High Resolution LC-MS Data Output and Analysis

(1)

High Resolution LC

-

MS

Data Output and Analysis

(2)

LC/MS

Sample Introduction Sample Preparation Ions Detection Ions Separation Ionization MS Interface UV Spectra Separation (column) Efficient Gradient

Your

Sample

Result

LC-MS

Data (computer) Today

(3)

Mass 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)

(4)

Mass Spectrometer

4. Structural information on metabolite

* fragmentation pattern

(5)

Alternative Ionization Modes

•

A

tmospheric

P

ressure

I

onization (

API

),

in LC-MS

•

E

lectro

s

pray

I

onisation (

ESI

): polar and semi-polar

•

A

tmospheric

P

ressure

C

hemical

I

onization (

APCI

): less

polar

ES

APCI

lipids

water

(6)

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)

(7)

Mass Analyzers

•

Ion Cyclotron

(FT-ICR-MS)

•

Time of Flight

(TOF)

•

Magnetic Sector

•

Quadrupole Ion Trap

•

Quadrupole

“High Resolution”

Instruments

“Low Resolution”

Instruments

(8)

Mass Analyzers

Mass

Accuracy

<1ppm

•

Ion Cyclotron

(FT-ICR-MS)

•

Time of Flight

(TOF)

3-10ppm

2-5ppm

•

Magnetic Sector

•

Quadrupole Ion Trap

•

Quadrupole

n/a

(9)

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

(10)

Spectra Obtained at Each Scan

MS-sample MS-reference (lock mass)

PDA-sample

(11)

Extracted chromatogram of a Specific Ion

TIC Extracted chromatogram at m/z 611 Extracted chromatogram at m/z 303

(12)

LC/UV/MS Chromatograms of Standard’s Mixture

(UV at 240nm, ES+ and ES-)

1 – Chlorogenic acid 2 – Caffeic acid 3 – Rutin 4 – p-Coumaric 5 – Quercetin-3-glucoside 6 – Ferulic acid 7 – Sinapic acid 8 – Benzoic acid

9 – Morin (Internal Standard) 10 – Tomatin 11 – Quercetin 12 – Cinnamic acid 13 – Naringenin Chalcone 14 – Naringenin 15 – Kaempferol MS (-) 09-Jan-2006 Standard Mix Time 1.50 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: 1.11 3.68 3.63 2.67 2.26 1.66 3.38 3.26 5.43 4.95 4.60 6.13 6.00 5.92

MixAllStand_b1 1: TOF MS ES+ TIC 1.18e4 5.16 3.65 3.50 2.28 1.69 3.28 2.69 3.71 4.98 4.54 6.02 5.45 5.93 6.10 6.46 1 2 3 3 4 5 6 6 7 8 9 11 12 13 1415 1 2 4 5 7 9 10 11 12 13 14 15 UV MS (+) 1 0 -J a n -2 0 0 6 T im e 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 % 9

ILM ixA llS tand-c4 1: T O F M S E S - T IC 8.88e3 6.07 5.99 3.37 2.68 2.2 7 2 .22 3.25 5.4 2 4.9 2 3.68 4.58 3.95 5.91 6.13 6.37 6 .72 6.77 1 2 4 5 6 9 11 12 13 14 15 3 7 8

(13)

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)

(14)

Chromatogram and Spectrum of Morin

ES(+)

ES(-) [M-H]

-[M+H]+

Chromatograms

(15)

Adduct Formation in Spectra (Na)

[M+H]+ [M+Na]+

Rutin C

₂₇

H

₃₀

O

₁₆ Na (22) C₂₇H₃₁O₁₆ C₂₇H₃₀O₁₆Na

(16)

S5

Typical Adducts formed in LCMS

Mass of Adduct Ion Source of Adduct Adducts Ion M+59 Acetic acid [ M+CH₃COO ] -M+35 Chlorinated solvent [ M+Cl ] -M+18 Ammonia [ M+NH₄] + M+23 Sodium Salt [ M+Na ]+ M+39 Potassium Salt [ M+K ]+ M+42 Acetonitrile [ M+CH₃CNH ]+ M+33 Methanol [ M+CH₃OHH ]+ M+19 Water [ M+H₂O]+

(17)

Isotopes in Spectra (

13

C)

[M+H]+ [M+Na]+

Rutin C

₂₇

H

₃₀

O

₁₆ C₂₇H₃₁O₁₆ C₂₇H₃₀O₁₆Na

(18)

(19)

An example of how isotopes can aid

in peak

identification

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.

