Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted
Nutraceutical Applications
Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted
Nutraceutical Applications
F. William CollinsF. William Collins
Eastern Cereals and Oilseeds Research CentreOttawa, ON.
Eastern Cereals and Oilseeds Research CentreOttawa, ON.
8th IOC Minneapolis, MN, June 20088th IOC Minneapolis, MN, June 2008
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OVERVIEWOVERVIEW
Group separation for reproducibility Group separation for reproducibility
2-D HPLC-MSMS techniques to map total avenanthramide diversity
2-D HPLC-MSMS techniques to map total avenanthramide diversity
Co-extracted compounds and their effects on quantitation
Co-extracted compounds and their effects on quantitation
UV Spectral properties of avenanthramides UV Spectral properties of avenanthramides
Need for improved methods of analysis Need for improved methods of analysis
22 Purification, Analysis, and Structures of Avenanthramides from Oats
Purification, Analysis, and Structures of Avenanthramides from Oats
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NEED FOR IMPROVED METHODSNEED FOR IMPROVED METHODS
Avenanthramide components are complex mixture of over 30 individual members
Avenanthramide components are complex mixture of over 30 individual members
Several other groups of compounds in oat extracts can interfere with UV-based HPLC estimation of avenanthramides
Several other groups of compounds in oat extracts can interfere with UV-based HPLC estimation of avenanthramides
Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more
accurate/robust analytical techniques
Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more
accurate/robust analytical techniques
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Purification, Analysis, and Structures of Avenanthramides from Oats
Purification, Analysis, and Structures of Avenanthramides from Oats
1. Stabilization and extraction with aqueous EtOH1. Stabilization and extraction with aqueous EtOH
SUMMARY OF PROCEDURESUMMARY OF PROCEDURE
2. Removal of lipophilic phenolics and prolamines using hydrophobic binding to Octyl
Sepharose
2. Removal of lipophilic phenolics and prolamines using hydrophobic binding to Octyl
Sepharose 3. Removal of flavonoids glycosides and avenacins
using aromatic absorption on Sephadex LH-203. Removal of flavonoids glycosides and avenacins
using aromatic absorption on Sephadex LH-20
4. Concentration of avenanthramides 4. Concentration of avenanthramides
5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A
5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A
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Anthranilic acid (2-aminobenzoic)
= AVENANTHRAMIDE: TYPE I
p-Coumaric acid (4-hydroxycinnamic)+
2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported
1. Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported
OH
NH
O
OHO
3
4
5
6
2'
3'
5'
6'
7'
8'
THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS…THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS…
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= AVENANTHRAMIDE: TYPE 2
Anthranilic acid (2-aminobenzoic)
Avenalumic acid 5-(4-hydroxyphenyl)-2,4-pentadienoic acid
+
1. Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported
…..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID
…..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID
2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported
9'
10' 8'
7'
OH
NH
O
OHO
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10 g ground oats
1. 75 mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH
Stir for 20 min with ambient cooling and decant into graduated glass column
Allow to settle and pack by gravity to form a percolation extraction bed, Vb
Procedure is scaleable
Denatures enzymes (e.g. peroxidases, esterases, etc)
Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
HPLC evaluation at this point, detection at 330nm
Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent
Drain extract and percolate a further 3xVb fresh solvent to give a “percolate”
Percolation is an extremely efficient extraction method
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Avenacosylate peaks (in part)
“Prolamine” peaks (in part)“Glycolipid” peaks (in part)“Phosphatide” peaks (in part)
Avenanthramide zone
HPLC Profiles During Group Separation Of Avenanthramides...
HPLC Profiles During Group Separation Of Avenanthramides...
