1. Purification, Analysis, and Structures of Avenanthramides from Oats: Unique Components for Targeted Nutraceutical Applications
F. William Collins
Eastern Cereals and Oilseeds Research Centre
Ottawa, ON.
8th IOC Minneapolis, MN, June 2008

2. Purification, Analysis, and Structures of Avenanthramides from Oats2
OVERVIEW
 Need for improved methods of analysis
 Group separation for reproducibility
 Co-extracted compounds and their effects on quantitation
 UV Spectral properties of avenanthramides
 2-D HPLC-MSMS techniques to map total avenanthramide diversity

3. NEED FOR IMPROVED METHODS3
NEED FOR IMPROVED METHODS
 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
 Bioactivity and bioavailability studies leading to possible health claims for avenanthramides in future oat products will require more accurate/robust analytical techniques

4. Purification, Analysis, and Structures of Avenanthramides from Oats4
SUMMARY OF PROCEDURE
Stabilization and extraction with aqueous EtOH
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-20
4. Concentration of avenanthramides
5. HPLC-PDA UV spectrometric standardization and quantization using avenanthramide A

5. THERE ARE TWO TYPES OF AVENANTHRAMIDES IN OATS…5
Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported
2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported
Anthranilic acid (2-aminobenzoic)
p-Coumaric acid (4-hydroxycinnamic) +
= AVENANTHRAMIDE: TYPE I

6. …..BASED ON THE CHAIN LENGTH OF THE CONJUGATING ACID6
Substitution with hydroxyl and or methoxy functions at C-4 and/or C-5 positions have been reported
Avenalumic acid
5-(4-hydroxyphenyl)-2,4-pentadienoic acid + 9'
10' 8' 7'
2. Substitution with hydroxyl and or methoxy functions at C-3’ have also been reported
Anthranilic acid (2-aminobenzoic)
= AVENANTHRAMIDE: TYPE 2

7. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...7
10 g ground oats
Procedure is scaleable
mL boiling, stirred, acidified (0.1% HOAc) 80% EtOH
Denatures enzymes (e.g. peroxidases, esterases, etc)
Stir for 20 min with ambient cooling and decant into graduated glass column
Cooling and sedimenting aids coalescing of insoluble and oil phases and forms bed with good flow-through properties
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”
Percolation is an extremely efficient extraction method
HPLC evaluation at this point, detection at 330nm
Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent

8. HPLC Profiles During Group Separation Of Avenanthramides...8 5 10 15 20 25 30 35 40 45 50 55 60 65 80
100
120
OD 330nm
(mAU)
Time (min)
VAO 48
(hulless)
Avenacosylate peaks (in part)
Crude EtOH extract
“Prolamine” peaks (in part)
“Glycolipid” peaks (in part)
“Phosphatide” peaks (in part)
Avenanthramide zone

9. AVENACOSYLATES: UV Interfering Components from Oats9
There are 3 major classes of avenacosylates in 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

10. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...10
10 g ground oats
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
Add ~ 3 mL of Octyl-Sepharose beads to percolate and evaporate solvent
Collins et al, US Patent #6,495,140 (2002)

11. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...11
Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column
(pre-equil. in acidified 50% 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
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
HPLC evaluation at this point, detection at 330nm
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

12. HPLC Profiles During Group Separation Of Avenanthramides...12 5 10 15 20 25 30 35 40 45 50 55 60 65 80
100
120
OD 330nm
(mAU)
Time (min)
VAO 48
(hulless)
Crude EtOH extract after removal of lipophilics
“flavone glycosides”
“Prolamine” peaks (in part)
“Avenacins”
Avenanthramide zone
luteolin aglycone
apigenin aglycone
tricin aglycone

13. Other UV Absorbing Components from Oats: The Avenacins13
Triterpene saponins with a chromophoric group with UV absorption max at ~ 325 nm (A-1, B-1)

14. Other UV Absorbing Components from Oats: Flavones14
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%?)

15. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...15
Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column
(pre-equil. in acidified 50% 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
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
HPLC analysis to look at profile
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

16. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...16
Re-suspend in acidified 50% EtOH and transfer with washings to top
of ~ 30 mL Octyl-Sepharose column
(pre-equil. in acidified 50% 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
Elute with 3xVb acidified 50% EtOH
Evaporate eluate to dryness and re-suspend in ~ 2mL acidified 40% EtOH
HPLC analysis to look at profile
3. Transfer with washings to top of a ~ 30 mL Sephadex LH-20 column pre-equilibrated in acidified 40% EtOH
All avenanthramides are bound to Sephadex LH-20 in 40% EtOH: saponins, sugars, amino acids, flavone-glycosides etc are not bound
Elute with 2xVb acidified 40% EtOH to remove polar components

17. EXTRACTION AND GROUP SEPARATION OF AVENANTHRAMIDES...17
All avenanthramides are not bound to Sephadex LH-20 in 95% EtOH
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
Procedure results in base-line resolution of all the avenanthramides and can be easily scaled up to gram quantities
HPLC-rapid scan (“diode array”)
HPLC-diode array-MSMS
Molecular Weight Range scanned between
daltons
Can also use different HPLC column:solvent configurations

18. HPLC Profiles During Group Separation Of Avenanthramides...18 5 10 15 20 25 30 35 40 45 50 55 60 65 80
100
120
OD 330nm
(mAU)
Time (min)
VAO 48
(hulless)
Final purified avenanthramide extract
Avenanthramide zone
Flat base line for ease of integration

19. Comparison of HPLC Profiles During Separation Of Avenanthramides...19
Avenanthramide zone 5 10 15 20 25 30 35 40 45 50 55 60 65 80
100
120
Time (min)
Crude EtOH extract
After Octyl Sepharose
After LH-20

20. Spectral analyses of Avenanthramides20
Spectral analyses of Avenanthramides 5 10 15 20 25 30 35 40 45 50 55 60 65 80
100
120
OD 330nm
(mAU)
VAO 48
(hulless)
Time (min)
High degree of peak resolution facilitates spectral analysis and identification

21. Spectral analyses of avenanthramides21
Spectral analyses of avenanthramides
OD 330nm
(mAU)
(mAU) B
m/z = 329
Retention Time (min)
250
300
350
275
325
375 λ
(nm)
400
200
100
(mAU)

22. Spectral analyses of avenanthramides22
Spectral analyses of avenanthramides
OD 330nm
(mAU)
m/z = 355 P
Retention Time (min)
250
300
350
275
325
375 λ
(nm)
400
200
100
(mAU)

23. Spectral analyses of avenanthramides23
Spectral analyses of avenanthramides
300s B
m/z = 329
335
350s
All Type I avenanthramides show a shoulder between 290 and 310nm
m/z = 355 P
360
340s
All Type II avenanthramides lack this shoulder

24. HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES24
Time A(%) B(%) C(%) Flow rate
mL/min
mL/min
mL/min
mL/min
mL/min
mL/min
mL/min
Hypersil® C18 5μ column:
Solvents
A: Methanol
B: H2O
C: 5% Acetic acid
Gradient

25. HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES25
Time A(%) B(%) C(%) Flow rate
mL/min
mL/min
mL/min
mL/min
mL/min
Polaris® Amide C18 5μ column:
Solvents
A: Methanol
B: H2O
C: 5% Formic acid
Gradient

26. HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES26
INSTRUMENTATION
HPLC:
Thermo Separation Products® (Thermo Finnigan)
P4000 pump
SN 4000 Controller
SpectraSystem UV3000 detector UV nm;
Chromquest software
Thermo Finnigan LCQ Advantage® with DA detector
Mode: electrospray ionization neg. mode
Source voltage: 4.5 Kvolts
Capillary voltage: -10v
Capillary temp: 300C Sheath gas flow = 80% full
Aux gas flow: 20% max. (no stream splitting)
MASS SPEC-MASS SPEC:
(MS-MS)

27. HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES27
OD 330nm
(mAU)
200
400
600
800
1000
1200
1400
1600
1800
2000
2200 30 40 20 50 10 60 70 80
Retention Time (min)
AC Baton
(hulless)
More than 30 peaks detectected

