high resolution lc-ms data output and analysis · glucose quercetin. spectra components ... rutin +...

High Resolution LCHigh Resolution LC--MS MS Data Output and AnalysisData Output and Analysis

LC/MS

Sample

Introduction

Sample

Preparation

IonsDetection

IonsSeparation

IonizationMS Interface

UVSpectra

Separation

(column)

Efficient

Gradient

Your Sample

Result

LC-MS

Data (computer)Today

Mass SpectrometerMass SpectrometerMass Spectrometer

1. Breaks up constituents into molecular ions and other fragments

2. The ions are separated according to their mass-to-charge ratio (m/z)

3. Measures masses

Mass SpectrometerMass SpectrometerMass Spectrometer

4. Structural information on metabolite

* fragmentation pattern

* accurate mass

Alternative Ionization ModesAlternative Ionization Modes

•Atmospheric Pressure Ionization (API), in LC-MS

• Electrospray Ionisation (ESI): polar and semi-polar

• Atmospheric Pressure Chemical Ionization (APCI): less polar

ESAPCI

lipids waterpolarity of analyte molecule

Positive or Negative Modes?Positive or Negative Modes?

The formation of positive or negative ions depends on the sign of the applied electrical field

ES+: (M+H)+

Good ionization of basic compounds (get proton)E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize)

ES-: (M-H)-

Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)

The formation of positive or negative ions depends on the sign of the applied electrical field

ES+: (M+H)+

Good ionization of basic compounds (get proton)E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize)

ES-: (M-H)-

Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)

Mass AnalyzersMass Analyzers•Ion Cyclotron(FT-ICR-MS)

•Time of Flight(TOF)

•Magnetic Sector

•Quadrupole Ion Trap

•Quadrupole

“High Resolution”Instruments

“Low Resolution”Instruments

Mass AnalyzersMass AnalyzersMass

Accuracy

<1ppm•Ion Cyclotron(FT-ICR-MS)

•Time of Flight(TOF)

3-10ppm

2-5ppm•Magnetic Sector

•Quadrupole Ion Trap

•Quadrupole

n/a

n/a

LC-PDA-QTOF-MS of a Tomato Sample

MS-sample - Total Ion Chromatogram

(TIC)

MS-reference (lock mass) injected every few scans (TIC)

PDA-sample

Spectra Obtained at Each Scan

MS-sample

MS-reference (lock mass)

PDA-sample

Enkephalin (556.2766 in ES+)

Extracted chromatogram of a Specific Ion

TIC

Extracted chromatogramat m/z 611

Extracted chromatogramat m/z 303

LC/UV/MS Chromatograms of Standard’s Mixture(UV at 240nm, ES+ and ES-)

1 – Chlorogenic acid2 – Caffeic acid3 – Rutin4 – p-Coumaric5 – Quercetin-3-glucoside6 – Ferulic acid7 – Sinapic acid8 – Benzoic acid9 – Morin (Internal Standard)10 – Tomatin11 – Quercetin12 – Cinnamic acid13 – Naringenin Chalcone14 – Naringenin15 – Kaempferol

MS (-)

09-Jan-2006Standard Mix

Time1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

17

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

MixAllStand_b1 3: Diode Array Range: 1.113.68

3.63

2.67

2.26

1.66

3.38

3.26

5.43

4.954.606.136.00

5.92

MixAllStand_b1 1: TOF MS ES+ TIC

1.18e45.16

3.653.502.28

1.69

3.282.69

3.71 4.98

4.54

6.02

5.455.93

6.10

6.46

1

2

3

3

4

5

6

6

7

89

11

12

1314

15

12

45 7 9

10

1112

1314

15

UV

MS (+)

10-Jan-2006

Tim e1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

9

ILMixAllStand-c4 1: TOF MS ES- T IC

8.88e36.07

5.99

3.372.68

2.272.22

3.25

5.42

4.92

3.68

4.58

3.95

5.91

6.13

6.376.72 6.77

1

2 4

56

9

11

12

13

14

15

3 7

8

In the Spectra: Molecular Ion- An unfragmented (parent) ion formed by loss or gain of an proton from a molecule

- Has the same mass as the metabolite being analyzed (+ H or - H)

The Molecular Ion

Chromatogram and Spectrum of Morin

ES(+)

ES(-) [M-H]-

[M+H]+

Chromatograms in ES(+) & ES(-) modes

Spectra'sin ES(+) & ES(-) modes

Adduct Formation in Spectra (Na)

[M+H]+

[M+Na]+

Rutin C27H30O16

Na (22)