VAO 48(hulless)
Crude EtOH extract
5 10 15 20 25 30 35 40 45 50 55 60 65
20
40
60
0
80
100
120 OD 330nm(mAU)
Time (min)
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There are 3 major classes of avenacosylates in oats
O
HO
ORO
O
HO
OR1O O
OH
OOR2
R = CH3; 1-O-feruloyl-n-hexacosan-1-ol
R = H; 1-O-caffeoyl-n-hexacosan-1-ol
(C26 alcohol)
(C26 alcohol)
R1 = R2 = CH3; 1,26-di-O-feruloyl-n- hexacosan-1,26-diol
(C26 α,ω -diol)
R1 = H; 1-O caffeoyl-26-O-feruloyl-n- R2 = CH3 hexacosan-1,26-diol
(C26 α,ω -diol)
R1 = R2 = CH3; 1,26-di-O-caffeoyl-n- hexacosan-1,26-diol
(C26 α,ω -diol)
O
HO
OR2O
O
O
O
OH
OOH
O
HO
OR1O
R1 = CH3; 1-O caffeoyl-2-O-feruloyl-3-O-(26'-O- R2 = OH caffeoyl)-n-hexacosanoyl-D-glycerol
AVENACOSYLATES: UV Interfering Components from Oats
AVENACOSYLATES: UV Interfering Components from Oats
Hydroxy-cinnamoyl esters of n-
alkanols
Hydroxy-cinnamoyl esters of n-alkan-
α,ω-diols
Mixed hydroxy-cinnamoyl esters of
ω-hydroxy-n-alkanoyl glycerides
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10 g ground oats
1. 75 mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH
Stir for 20 min with ambient cooling and decant into graduated glass column
Allow to settle and pack by gravity to form a percolation extraction bed, Vb
Drain extract and percolate a further 3xVb fresh solvent to give a “percolate”
Procedure is scaleable
Denatures enzymes (e.g. peroxidases, esterases, etc)
Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties
Percolation is an extremely efficient extraction method
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...1010
Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent
Collins et al, US Patent #6,495,140 (2002)
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2. Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% EtOH)
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
Prolamines and lipophilic phenolics are bound to the beads in 50% EtOH
All avenanthramides and hydrophilic components are not bound to Octyl Sepharose in 50% EtOH
3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH
Elute with 2xVb acidified 40% EtOH to remove polar components
All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
HPLC evaluation at this point, detection at 330nm
1111
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Avenanthramide zone
VAO 48(hulless)
Crude EtOH extract after removal of lipophilics
“Prolamine” peaks (in part)“flavone glycosides”
luteolin aglycone apigenin aglycone
tricin aglycone
5 10 15 20 25 30 35 40 45 50 55 60 65
20
40
60
0
80
100
120 OD 330nm(mAU)
Time (min)
1212 HPLC Profiles During Group Separation Of Avenanthramides...
HPLC Profiles During Group Separation Of Avenanthramides...
“Avenacins”
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Other UV Absorbing Components from Oats: The Avenacins
Other UV Absorbing Components from Oats: The Avenacins
O
O
OH
OHH
O
H
O
O
HO
O
O
OHHO
HO
HO
O
OHHO
HOHO
R
O HNCH3
OR =
Avenacin A-1
O
OR =
Avenacin A-2
R
O
O
OH
O
H3C H
O
H
O
O
HO
O
O
OHHO
HO
HO
O
OHHO
HOHO O HN
CH3
O
Avenacin B-1
R =
O
O
Avenacin B-2
R =
1313
Triterpene saponins with a chromophoric group with UV
absorption max at ~ 325 nm (A-1, B-1)
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O
OH
OH
HO
O
4',5,7-(OH)4-flavone(Apigenin)
O
OH
OH
HO
O
OH
3',4',5,7-(OH)4-flavone(Luteolin)
O
OH
OH
HO
O
4',5,7'-(OH)3-3',5'-(OMe)2-flavone
(Tricin)
OCH3
OCH3
O
OH
OH
H3CO
O
O
HOHOHO
H3C
OOH
HO
OHO
Isoswertisin-2"-O-rhamnoside(Apigenin-8-C-(2"-O-α-L-rhamno-pyranosyl)-β-D-glucoside-7-O-
methyl ether)
O
OHHO
HOHO O
OH
OH
HO
O
IsovitexinApigenin-6-C-β-D-glucoside
1414 Other UV Absorbing Components from Oats: Flavones
Other UV Absorbing Components from Oats: Flavones
• UV absorption spectral maxima similar to those of avenanthramides
• Chromatographic properties overlap with avenanthramides
• However, flavone derivatives appear to be minor components in oat groat tissues (~5%?)
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2. Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% EtOH)
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH
Elute with 2xVb acidified 40% EtOH to remove polar components
Prolamines and lipophilic phenolics are bound to the beads in 50% EtOH
All avenanthramides and hydrophilic components are not bound to Octyl Sepharose in 50% EtOH
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
HPLC analysis to look at profile
1515
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2. Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column (pre-equil. in acidified 50% EtOH)
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH
Elute with 2xVb acidified 40% EtOH to remove polar components
All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
All avenanthramides and hydrophilic components are not bound to Octyl Sepharose in 50% EtOH
HPLC analysis to look at profile
1616
Prolamines and lipophilic phenolics are bound to the beads in 50% EtOH
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Recover avenanthramides by eluting column with 3xVb 95% EtOH
4. Evaporate eluate to dryness, take up in known volume of 50% EtOH (~ 2-5 mL),
pass sub-sample through 0.45μ filter
HPLC-rapid scan (“diode array”)
HPLC-diode array-MSMS
Can also use different HPLC column:solvent configurations
All avenanthramides are not bound to Sephadex LH-20 in 95% EtOH
Procedure results in base-line resolution of all the avenanthramides and can be easily scaled up to gram quantities
Molecular Weight Range scanned between 100-700daltons
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...
EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...1717
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Avenanthramide zone
VAO 48(hulless)
5 10 15 20 25 30 35 40 45 50 55 60 65
20
40
60
0
80
100
120 OD 330nm(mAU)
Time (min)
1818 HPLC Profiles During Group Separation Of Avenanthramides...
HPLC Profiles During Group Separation Of Avenanthramides...
Final purified avenanthramide
extract
Flat base line for ease of
integration
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1919
Avenanthramide zone
After LH-20
After Octyl Sepharose
Crude EtOH extract
Comparison of HPLC Profiles During Separation Of Avenanthramides...
Comparison of HPLC Profiles During Separation Of Avenanthramides...
5 10 15 20 25 30 35 40 45 50 55 60 65
20
40
60
0
80
100
120
Time (min)
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5 10 15 20 25 30 35 40 45 50 55 60 65
20
40
60
0
80
100
120 OD 330nm(mAU)
VAO 48(hulless)
Time (min)
2020 Spectral analyses of AvenanthramidesSpectral analyses of Avenanthramides
High degree of peak
resolution facilitates spectral
analysis and identification
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OD 330nm(mAU)
Retention Time (min)
250
300
350
275
325
375
λ(nm)
400
300
200
100
0
(mAU)
BOH
OCH3NH
O
OHO
HO
m/z = 329
2121 Spectral analyses of avenanthramidesSpectral analyses of avenanthramides
(mAU)
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OD 330nm(mAU)
(mAU)
2222 Spectral analyses of avenanthramidesSpectral analyses of avenanthramides
OH
NH
O
OHO
HO
OCH3
m/z = 355
P
Retention Time (min)
250
300
350
275
325
375
λ(nm)
400
300
200
100
0
(mAU)
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BOH
OCH3NH
O
OHO
HO
m/z = 329
OH
NH
O
OHO
HO
OCH3
m/z = 355
P
2323
300s
335 350s
Spectral analyses of avenanthramidesSpectral analyses of avenanthramides
All Type I avenanthramides show a shoulder between 290
and 310nm
All Type II avenanthramides lack this shoulder
360340s
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Time A(%) B(%) C(%) Flow rate
0 40 55 5 0.8 mL/min 80 60 35 5 0.8 mL/min 85 80 15 5 0.8
mL/min 88 100 0 0 0.8 mL/min 91 100 0 0 0.8 mL/min 94 40 55 5 0.8 mL/min105 40 55 5 0.8
mL/min
Hypersil® C18 5μ column:
SolventsA: MethanolB: H2OC: 5% Acetic acid
Gradient
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
2424
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Time A(%) B(%) C(%) Flow rate
0 55 40 5 0.8 mL/min
96 95 0 5 0.8 mL/min 100 95 0 5 0.8 mL/min 105 55 40 5 0.8
mL/min 115 55 40 5 0.8
mL/min
Polaris® Amide C18 5μ column:
SolventsA: MethanolB: H2OC: 5% Formic acid
Gradient
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
2525
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HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
Thermo Finnigan LCQ Advantage® with DA detectorMode: electrospray ionization neg. modeSource voltage: 4.5 KvoltsCapillary voltage: -10vCapillary temp: 300C Sheath gas flow = 80% fullAux gas flow: 20% max. (no stream splitting)
MASS SPEC-MASS SPEC:(MS-MS)
INSTRUMENTATIONINSTRUMENTATION HPLC:
Thermo Separation Products® (Thermo Finnigan)
P4000 pumpSN 4000 ControllerSpectraSystem UV3000 detector UV 250-400nm; Chromquest software
2626
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OD 330nm(mAU)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
0 30 4020 5010 60 70 80Retention Time (min)
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
More than 30 peaks
detectected
AC Baton(hulless)
2727
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OD 330nm(mAU)
0
100
200
300
400
500
600
700
800
900
1000
0 30 4020 5010 60 70 80Retention Time (min)
90 100
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES
More than 20 peaks
detectected
AC Baton(hulless)
2828
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Parent ion [M-1]-
nn
-
RRRR
RR
OH
NH
O
OO
KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m2
KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m2
Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide
Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide
MS parameters optimized for
generation of [M-1] -
MS parameters optimized for
generation of [M-1] -
Daughter m2 - gives
molecular weight of both ring moieties
Daughter m2 - gives
molecular weight of both ring moieties