28. HPLC DIODE ARRAY-MSMS OF AVENANTHRAMIDES28
100
200
300
400
500
600
700
800
900
1000
OD 330nm
(mAU)
AC Baton
(hulless)
More than 20 peaks detectected 10 20 30 40 50 60 70 80 90
100
Retention Time (min)

29. KEY TO AVENANTHRAMIDE ID IS THE MS-MS IONS m1 AND m229
MS parameters optimized for generation of [M-1] -
Parent ion [M-1]- n - R
Parent ion [M-1]- - R n
Parent ion M-1 – (always even number) gives molecular weight of the avenanthramide R
Daughter ion m1 - -
Daughter ion m2 - n R -
Daughter m1 - gives molecular weight of the anthranilic moiety
Daughter m2 - gives molecular weight of both ring moieties

30. STRUCTURES OF TYPE I AVENANTHRAMIDES30
STRUCTURES OF TYPE I AVENANTHRAMIDES
R = H
R = OCH3
R = OH D E F A B C G H K AA BB CC X Y Z

31. STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES31
TYPE 1 AVENANTHRAMIDES 20 40 60 80
100
SUMMARY
5 Different anthranilic acid amides with either
p-coumaric, ferulic, or caffeic acids
anthranilic
Rt (min) Amide
Rt (min) C18
5-OH anthranilic
4-OH anthranilic
4-OH,5-OMe anthranilic
4,5-di(OH) anthranilic

32. STRUCTURES OF TYPE II AVENANTHRAMIDES32
STRUCTURES OF TYPE II AVENANTHRAMIDES
R = H
R = OCH3
R = OH L M N O P Q R S T OO PP QQ U V W

33. STRUCTURES AND CHROMATOGRAPHIC PROPERTIES OF AVENANTHRAMIDES33
TYPE 2 AVENANTHRAMIDES 20 40 60 80
100
SUMMARY
5 Different anthranilic acid amides with either avenalumic, 3’-OMe-avenalumic, or 3’-OH-avenalumic acids
Rt (min) Amide
Rt (min) C18
5-OH anthranilic
4-OH anthranilic
4,5-di(OH) anthranilic
4-OH,5-OMe anthranilic

34. 2-D HPLC MS-MS “MAPPING”OF AVENANTHRAMIDES34
Based on:
1) Rt in 2 solvents
2) UV spectra
3) MS pattern 20 40 60 80
100
Rt (min) Amide
Rt (min) C18 K G H A B C D E F L M N CC AA BB X Y Z O P Q QQ OO PP V U W R S T
There are about 30 different avenanthramides not including dimeric forms
Each one identified by an alphabetic designation
Agri-Food Canada
Agriculture and
Agriculture et
Agroalimentaire Canada

35. RELATIVE AMOUNTS OF EACH AVENANTHRAMIDE…..35
Rt (min) Amide
OD (AU)
0.5
1.0
1.5
2.0
2.5
AC Baton
(hulless)
Relative quantitative estimations: 20 40 60 80
100
Major Avenanthramides
A, B, C
O, P, Q
OO, PP, QQ
AA, BB, CC
Rt (min) C18

36. GENOTYPIC VARIATION IN AVENANTHRAMIDE LEVELS36
CONCENTRATION IN PPM 50
100
200
300
400
500
VARIETIES / LINES
AV A
AV B
AV C TOTAL
Strong genotype x environment interaction as well

37. TEAM CONTRIBUTORS AND ACKNOWLEDGEMENTS….37
USDA TUFTS UNIVERSITY
Mohsen Medani
Liping Liu
Ligia Zubik
Melissa Marko
Weimin Guo
Vascular
Biology Lab:
In vitro studies
AAFC
F. William Collins
Vern Burrows
Winson Orr
Dwight Colley
Nicole Fillion
Synthesis ,
GMP Purification, Analytical,
Germplasm
Jeffrey Blumberg
Paul Milbury
Chung-Yen Chen
Ting Li
Jennifer O’Leary
Antioxidant
Research Lab:
In vivo studies