(20)

Fragments of a Metabolite in Spectra

[M+H]+

[M+Na]+

Glucose (162)

Rhamnose (146)

MS spectrum of Rutin in ES(+)

Rhamnose

Glucose Quercetin

(21)

Spectra Components

Molecular Ion

Fragments of Metabolite

Adducts (from solvent or di

-

tri

-

mers)

(22)

Spectrum of Quercetin Trisaccharide

(a Rutin Derivative)

Rutin Quercetin [M+H]+ [M+Na]+ m/z 100 200 300 400 500 600 700 800 900 1000 1100 % 0 100 765.1812 743.1995 303.0515 76.0834 469.1320 391.1980 _611.1645 470.1434 471.1271 612.1766 613.1899 766.1797 767.1795 827.1555 828.1354 1133.4736 743

loss of pentose residue 743.19 – 611.16 = 132.03

UV spectra of Rutin and Quercetin Trisaccharide

Rutin

(23)

Mass Accuracy and Structure Elucidation

High mass accuracy

(24)

Mass Accuracy and Structure Elucidation

Selected natural compounds

with formula C

₁₈

H

₁₉

NO

(monoisotopic mass

313.1314 Da)

(25)

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

(26)

Output Data For High Res. LC-MS Based

Metabolomics

Accurate Nominal

(27)

Software for comparing full-scan datasets

MetAlign method

Peak picking

Alignment

(28)

Software for comparing full-scan datasets

MarkerLynx method

(29)

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)

(30)

A Case Study in Tomato

Small Green Big Green Middle Green

(31)

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 (-) ]

(32)

Experimental Set-Up for LC-MS Analysis:

PEEL

and

FLESH

during tomato fruit development

Peel Flesh ES-ES+ ES-ES+ Peel Flesh ES-ES+ ES-ES+

Green

stage

Breaker

stage

Orange

stage

Red

stage

(33)

UPLC-Q-Tof-MS Analysis of Four

Stages of Tomato Fruit Development

09-Jan-2006 Time 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 1.5 2.00 4.00 6.00 8.00 AU 0.0 5.0e-1 1.0 1.5

Tom_24_WTa7 3: Diode Array Range: 4.306 5.98 0.80 3.25 2.91 2.26 2.04 4.94 3.68 4.13 _5.37

Tom_22_WTa6 3: Diode Array

Range: 6.418 5.97 0.80 3.24 2.25 1.02 2.90 4.94 3.674.21 5.36

Tom_20_WTa5 3: Diode Array

Range: 2.955 0.80 5.99 3.25 2.91 2.26 1.02 4.95 3.68 4.13 7.67

Tom_18_WTa4 3: Diode Array Range: 3.2 0.80 3.26 2.26 1.02 2.91 4.95 3.68 7.67 09-Jan-2006 Time 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 % 0 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 % 0 100

Tom_24_WTa7 1: TOF MS ES+ TIC 5.00e4 6.01 0.84 0.70 1.09 5.17 1.81 2.40 3.78 4.23 8.33

Tom_22_WTa6 1: TOF MS ES+

TIC 5.00e4 6.00 0.85 0.70 1.09 5.16 2.40 1.81 3.263.784.23 7.71

TIC 5.00e4 5.13 5.09 0.85 0.70 _1.09 _4.22 3.78 2.40_2.59 5.00 6.02 7.39_7.69_7.86

TIC 5.00e4 5.13 0.84 0.70 1.09 5.00 1.82 2.41 3.28 3.78 6.136.517.387.697.94 UV (240 nm) _{MS (ES(+),TIC)} Red Red Orange Breaker Green Orange Breaker Green

