AVEQAVEQ
Analysis of Tocopherols/Tocotrienols Analysis of Tocopherols/Tocotrienols
and Avenanthramides in Oats and Avenanthramides in Oats
WP 8
Rita Redaelli and Lena Dimberg
Tocopherols/ TocotrienolsTocopherols/ Tocotrienols
Tocopherols
Tocotrienols
Isomers R' R''
α CH3 CH3
β CH3 H
γ H CH3
δ H H
Pisacane et al., 2004Solvent: MethanolFlour/solvent ratio: 1 g/14 ml Time: 2*1 minSolubilized: Methanol(or hexane/1,4-dioxane, 95/5)
SLU-labSolvent - MethanolFlour/solvent ratio:(1 g/21 ml)Time: 3*20 min Solubilized - Hexane
Peterson et al., 2007Solvent: Methanol (or saponified KOH in ethanol + antioxidant)Flour/solvent ratio: 1 g/28 mlTime: 2* 20 minSolubilized: Hexane(or hexane/2-propanol, 99.5/0.5)
Extraction of Tocopherols/Tocotrienols
Nielsen and Hansen, 2008Solvent: HexaneFlour/solvent ratio: 1 g/30 mlTime: 1* 10 minSolubilized: Hexane
SLU-labNP- column: 250 mmIsocratic solvent:Hexane/1,4-dioxane (96:4 v/v)Flow: 1.5 ml/minTime: 21 minFluorescence detector: ex 294 nm; em 326 nmReference: 8 isomers
Nielsen and Hansen, 2008NP- column: 125 mmIsocratic solvent:Hexane/ethyl acetate/acetic acid (94.6/3.6/1.8)Flow: 1 ml/minTime: 16 minFluorescence detector: ex 290 nm; em 330 nmReferences: 8 isomers
Pisacane et al., 2004NP- column: 250 mmIsocratic solvent:Hexane/1,4-dioxane (95:5 v/v)Flow: 1,5 ml/min Time: 11 minFluorescence detector: ex 294 nm; em 326 nmReference: 8 isomers
Peterson et al., 2007NP- column: 250 mmIsocratic solvent:Hexane/2-propanol (99.5/0.5 v/v)Flow: 1 ml/min Time:?Fluorescence detector: ex 295 nm; em 330 nmReferences: α-tocopherol
HPLC analysis of Tocopherols/ Tocotrienols
0.0475
Volts
0.0225
0.0250
0.0275
0.0300
0.0325
0.0350
0.0375
0.0400
0.0425
0.0450
0.0475
Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12
volt
s
0.0225
0.0250
0.0275
0.0300
0.0325
0.0350
0.0375
0.0400
0.0425
0.0450
αT
αT3
βT
γT
βT3
γT3
δT
δT3
Pisacane et al. 2004
NP-HPLC OF TOCOLSNP-HPLC OF TOCOLS
AvenanthramidesAvenanthramides
R
R
NH
O
OH
R
R
COOH
n
R= H, OH or OCHR= H, OH or OCH33
n= 1 or 2n= 1 or 2
AvenanthramidesAvenanthramides(ca 30 different compounds in oats)(ca 30 different compounds in oats)
1 2 3
p N
O
OHCOOHH
N
O
OHCOOHH
HO
N
O
OHCOOHH
HO
OMe
f N
O
OHCOOHH
OMe
N
O
OHCOOHH
OMe
HO
N
O
OHCOOHH
OMe
HO
OMe
c N
O
COOHH
OH
OH
N
O
COOHH
HO
OH
OH N
O
COOHH
HO
OH
OH
OMe
HPLC-chromatogram (UV 340 nm) HPLC-chromatogram (UV 340 nm) oat extract and reference compoundsoat extract and reference compounds
0
10
20
30
40
50
15 20 25 30 35 40
Time (min)
Ab
so
rba
nc
e (
mA
U)
a
2c
2p2f
2fd
3f
2pd
0
40
80
120
160
15 20 25 30 35 40
Time (min)
Ab
so
rba
nc
e (
mA
U)
b
2c3c
2p
p
2f
3p
3f1c 2fd
1p1f
Tran
AvenanthramidesAvenanthramidesUV-spectraUV-spectra
0
20
40
60
80
100
120
140
160
200 250 300 350 400
Wavelength (nm)
Ab
sorb
ance
(mA
U)
2c
0
20
40
60
80
100
120
140
200 250 300 350 400
Wavelength (nm)
Ab
sorb
ance
(mA
U)
3c
0
50
100
150
200
250
300
200 220 240 260 280 300 320 340 360 380 400
Wavelength (nm)
Ab
sorb
ance
(mA
U)
2p
0
50
100
150
200
250
200 250 300 350 400
Wavelength (nm)
Ab
so
rba
nc
e (
mA
U)
2f
0
50
100
