Presentación de PowerPoint - Inra Bordeaux · AgilentAgilent QTOF 6550 Quadrupole + Time of flight...

6/8/2015

1

FA1104 Training School

Determination of phytochemical components with advanced analytical methods


Qualitative, physicochemical and phytochemical indicators of cherry fruit quality

[2-4 June 2015]

Francisco Tomás Barberán

CEBAS – CSIC, Murcia, Spain

[email protected]

Determination of phytochemical components with

advanced analytical methods – Part II



Individual compounds

Phytochemical analysis

HPLC-DAD: identification of individual

compounds.

HPLC-MSn (Ion Tramp): Isolates and

fragments individual compounds

HPLC-MS (Triple Quadrupole: TARGETED

METABOLOMICS. High sensitivity. Needs

pure standards

HPLC-MS (Quadrupole + Time of flight): UN-

TARGETED METABOLOMICS. Give

molecular formula. High sensitivity. Needs

pure standards for quantification.

Agilent QTOF 6550

Quadrupole + Time of flight

HPLC-MS Ion tramp

Agilent QqQ 6460

Triple Quadrupole



Determining individual compounds


NMR-QTOF: Nuclear Magnetic Resonance +

Quadrupole + Time of flight (QTOF).

The most advanced equipment for the

identification of metabolites.

GC-MS: identification of volatiles

compounds.

GC-QTOF: non-targeted volatile metabolites.

Bruker NMR-QTOF UNTARGETED METABOLOMICS

Agilent GC-MS 5975C

TARGETED METABOLOMICS



HPLC-DAD-ESI-MS-MS



Sequential (MS-MS)n analysis for structure characterization

MS

MS2

MS3



MS4

MS5

MS6

Sequential (MS-MS)n analysis for structure characterization

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HPLC-DAD-MS



Citrus

• 1 N-glc-rha + N-glc

• 2 H-glc-rha + H-glc

• 3 D-glc-rha + D-glc

1

1a

2

1b

2a

2b

3

3a

3b

1

1a

2

1b

2a

2b

3

3a

3b

(A)

(B)

0

500

1000

0246x106

0.0

0.5

1.0

0

2

4

0

1

2

3

0

2

4

0.0

0.5

1.0

1.5

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

Intens.

mAbs.

x105

x106

x105

x106

x105

EIC at 579 m/z-

EIC at 433 m/z-

EIC at 609 m/z-

EIC at 463 m/z-

EIC at 593 m/z-

EIC at 447 m/z-

0

500

1000

0

2

4

6

0.0

0.5

1.0

0

2

4

0

1

2

3

0

2

4

0.0

0.5

1.0

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

mAbs.

x106

x105

x106

x105

x106

x105

Intens.

EIC at 579 m/z-

EIC at 433 m/z-

EIC at 609 m/z-

EIC at 463 m/z-

EIC at 593 m/z-

EIC at 447 m/z-

UV Chromatogram at 280 nm

UV Chromatogram at 280 nm

Hplc MS-MS Control

Rhamnosidases



UPLC-QQQ



PHASE II METABOLITES ANALYSED BY UHPLC-QQQ

HESPERETIN SULPHATE

NARINGENIN GLUCURONIDE

HESPERETIN GLUCURONIDE

HESPERETIN SULFOGLUCURONIDE

NARINGENIN DIGLUCURONIDE

HESPERETIN DIGLUCURONIDE



UPLC-QTOF



Relevant identified compounds (102)

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Volcano plot



PCA analysis





UPLC-QTOF-SPE-NMR



Standard

no

available

Picture by O.Yanes

CHALLENGE: NEW METABOLITES



UV detectors (DAD, PDA)


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O

O

OH

OH

HO

+

glc

O

OH

O

O

OH

HO

glur

O

O

OO

O

OO

O

O

O

HO

HO

OHOH

OH

HO

OH

OH

HOOH

HO

HO

O

OH

OH

OHO

O

HO

OH

OH

O

O O

rhamn

HO

O

O glc

Pelargonidin-glucoside Kaempferol-3-glucuronide

p-Coumaroyl-glucose

Ellagic acid rhamnoside

Sanguiin

Phenolics of strawberry

Example



-200

0

200

400

600

800

Intens.