C27H31O16

C27H30O16Na

S5

Typical Adducts formed in LCMSTypical Adducts formed in LCMS

Mass of Adduct IonSource of AdductAdducts Ion

M+59Acetic acid[ M+CH3COO ]-

M+35Chlorinated solvent[ M+Cl ] -

M+18Ammonia[ M+NH4 ] +

M+23Sodium Salt[ M+Na ]+

M+39Potassium Salt[ M+K ]+

M+42Acetonitrile[ M+CH3CNH ]+

M+33Methanol[ M+CH3OHH ]+

M+19Water[ M+H2O]+

Isotopes in Spectra (13C)

[M+H]+

[M+Na]+Rutin C27H30O16

C27H31O16

C27H30O16Na

13C (little from other isotopes H, O)

Abundance of Isotopes (in Spectra)

An example of how isotopes can aid An example of how isotopes can aid in peak in peak identificationidentification

A (50.6%) A + 2 (49.3%)

The ratio of peaks containing 79 Br and its isotope 81 Br (100/98) confirms the presence of bromine in the compound.

Fragments of a Metabolite in Spectra

[M+H]+

[M+Na]+

Glucose (162)

Rhamnose (146)

MS spectrum of Rutin in ES(+)

Rhamnose

Glucose

Quercetin

Spectra ComponentsSpectra Components

Molecular IonMolecular IonFragments of MetaboliteFragments of MetaboliteAdducts (from solvent or diAdducts (from solvent or di--tritri--mers)mers)IsotopesIsotopes

Spectrum of Quercetin Trisaccharide(a Rutin Derivative)

Rutin

Quercetin[M+H]+

[M+Na]+

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0

100 765.1812

743.1995

303.0515

76.0834

469.1320391.1980 611.1645

470.1434

471.1271

612.1766

613.1899

766.1797

767.1795

827.1555828.1354 1133.4736

743

loss of pentose residue743.19 – 611.16 = 132.03

UV spectra of Rutinand Quercetin Trisaccharide

Rutin

+ sugar

Mass Accuracy and Structure Elucidation

High mass accuracy(1 mDa)

Low mass accuracy(100 mDa)

Mass Accuracy and Structure Elucidation

Selected natural compounds with formula C18H19NO (monoisotopic mass 313.1314 Da)

Performing MS-MS: MS-MS of m/z 435 at different collision energies

CE=5 eV

CE=10 eV

CE=15 eV

CE=30 eV

Output Data For High Res. LC-MS Based Metabolomics

AccurateNominal

Software for comparing full-scan datasetsMetAlign method

Peak picking

Alignment

Statistics

Software for comparing full-scan datasets

MarkerLynx method

Out-put from the MarkerLynx (Waters) Program :Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)

Principle Components Analysis

Markers Detected

Samples Processed

TIC of currently selected sample

Response of a marker across

all samples

Markers responsible for clustering (by PCA)

A Case Study in Tomato

Small Green

Big Green

Middle Green

Breaker Orange Red

Sample Preparation for Tomato Fruit Metabolome Analysis

Harvesting tomato fruit at different developmental stages

Separation of peel and flesh tissues

Immediate freezing in liquid nitrogen

Grinding to fine powder

Weighing frozen samples

Extraction in 100% MeOH by vortexing and sonication

Analyzing with UPLC-MS [ ES (+) and ES (-) ]

Experimental Set-Up for LC-MS Analysis:PEEL and FLESH during tomato fruit development

Peel

FleshES-

ES+

ES-

ES+Peel

FleshES-

ES+

ES-

ES+

Greenstage

Breakerstage

Peel

FleshES-

ES+

ES-

ES+Peel

FleshES-

ES+

ES-

ES+

Orangestage Red

stage

UPLC-Q-Tof-MS Analysis of Four Stages of Tomato Fruit Development

09-Jan-2006

Time2.00 4.00 6.00 8.00

AU

0.0

5.0e-1

1.0

1.5

2.00 4.00 6.00 8.00

AU

0.0

5.0e-1

1.0

1.5

2.00 4.00 6.00 8.00

AU

0.0

5.0e-1

1.0

1.5

2.00 4.00 6.00 8.00

AU

0.0

5.0e-1

1.0

1.5Tom_24_WTa7 3: Diode Array

Range: 4.3065.980.80

3.25

2.912.262.044.94

3.68 4.135.37

Tom_22_WTa6 3: Diode Array Range: 6.4185.970.80

3.24

2.251.02 2.90 4.943.674.21 5.36

Tom_20_WTa5 3: Diode Array Range: 2.9550.80 5.99

3.25

2.912.261.02

4.953.68 4.13 7.67

Tom_18_WTa4 3: Diode Array Range: 3.20.80

3.26

2.261.02 2.914.95

3.68 7.67

09-Jan-2006

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100Tom_24_WTa7 1: TOF MS ES+