(34)

Out-put from the MarkerLynx (Waters) Program :

(35)

Peel-Flesh Profiles During Fruit Development

Green peel Breaker peel

Breaker flesh

Green flesh

Red peel Orange peel Orange flesh

Red flesh

Component 2

(36)

Wild-type Tomato Peel, Different Developmental Stages

09-Jan-2006 Time 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 % 0 100 1.00 2.00 3.00 4.00 % 0 100

Tom_24_WTa7 1: TOF MS ES+ 303 1000 Area 3.27 42 2.93 8

Tom_18_WTa4 1: TOF MS ES+ 303 1000 Area 3.27 35 2.93 7 09-Jan-2006 Time 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 % 0 100

Tom_24_WTa7 1: TOF MS ES+ 273 2.00e4 Area

6.01 731

6.00 1158

6.03 357

Tom_18_WTa4 1: TOF MS ES+ 273 2.00e4 Area 09-Jan-2006 Time 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 % 0 100

Tom_24_WTa7 1: TOF MS ES+ 578.4 1.00e4 Area 5.17 48 4.31 3 3.63 1

Tom_22_WTa6 1: TOF MS ES+ 578.4 1.00e4 Area 5.16 90 4.31 4

Tom_20_WTa5 1: TOF MS ES+ 578.4 1.00e4 Area 5.13 362 5.07 8

Tom_18_WTa4 1: TOF MS ES+ 578.4 1.00e4 Area 5.12 509 5.24 10 Red Orange Breaker Green

Tomatine Rutin & Quercetin trisacharide Naringenin chalcone

(37)

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

(38)

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

(39)

Experimental Set-Up for LC-MS Analysis:

PEEL

and

FLESH

during “Y” fruit development

“Y”

Breaker

stage

“Y”

Green

stage

“Y”

Orange

stage

“Y”

Red

stage

(40)

HPLC

-

QTOF

-

MS (Ultima

-

PRI)

-

-Wild Type and

Wild Type and

“

Y

”

Peels

WT-Orange “Y”-Red WT-Red “Y”-Orange All breaker All green 1st _{principal component (X axis) - developmental stage (47.1%)}

(41)

UV and MS Chromatograms of Red

Tomato Peel (Wild Type and Mutant “Y”)

09-Jan-2006 Time 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

TIC 2.11e4 0.84 0.70 0.89 1.09 1.81 5.18 2.40 3.78 3.64 2.60 4.24 4.98 4.86 8.02 7.95 5.27 5.75 8.31

TIC 2.12e4 0.85 0.70 6.02 1.81 1.09 5.16 2.41 3.283.62 2.94 3.77 4.23 4.98 5.43 7.90 7.63 6.46 8.32 Wild Type 09-Jan-2006 Time 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 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

Tom_56_WTa15 3: Diode Array Range: 2.916 0.80 3.25 2.91 2.04 1.90 2.20 4.95 3.68

Tom_8_WTa3 3: Diode Array Range: 3.022 0.80 3.25 2.90 2.33 2.03 1.02 5.99 4.94 3.684.13 5.37 Wild Type

Mutant “Y” Mutant “Y”

UV (240 nm)

MS (ES(+),TIC)

(42)

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 acid 13 – Naringenin Chalcone 14 – Naringenin 15 – Kaempferol m/z 100 200 300 400 500 600 700 800 900 1000 1100 % 0 100 % 0 100 1: TOF MS ES+ 2.35e4 273.0797 147.0500 274.0554 275.0623 357.0263 583.1375629.0711 855.1465 987.4974 1151.2333 1: TOF MS ES+ 7.90e3 273.0789 153.0202 274.0617 357.0297 583.1320651.2269 855.1419 1011.27051060.0111 Time 5.70 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 % 0 100

Tom_8_WTa3 1: TOF MS ES+ TIC 1.37e4

6.02

5.75

MixAllStand_b1 1: TOF MS ES+ TIC 8.16e3 6.02 5.93 5.69 6.10 6.15 6.46 6.41

(43)

Unknown Mass peak (RT=3.51 min)

