08

33.92465.10

33.15465.10

35.92465.10

39.46465.10

46.85465.10

29.69465.10

10.32465.10

22.34465.10

4.95464.92

19.90465.07

16.14465.10

NL: 1.94E8

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

NL: 1.34E5

m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00] MS MixSTD1_060412

NL: 4.20E7

m/z= 464.50-465.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

RT: 0.00 - 44.91 SM: 11G

0 5 10 15 20 25 30 35 40

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.43479.08

44.86668.4437.66

493.1033.48465.106.11

294.1525.59163.04

22.49641.14

7.64371.10

12.75371.10

35.87303.02

33.48465.10

34.41465.10

31.52465.10

39.29465.10

5.99465.21

22.49465.10

20.26465.10

27.66465.14

9.80465.07

15.91465.07

NL: 1.41E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 7.40E3

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00] MS panserinaunisa120ngulposddep060412

NL: 7.84E6

m/z= 464.50-465.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

MixSTD1_060412 #3112 RT: 34.35 AV: 1 NL: 5.59E5F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

303.08

447.07345.07 399.13 429.08369.11315.04144.92 229.05 247.36 285.14162.96

panserinaunisa120ngulposddep060412 #3000 RT: 34.33 AV: 1 NL: 2.30E4F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

303.04

319.10 447.21429.18345.14 369.14 399.11289.16277.08237.36163.20 199.17147.09

A

B

C

panserinaunisa120ngulposddep060412 07/04/2012 05:58:27

RT: 29.46 - 41.82 SM: 11G

30 31 32 33 34 35 36 37 38 39 40 41

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.29479.08

36.83449.11

37.56493.10

33.56451.09

31.50451.09

34.82509.09

38.12517.13

29.64371.10

32.14371.10

39.84371.10

40.55371.10

36.59449.11 37.43

449.11

38.39449.11

35.76449.11

33.15449.11

34.87449.11

39.49449.11

41.34449.11

31.04449.11

29.55449.11

31.76449.11

NL: 3.03E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS mixstd2_060412

NL: 7.92E3

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 449.11@cid40.00 [110.00-460.00] MS MixSTD1_060412

NL: 2.38E7

m/z= 448.50-449.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS MixSTD1_060412

RT: 24.96 - 41.99 SM: 11G

26 28 30 32 34 36 38 40

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.43479.08 37.66

493.1033.48465.10

41.77593.19

36.78287.05

30.65495.08 38.58

648.4125.59163.04

29.67565.16

26.37595.17

34.35287.03

36.80449.11

29.85449.11 33.08

449.0734.41449.11

38.87449.11

30.42449.11

39.70449.11

25.20449.11

29.18449.11

NL: 1.41E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 1.10E4

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 449.07@cid40.00 [110.00-460.00] MS panserinaunisa120ngulposddep060412

NL: 4.95E6

m/z= 448.50-449.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

MixSTD1_060412 #3242 RT: 35.77 AV: 1 NL: 9.48E3F: ITMS + c NSI d Full ms2 449.11@cid40.00 [110.00-460.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

287.06

433.13303.05 345.01 383.14329.07 419.11185.30 223.32 238.90153.89 278.99

panserinaunisa120ngulposddep060412 #3136 RT: 35.85 AV: 1 NL: 3.60E3F: ITMS + c NSI d Full ms2 449.07@cid40.00 [110.00-460.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

287.04

433.10329.17 345.06 403.18383.18315.08285.14177.68 256.43 440.40227.11205.27147.10panserinaunisa120ngulposddep060412 07/04/2012 05:58:27

RT: 30.72 - 37.03 SM: 11G

31 32 33 34 35 36 37

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

33.46465.10

36.66435.0934.02

465.1034.45465.10

35.38303.05

31.40371.10

31.71371.10

32.32371.10

35.71371.10

33.46465.10

34.02465.10

34.45465.10

35.90465.10

35.49465.10

32.41465.10

36.18465.10

31.16465.10

31.56465.10

NL: 1.48E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS mixstd3_060412

NL: 0

Base Peak m/z= 49.50-50.50 F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00] MS mixstd3_060412

NL: 1.54E7

m/z= 464.50-465.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS mixstd3_060412

RT: 30.66 - 36.18 SM: 11G

31 32 33 34 35 36

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.43479.08

33.48465.10 35.34

303.0531.52597.15

35.86303.05

32.99451.09

31.79371.10

33.48465.10

34.41465.10

33.98465.10

31.52465.10

35.86465.10

31.13465.10

32.90465.10

32.44465.10

NL: 1.41E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 7.40E3

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00] MS panserinaunisa120ngulposddep060412

NL: 7.84E6

m/z= 464.50-465.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

mixstd3_060412 #2804 RT: 33.10 AV: 1 NL: 2.44E3F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

319.01

447.29303.06 429.02329.01 361.10 411.13262.21 459.99178.08 236.98

panserinaunisa120ngulposddep060412 #2910 RT: 33.31 AV: 1 NL: 2.69E3F: ITMS + c NSI d Full ms2 465.10@cid40.00 [115.00-480.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

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bundance

319.04

303.06

447.16428.91329.07 361.11 403.12285.09147.07 240.29 259.15 449.99208.93165.91e:\data\orbi\orbistdmix\mixstd3_060412 06/04/2012 22:19:43

RT: 30.72 - 37.03 SM: 11G

31 32 33 34 35 36 37

Time (min)

0

50

100

0

50

100

Relativ

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bundance 0

50

100

33.46465.10

36.66435.0934.02

465.1034.45465.10

35.38303.05

31.40371.10

31.71371.10

32.32371.10

35.71371.10

34.47479.08

36.66479.06

35.33479.08

31.68479.08

33.23479.08

36.09479.08

30.83479.08

32.48479.12

NL: 1.48E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS mixstd3_060412

NL: 0

Base Peak m/z= 49.50-50.50 F: ITMS + c NSI d Full ms2 479.08@cid40.00 [120.00-490.00] MS mixstd3_060412

NL: 7.13E5

m/z= 478.50-479.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS mixstd3_060412

RT: 30.66 - 36.18 SM: 11G

31 32 33 34 35 36

Time (min)

0

50

100

0

50

100

Relativ

e A

bundance 0

50

100

34.43479.08

33.48465.10 35.34

303.0531.52597.15

35.86303.05

32.99451.09

31.79371.10

34.43479.08

34.00479.12

35.50479.08

33.01479.15

31.10479.08

32.39479.15

31.65479.08

NL: 1.41E7

Base Peak F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

NL: 6.26E3

Base Peak m/z= 50.00-2000.00 F: ITMS + c NSI d Full ms2 479.08@cid40.00 [120.00-490.00] MS panserinaunisa120ngulposddep060412

NL: 1.46E7

m/z= 478.50-479.50 F: FTMS + p NSI Full ms [150.00-1000.00] MS panserinaunisa120ngulposddep060412

mixstd3_060412 #2910 RT: 34.34 AV: 1 NL: 4.00E3F: ITMS + c NSI d Full ms2 479.08@cid40.00 [120.00-490.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

303.06

461.01398.90 443.25345.10 380.91317.00262.80241.21 275.01217.29158.56

panserinaunisa120ngulposddep060412 #2982 RT: 34.13 AV: 1 NL: 5.98E3F: ITMS + c NSI d Full ms2 479.08@cid40.00 [120.00-490.00]

150 200 250 300 350 400 450

m/z

0

10

20

30

40

50

60

70

80

90

100

Relativ

e A

bundance

303.06

317.07461.08438.03424.91399.04299.16 347.01147.12 369.16256.93163.05 239.11183.20

D

E

F