0

250

500

750

1000

1250

1500

0

50

100

150

200

0

500

1000

1500

2000

10 20 30 40 50 60 Time [min]

280 nm

320 nm

360 nm

510 nm

Proanthocyanidins

Ets (Et)

Anthocyanins (An)

Flavonols (Fl)

Hydroxycinnamates (Hc)

An Fl Hc

Et



Phenolic content (mg/100g f.w.)

Anthocyanins 20-47

Flavonols 1-4

Hydroxycinnamates 1-7

Ellagitannins 10-25

Proanthocyanidins 50-170

Vitamin C 30-50

0

50

100

150

200

ANTH FOL HCA ETS PAC

lowest

highest



• Hydroxycinnamic acid derivatives (HPLC-DAD) • Flavonols (HPLC-DAD) • Anthocyanins (HPLC-DAD) • Proanthocyanidins (HPLC-MS, DAD, Fluorescence) • Carotenoids (HPLC-DAD) • Melatonin and serotonin (HPLC-QQQ)



UV spectra for phytochemical characterization 320 nm

1

1

2 3

4

2

3 4



UV spectra for phytochemical characterization

360 nm

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5

6

5 6

360 nm




510 nm



UV spectra for phytochemical characterization 510 nm

7

8

9

7 8

9




280 nm




280 nm *

10

10



MS for phytochemical characterization

ESI- ION TRAP MS-MS DETECTORS

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MS spectra for phytochemical characterization















MS-MS spectra for phytochemical characterization

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MS spectra for phytochemical characterization flavan-3-ols and proanthocyanidins



epicatechin-adduct

Catequin adduct

epicatechin

catechin

Terminal unit

Extension unit

Extension unit

Drupe NON-extractable

phenolic compounds

Mean degree of polymerization

Analysis of non-extractable proanthocyanidins by phloroglucinolysis

Kennedy & Jones, 2001, JAFC



MS spectra for phytochemical characterization Phloroglucinolysis



MS spectra for phytochemical characterization Phloroglucinolysis

m/z 289 11.3 min 245, 225, 203, 163, 145, 125 14.0 min 271, 245, 231, 205, 179, 161, 125 17.8 min 271, 245, 231, 205, 179, 161, 125 m/z 413 7.4 min 287, 167 10.9 min 287, 261, 161, 125 12.6 min 377, 359, 329



1 2 3 4 5 6 7

0.00 0.25 0.50 0.75 1.00 1.25

6 x10 Intens.

0

1 2

3 4

5 6 x10

0

2

4

6

6 x10

0.0 0.5 1.0 1.5 2.0 2.5 3.0

6 x10

0

1

2

3

5 x10

10 20 30 40 50 60 Time [min]

m/z 289

m/z 577

m/z 865

m/z 1153

m/z 1441

1

2

3 6

4 8

5 10

Strawberry

MS of procyanidin oligomers

Normal Phase. Fluorescence detection



Databases to estimate phenolic content

USDA

Proanthocyanidin content 2004

Flavonoid content 2011 (flavonols, flavones, flavanones,

flavan 3-ols, anthocyanins)

Eurofir

Phenol Explorer

www.ars.usda.gov (printed pdf)

www.eurofir.net

www.phenol-explorer.eu

http://www.ars.usda.gov/

http://www.eurofir.net/

6/8/2015

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www.phenol-explorer.eu

Incomplete databases ?

The problem of non-extractable polyphenols.

Search

Polyphenols

Foods

Metabolites

Graphics

Pharmacokinetics

Neveu et al., 2010, Database

502 polyphenols in 452 foods Based in published papers

Augustin Scalbert ICPH 2011









[email protected]

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