TIC5.00e4

6.010.840.70 1.09

5.171.81 2.403.78 4.23 8.33

Tom_22_WTa6 1: TOF MS ES+ TIC

5.00e4

6.000.85

0.70 5.161.09

2.401.81 3.783.26 4.23 7.71


5.00e45.13

5.090.850.70 4.221.09 3.78

2.40 2.595.00 6.02 7.697.39 7.86


5.00e45.13

0.840.70 1.09

5.001.82 2.41 3.28 3.78 6.13 7.697.386.51 7.94

UV (240 nm) MS (ES(+),TIC)

RedRed

Orange

Breaker

Green

Orange

Breaker

Green

Out-put from the MarkerLynx (Waters) Program :Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)

Peel-Flesh Profiles During Fruit Development

Green peel

Breaker peelBreaker flesh

Green flesh

Red peelOrange peel

Orange flesh

Red flesh

Com

pone

nt 2

Comp. 1

Wild-type Tomato Peel, Different Developmental Stages

09-Jan-2006

Time1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0

100

1.00 2.00 3.00 4.00

%

0


3031000Area

3.2742

2.938

Tom_22_WTa6 1: TOF MS ES+ 303

1000Area

3.2740

2.947


1000Area

3.2836

2.9311


1000Area

3.2735

2.937

09-Jan-2006

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0


2732.00e4

Area6.01731


2.00e4Area

6.001158


2.00e4Area

6.03357


2.00e4Area

09-Jan-2006

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0


578.41.00e4

Area

5.17484.31

3

3.631

Tom_22_WTa6 1: TOF MS ES+ 578.4

1.00e4Area

5.1690

4.314


1.00e4Area5.13

362

5.078


1.00e4Area

5.12509

5.2410

Red

Orange

Breaker

Green

Tomatine Rutin & Quercetin trisacharide Naringenin chalcone

DOWN NO CHANGE UP

Tomato (var. Ailsa Craig) and the Mutant “Y”,at different ripening stages

Green Breaker Orange Red

Tomato peel

of Mutant “Y” and Wild-type

Wild-Type

Mutant “Y”

Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway

Naringenin chalcone

Cinnamate

Chalcone synthase (CHS)

Anthocyanines

Condensed tannins

Coumarate

Coumaroyl - CoA

Lignins,Coumarins,Chlorogenic-acid

Flavonols

Phenylalanine

Naringenin

Experimental Set-Up for LC-MS Analysis:PEEL and FLESH during “Y” fruit development

Peel

FleshES-

ES+

ES-

ES+Peel

FleshES-

ES+

ES-

ES+

“Y”Breaker

stage

“Y”Greenstage

Peel

FleshES-

ES+

ES-

ES+Peel

FleshES-

ES+

ES-

ES+

“Y”Orange

stage

“Y”Redstage

HPLCHPLC--QTOFQTOF--MS (UltimaMS (Ultima--PRI) PRI) --Wild Type and Wild Type and ““YY”” PeelsPeels

WT-Orange

“Y”-Red

WT-Red

“Y”-Orange

All breaker

All green

1st principal component (X axis) - developmental stage (47.1%) 2nd principal component (Y axis) – mutation (9.4%)

UV and MS Chromatograms of Red Tomato Peel (Wild Type and Mutant “Y”)

09-Jan-2006

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

%

0


TIC2.11e4

0.84

0.70

0.89

1.09

1.815.18

2.403.78

3.642.604.24 4.98

4.86

8.027.95

5.27 5.75

8.31


2.12e40.85

0.70

6.021.811.095.16

2.41 3.623.28

2.94

3.77

4.23 4.98

5.43

7.907.63

6.46

8.32

Wild Type

09-Jan-2006

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

AU

0.0

5.0e-1

1.0

1.5

2.0

2.5

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

AU

0.0

5.0e-1

1.0

1.5

2.0

2.5


3.25

2.912.041.90 2.20

4.953.68


3.25

2.902.332.031.02

5.99

4.943.684.13 5.37

Wild Type

Mutant “Y” Mutant “Y”

UV (240 nm) MS (ES(+),TIC)