Detected Only in Wild Type

09-Jan-2006 Time 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 % 0 100 2.00 4.00 6.00 8.00 % 0 100

Tom_72_WTa19 1: TOF MS ES+ 621.1 300

7.44 8.39

Tom_56_WTa15 1: TOF MS ES+ 621.1 300 5.75 5.47 5.07 3.08 0.74 7.69 7.868.40

621.1 300

3.51

2.35 5.075.47 7.37

Tom_24_WTa7 1: TOF MS ES+ 621.1 300 3.51 7.50 3.64 5.48 8.39

Tom_8_WTa3 1: TOF MS ES+ 621.1 300 3.51 2.34 0.76 7.54 Wild Type Mutant “Y” Mutant “Y” Wild Type Wild Type

Extracted mass

chromatogram at

m/z 621.1 Da

(44)

Unknown Mass peak Detected in

Higher Levels in “Y”

09-Jan-2006 Time 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 % 0 100 1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15 % 0 100

Tom_72_WTa19 1: TOF MS ES+ 265.1 2.00e3 Area

2.09 70

2.10 67

2.10 11

2.09 11

Tom_8_WTa3 1: TOF MS ES+ 265.1 2.00e3 Area 2.10 10 Wild type Wild type Wild-type Mut. “Y”

Mut. “Y”

Extracted mass

chromatogram at

m/z 265.1 Da

(45)

In-depth Analysis of Red Tomato Peel (ES+)

Wild-type vs. the “Y” Mutant

MarkerLynx

Differential Markers

- Filtering out non-replicated data

- Identifying molecular ions, isotopes, adducts,

fragments and passing from Markers to Metabolites

(46)

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

(47)

Differential Metabolites between the "Y" and

Wild Type Peel UPLC-QTOF-MS in ES- Mode

Differential metabolites (ES

-

)

"Y" > WT

WT > "Y"

11

10

R

only

16

12

O

only

4

26

Both

R

&

O

31

48

Total

(48)

Differential Metabolites between the "Y" and

Wild Type Peel UPLC-QTOF-MS in ES+ Mode

Differential metabolites (ES+)

"Y" > WT

WT >"Y"

2

3

R

only

2

0

O

only

8

16

Both

R

&

O

12

19

Total

(49)

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.

(50)

Biosynthesis of Naringenin Chalcone

in the Flavonoid Pathway

Naringenin chalcone

Cinnamate

Anthocyanines

Condensed tannins

Coumarate

Coumaroyl - CoA

Lignins,

Coumarins,

Chlorogenic-acid

Flavonols

Phenylalanine

Naringenin

(51)

Hydroxylated

Naringenin &

Naringenin- Chalcone

Ilana w t-tom rp 23 T im e 27.50 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 % 0 100 M 05983 1: T O F M S E S + 289.07 0.05D a 875 26.52 1270.5037 M 05982 1: T O F M S E S + 289.07 0.05D a 875 37.05 289.0717 35.79 289.0709 29.95 949.2509 28.34 580.7736 _42.64 273.0382

HPLC-QTOF

“Y”

WT

(52)

UV Spectra of Differential Peaks

(35.7 min. and 37.05 min.)

n m 2 2 5 2 5 0 2 7 5 3 0 0 3 2 5 3 5 0 3 7 5 4 0 0 4 2 5 4 5 0 4 7 5 AU 0 .0 5 .0 e -4 1 .0 e -3 1 .5 e -3 2 .0 e -3 AU 0 .0 2 .0 e -3 4 .0 e -3 6 .0 e -3 8 .0 e -3 1 .0 e -2 1 .1 2 6 e -2 2 25 .6 2 8 7 .6 M 0 5 9 8 2 2 1 3 5 (3 5 .6 6 7 ) C m (2 1 3 1 :2 1 3 8 -2 1 4 9 :2 1 6 0 ) 3 : D io d e A rra y 2 .1 9 5 e -3 2 86 .6