RR
Daughter ion m1 -
N
O
OO
-
RRDaughter m1
- gives molecular weight of the anthranilic moiety
Daughter m1 - gives
molecular weight of the anthranilic moiety
Parent ion [M-1]-
-
RRRR
nnRR
OH
NH
O
OO
Daughter ion m2 -
nn
RR
-
RR
RR
OH
NH
O
2929
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OH
NH
O
OHO
R
DD EE FF
OH
NH
O
OHO
HO
RAA BB CC
OH
NH
O
OHO
R
OH
GG HH KK
OH
NH
O
OHO
HO
R
OH
AAAA BBBB CCCC
OH
NH
O
OHO
HO
R
OCH3
XX YY ZZ
R = HR = H R = OCH3R = OCH3 R = OHR = OH
STRUCTURES OF TYPE I AVENANTHRAMIDES
STRUCTURES OF TYPE I AVENANTHRAMIDES
3030
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Rt (min) Amide
Rt (min) C18
0
0
20
20
40
40
60
60 80
80
100
STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES
STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES
DDEEFF
BBCCAA
KKGG
HHCCCC
AAAA BBBB
TYPE 1 AVENANTHRAMIDESSUMMARY
5 Different anthranilic acid amides with either p-coumaric, ferulic, or caffeic acids
SUMMARY
5 Different anthranilic acid amides with either p-coumaric, ferulic, or caffeic acids
5-OH anthranilic
4-OH anthranilic
anthranilic
4,5-di(OH) anthranilic
ZZXX YY
4-OH,5-OMe anthranilic
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OH
NH
O
OHO
R
R = HR = H R = OCH3R = OCH3 R = OHR = OH
LL MM NN
OH
NH
O
OHO
R
HO
OO PP QQ
OH
NH
O
OHO
R
OH
RR SS TT
OH
NH
O
OHO
R
OH
HO
OOOO PPPP QQQQ
OH
NH
O
OHO
R
OCH3
HO
UU VV WW
STRUCTURES OF TYPE II AVENANTHRAMIDES
STRUCTURES OF TYPE II AVENANTHRAMIDES
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Rt (min) Amide
Rt (min) C18
0
0
20
20
40
40
60
60 80
80
100
STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES
STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES
OOPP
MMNN
LL
QQQQOOOO
PPPP
4,5-di(OH) anthranilic
WWUU
VV
SSRRTT
SUMMARY
5 Different anthranilic acid amides with either avenalumic, 3’-OMe-avenalumic, or 3’-OH-avenalumic acids
TYPE 2 AVENANTHRAMIDESanthranilic
5-OH anthranilic
4-OH anthranilic
4-OH,5-OMe anthranilic
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Rt (min) Amide
Rt (min) C18
0
0
20
20
40
40
60
60 80
80
100
KK
GG
HH
AA
BBCC
DD
EEFF
LL
MMNN
CCCCAAAA
BBBB
XX
YY
ZZ
OO
PPQQ
QQQQ
OOOOPPPP
VV
UU
WW
RR
SSTT
2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES
2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES
Based on:1) Rt in 2 solvents2) UV spectra3) MS pattern
Based on:1) Rt in 2 solvents2) UV spectra3) MS pattern
There are about 30 different avenanthramides not including dimeric forms
There are about 30 different avenanthramides not including dimeric forms
Each one identified by an alphabetic designation
Each one identified by an alphabetic designation
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Rt (min) Amide
Rt (min) C18
0
0
20
20
40
40
60
60 80
80
100
OD (AU)
0
0.5
1.0
1.5
2.0
2.5 Relative quantitative estimations:
Relative quantitative estimations:
Major Avenanthramides
Major Avenanthramides
A, B, CA, B, C
O, P, QO, P, Q
OO, PP, QQOO, PP, QQ
AA, BB, CCAA, BB, CC
AC Baton(hulless)
RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE…..
RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE…..
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CO
NC
EN
TR
AT
ION
IN
PP
M
0
50
100
200
300
400
500
VARIETIES / LINES AV AAV BAV C TOTAL
AV AAV BAV C TOTAL
3636 GENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELSGENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELS
Strong genotype x environment interaction as wellStrong genotype x environment interaction as well
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TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS….
TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS….
AAFCAAFC
F. William CollinsVern BurrowsWinson OrrDwight ColleyNicole Fillion
F. William CollinsVern BurrowsWinson OrrDwight ColleyNicole Fillion
Synthesis ,GMP Purification, Analytical,Germplasm
USDA TUFTS UNIVERSITYUSDA TUFTS UNIVERSITY
Mohsen MedaniLiping LiuLigia Zubik
Melissa MarkoWeimin Guo
Mohsen MedaniLiping LiuLigia Zubik
Melissa MarkoWeimin Guo
Vascular Biology Lab:In vitro studies
Jeffrey BlumbergPaul MilburyChung-Yen ChenTing LiJennifer O’Leary
Jeffrey BlumbergPaul MilburyChung-Yen ChenTing LiJennifer O’Leary
Antioxidant Research Lab:In vivo studies
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