IS

The Yellow Pigment Absent in the “Y”Mutant Fruit Peel is Naringenin Chalcone

Tomato peel

s mixture’Standard Naringenin Chalcone Standard

12

13

13 14

15

Tomato peel

12 – Cinnamic acid13 – Naringenin Chalcone14 – Naringenin15 – Kaempferol

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0

100

%

0

1001: TOF MS ES+

2.35e4273.0797

147.0500

274.0554

275.0623357.0263 583.1375 855.1465629.0711 987.4974 1151.2333

1: TOF MS ES+ 7.90e3273.0789

153.0202

274.0617

357.0297 583.1320 855.1419651.2269 1011.27051060.0111Time5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40

%

0

100

5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40

%

0


TIC1.37e4

6.02

5.75

MixAllStand_b1 1: TOF MS ES+ TIC

8.16e36.02

5.93

5.69

6.106.15

6.466.41

Unknown Mass peak (RT=3.51 min) Detected Only in Wild Type

09-Jan-2006

Time2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0

100

2.00 4.00 6.00 8.00

%

0


621.1300

7.44 8.39


300

5.755.475.073.080.74 7.69 7.868.40


3003.51

2.35 7.375.07 5.47


3003.51

7.503.64 5.48 8.39


3003.51

2.340.76 7.54

Wild Type

Mutant “Y”

Mutant “Y”

Wild Type

Wild Type

Extracted mass chromatogram at

m/z 621.1 Da

Unknown Mass peak Detected in Higher Levels in “Y”

09-Jan-2006

Time1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15

%

0

100

1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15

%

0

100

1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15

%

0

100

1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15

%

0

100

1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15

%

0


265.12.00e3

Area

2.0970


2.00e3Area

2.1067


2.00e3Area

2.1011


2.00e3Area

2.0911


2.00e3Area

2.1010

Wild type

Wild type

Wild-type

Mut. “Y”

Mut. “Y” Extracted mass chromatogram at m/z 265.1 Da

In-depth Analysis of Red Tomato Peel (ES+)Wild-type vs. the “Y” Mutant

MarkerLynxDifferential Markers

- Filtering out non-replicated data- Identifying molecular ions, isotopes, adducts, fragments and passing from Markers to Metabolites- Analysis of differential metabolites and identification

What are the Differentially Expressed Metabolites between WT and “Y” ?

Peak assignment by:- Accurate mass and elemental composition

(MassLynx and natural products database)- MS-MS fragmentation- UV spectrum- Literature - UV & MS spectra databases(e.g. for tomato in PRI)

Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES- Mode

Differential metabolites (ESDifferential metabolites (ES--))

"Y" > WT"Y" > WTWT > "Y"WT > "Y"

11111010RR onlyonly

16161212OO onlyonly

442626Both Both RR & & OO

31314848TotalTotal

Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES+ Mode

Differential metabolites (ES+)Differential metabolites (ES+)

"Y" > WT"Y" > WTWT >"Y"WT >"Y"

2233RR onlyonly

2200OO onlyonly

881616Both Both RR & & OO

12121919TotalTotal

What are the Differentially Expressed Metabolites between WT and “Y” ?

Most mass peaks showing higher levels in WT than in the “Y” mutant are derivatives of Naringenin Chalcone or Naringenin.


Naringenin chalcone

Cinnamate


Anthocyanines

Condensed tannins

Coumarate

Coumaroyl - CoA


Flavonols

Phenylalanine

NaringeninIsom

ers

(iden

tical

mas

s)

Hydroxylated Naringenin & Naringenin- Chalcone

Ilana w t-tom rp 23

Tim e27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

100

27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

100M05983 1: TOF MS ES+

289.07 0.05Da875

26.521270.5037

M05982 1: TOF MS ES+ 289.07 0.05Da

87537.05

289.0717

35.79289.0709

29.95949.2509

28.34580.7736 42.64

273.0382

HPLC-QTOF

“Y”

WT

UV Spectra of Differential Peaks (35.7 min. and 37.05 min.)

nm225 250 275 300 325 350 375 400 425 450 475

AU

0 .0

5.0e-4

1.0e-3

1.5e-3

2.0e-3

AU

0 .0

2.0e-3

4.0e-3

6.0e-3

8.0e-3

1.0e-2

1.126e-2225.6

287.6

M05982 2135 (35.667) Cm (2131:2138-2149:2160) 3: D iode Array 2.195e-3286.6

Peak at 35.7 min

Naringenin

nm250 300 350 400 450 500 550 600

AU

0.0

2.0e-3

4.0e-3A

U

0.0

2.0e-1

4.0e-1

6.0e-16.238e-1366.5740

211.5740

M05982 2209 (36.900) Cm (2209-(2223+2196)) 3: Diode Array 5.444e-3211.5740

376.5740

Naringenin Chalcone

Peak at 37.05 min

Spectra of differential compounds at RT=35.79 min and 37.05 min

RT=37.05 min

RT=35.79 min

Elemental Composition (289.0712)