Peak at

35.7 min

Naringenin

nm 250 300 350 400 450 500 550 600 AU 0.0 2.0e-3 4.0e-3 AU 0.0 2.0e-1 4.0e-1 6.0e-1 6.238e-1 366.5740 211.5740 M05982 2209 (36.900) Cm (2209-(2223+2196)) 3: Diode Array 5.444e-3 211.5740 376.5740

Naringenin

Chalcone

Peak at

37.05 min

(53)

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

RT=37.05 min RT=35.79 min

(54)

Elemental Composition (289.0712)

Elemental composition:

C

₁₅

H

₁₂

O

₆

for neutral compound

Elemental composition of Naringenin:

C

₁₅

H

₁₂

O

₅

(55)

Suggested Metabolite

MS-MS Fragmentation of m/z 289.07

m/z 100 125 150 175 200 225 250 275 300 325 % 0 100 289.0717 274 163.0419 153.0219 179.0395 _271.0564 290.0741

MS-MS fragmentation

of m/z 289.0717

Eriodictyol

(C

₁₅

H

₁₂

O

₆

)

Main fragmentations in reference:

I. J. M. S. 247 (2005) 93–100

(56)

Naringenin and Naringenin Chalcone

Glycosides

HPLC-QTOF

“Y”

WT

Ilana Y-tom rp 55 Time 28.00 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 32.79 457.2086 28.72 929.2811 M05982 1: TOF MS ES+ 435.12 0.10Da 1.41e3 28.72 435.1274 30.53 517.1318 29.22 565.7620 33.42 528.7610 32.80 457.2038

(57)

UV Spectra of Peak at 32.8 min.

and Naringenin Chalcone

nm 250 300 350 400 450 500 550 600 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-1 366.6 211.6 M05982 1954 (32.650) Cm (1954-(1965+1944)) 3: Diode Array 3.933e-3 366.6 211.6 555.6

Peak at 32.8 min

Naringenin Chalcone

(58)

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

(59)

MS-MS Fragmentation of m/z 435.12 for

Peak at RT=32.8

m/z 100 150 200 250 300 350 400 450 % 0 100 M06007 407 (33.469) Cm (405:411) 2: TOF MSMS 435.13ES+ 1.59e3 273.0735 153.0214 154.0275 435.1260 274.0769 275.0669 436.1255 437.1289

Hexose (m/z 162)

Peak at 32.8

Naringenin

Chalcone

Naringenin

Chalcone-hexose

(60)

Mass Peak at RT=43.40min (>WT)

Selected Mass Chromatogram m/z 303.08 Da

MS spectra at RT=43.40min _{TIC chromatogram}

Mut _Mut Mut

(61)

Methyl ether of Hydroxylated Naringenin

and 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

(62)

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

179 153 177

Hesperetin

15.00000000 Ilana wt-tom rp 23 msms m/z 100 120 140 160 180 200 220 240 260 280 300 320 % 0 100 M06007 505 (43.325) Cm (505:506) 2: TOF MSMS 303.08ES+ 369 303.0883 153.0225 145.0342 117.0441 177.0581 154.0266 179.0368 180.0453 285.0826 304.0926 305.0851 [M+H]+ [M+H-H₂O]+

(63)

Proposed Scheme of Naringenin & Naringenin Chalcone

Hydroxylation, Methylation and Glycosylation

OH O H OH OH O OH O O H OH O A C B

Naringenin Chalcone Naringenin

OH O H OH OH O OH

Hydroxylated Naringenin Chalcone

OH O H OH O OH OMe

Methyl ether of Hydroxylated Naringenin Chalcone

O O H OH O OH OH Hydroxylated Naringenin

e.g. Eriodyctyol or a B-ring isomer

O O H OH O OH OMe

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

O OH OH OH OH OH O O OH O Glycosylation

e.g. Naringenin glucoside Glycosylation

Glycosylation

(64)

Biosynthesis of Naringenin Chalcone

in the Flavonoid Pathway

Naringenin chalcone

Cinnamate

Anthocyanines

Condensed tannins

Coumarate

Coumaroyl - CoA

Lignins,

Coumarins,

Chlorogenic-acid

Flavonols

Phenylalanine

Naringenin