Elemental composition: C15H12O6 for neutral compound Elemental composition of Naringenin: C15H12O5Difference: OH group instead of H

Suggested Metabolite

MS-MS Fragmentation of m/z 289.07

m/z100 125 150 175 200 225 250 275 300 325

%0

100 274289.0717

163.0419

153.0219

179.0395 271.0564

290.0741

MS-MS fragmentation of m/z 289.0717Eriodictyol (C15H12O6)

Main fragmentations in reference:I. J. M. S. 247 (2005) 93–100

Naringenin and Naringenin ChalconeGlycosides

HPLC-QTOF

“Y”

WT

Ilana Y-tom rp 55

Time28.00 30.00 32.00 34.00

%

0

100

28.00 30.00 32.00 34.00

%

0

100

M05983 1: TOF MS ES+ 435.12 0.10Da

1.41e3

32.79457.2086

28.72929.2811

M05982 1: TOF MS ES+ 435.12 0.10Da

1.41e328.72

435.127430.53

517.1318

29.22565.7620

33.42528.761032.80

457.2038

UV Spectra of Peak at 32.8 min. and Naringenin Chalcone

nm250 300 350 400 450 500 550 600

AU

0.0

1.0e-3

2.0e-3

3.0e-3

AU

0.0

2.0e-1

4.0e-1

6.0e-1

M05982 2544 (42.483) Cm (2544-(2562+2526)) 3: Diode Array 6.774e-1366.6

211.6

M05982 1954 (32.650) Cm (1954-(1965+1944)) 3: Diode Array 3.933e-3366.6211.6

555.6

Peak at 32.8 min

Naringenin Chalcone

MS spectra at RT=28, 30 32 and 33 min

RT=28.7 min

RT=30.5 min

RT=32.8 min

RT=33.4 min

For MS-MS

Exact mass of 435.1273 shows elemental composition of C21H22O10

MS-MS Fragmentation of m/z 435.12 for Peak at RT=32.8

m/z100 150 200 250 300 350 400 450

%

0

100M06007 407 (33.469) Cm (405:411) 2: TOF MSMS 435.13ES+

1.59e3273.0735

153.0214154.0275

435.1260

274.0769

275.0669

436.1255

437.1289

Hexose (m/z 162)

Peak at 32.8 Naringenin Chalcone Naringenin

Chalcone-hexose

Mass Peak at RT=43.40min (>WT)

Selected MassChromatogramm/z 303.08 Da

MS spectra at RT=43.40min TIC chromatogram

Mut MutMut

WT WTWT

Methyl ether of Hydroxylated Naringeninand Naringenin Chalcone

Peaks at 43.4 min and 44.06 min

- Calculated formula is C16H14O6 based on exact mass- Proposed structure for the peak at 43.4 min: Hesperetin or a B-ring positional isomer

MS-MS Fragmentation of m/z 303.08 (43.4 min.)

179153

177

Hesperetin

15.00000000Ilana wt-tom rp 23 msms

m/z100 120 140 160 180 200 220 240 260 280 300 320

%

0

100M06007 505 (43.325) Cm (505:506) 2: TOF MSMS 303.08ES+

369303.0883

153.0225

145.0342

117.0441

177.0581

154.0266179.0368

180.0453 285.0826

304.0926

305.0851

[M+H]+

[M+H-H2O]+

Proposed Scheme of Naringenin & Naringenin Chalcone Hydroxylation, Methylation and Glycosylation

OHOH

OH

OH

O

OH

OOH

OH O

A C

B

Naringenin Chalcone Naringenin

OHOH

OH

OH

O

OH

Hydroxylated Naringenin Chalcone

OHOH

OH O

OHOMe

Methyl ether of Hydroxylated Naringenin Chalcone

OOH

OH O

OHOH

Hydroxylated Naringenine.g. Eriodyctyol or a B-ring isomer

OOH

OH O

OHOMe

Methyl ether of Hydroxylated Naringenine.g.Hesperetin or a B-ring isomer

OOH

OHOH

OH

OH

OO

OH O

Glycosylatione.g. Naringenin glucoside

Glycosylation

Glycosylation

Glycosylation

Glycosylation

Glycosylation


Naringenin chalcone

Cinnamate


Anthocyanines

Condensed tannins

Coumarate

Coumaroyl - CoA


Flavonols

Phenylalanine

Naringenin

high resolution lc-ms data output and analysis · glucose quercetin. spectra components ... rutin +...

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