150
200
250
200 250 300 350 400
Wavelength (nm)
Ab
sorb
ance
(mA
U)
3p
0
20
40
60
80
100
120
140
200 250 300 350 400
Wavelength (nm)
Ab
so
rba
nc
e (
mA
U)
3f
0
20
40
60
80
100
120
140
160
200 250 300 350 400
Vawelength (nm)
Ab
so
rba
nc
e (
mA
U)
1c
020406080
100120140160180200
200 250 300 350 400
Wavelength (nm)
Ab
so
rba
nc
e (
mA
U)
2fd
0
50
100
150
200
250
200 250 300 350 400
Vawelength (nm)
Ab
sorb
ance
(mA
U)
1p
0
50
100
150
200
250
200 250 300 350 400
Wavelength (nm)
Ab
so
rba
nc
e (
mA
U)
1f
020406080
100120140160180200
200 250 300 350 400
Wavelength (nm)
Ab
sorb
ance
(nm
)
Tran
SLU-labSolvent - 80% Ethanol (pH 2)Flour/solvent ratio:(1 g/21 ml)Temperature: 50 CTime: 3*20 min Solubilized - Methanol
Extraction of Avenanthramides
Wise et al., 2009Solvent: 80% EthanolFlour/solvent ratio: 1 g/30 ml Temperature: 50 CTime: 3*20 minSolubilized: Methanol
Matsukawa et al., 2000Solvent - MethanolFlour/solvent ratio:(1 g/30 ml)Temperature: ambient?Time: 3*? min Solubilized -Methanol
CollinsSolvent - 80% Ethanol (acidified)Flour/solvent ratio:(1 g/25 ml)Temperature: 100 CTime: 3*20 minPurification: Octyl-sepharose,
Sephadex-LH20 Solubilized – 50% Ethanol
SLU-labRP C-18 column: 125 mmSolvent: A. = 0.01 M formic acid
(pH 2.9), 5% acetonitrileB. acetonitrile
Gradient: 5 to 38% B in A in 35 minFlow: 1 ml/minTime: 30 minDiod array: 340 nmReference: 17 compounds
Wise et al., 2009RP C-18 : 50 mmSolvent: A. 0.1% formic acid, 5% acetonitrile
B. acetonitrile, 0.1% formaric acidGradient: 13-30% B in A for 20 minFlow: 1 ml/minTime: 20 minDiode array detector: 280 and 330 nm References: 3 compounds
Collins et al., 2004RP C-18 column: ? mmSolvent: A. 5% acetic acid
B. methanolGradient: 40 to 100% B in A for 88 minFlow: 0.8 ml/minTime: 50 minDiod array: 330 nmReference: ? compounds
Matsukawa et al., 2000RP C-18 column: 150 mmSolvent: A. 0.5% TFA,
B. acetonitrileGradient: 15 to 30% B in A for 60 minFlow: ? ml/minTime: ? minDiod array: 340 nmReference ? compounds
HPLC analysis of Avenanthramides
a
b c d e f g
2c
2p
2f
3f
2pd
2fd
2c
2p
2f
3f
2pd
15 17 19 21 23 25 27 29
Time (min)
2fd
Analysis of AvenanthramidesAnalysis of AvenanthramidesLC-MSLC-MS
b-g Selective ion monitoring (SIM) detection[M+H]+
for 2c, 2p, 2f, 3f, 2pd and 2fd, respectively
aUV detection, 340 nm
Molecular weights of avenanthramidesMolecular weights of avenanthramides
anthranilic
acid 5-OH 5-OH, 4-MeOH 4-OH 4-OH, 5-OH
1 2 3 4 5
p-coumaric p 283 299 329 299 315
ferulic f 313 329 359 329 345
sinapic s 343 359 389 359 375
caffeic c 299 315 345 315 331
p-coumaric p diene 309 325 355 325 341
ferulic f diene 339 355 385 355 371
sinapic s diene 369 385 415 385 401
caffeic c diene 325 341 371 341 357
0
2
4
6
8
10
2 4 6 8 10 12 14 16 18 20
Time (min)
Pea
k ar
ea (
AU
)
120 h germination Raw grains
1
2
32c
567 8
2p
2f
1112
13
14=3f
15
16
171819 20
21
Effect of germinationEffect of germination
Thank youThank you
Antioxidant activity of different Antioxidant activity of different avenanthramidesavenanthramides
0
400
800
1200
1600
0 30 60 90 120
Time (min)C
on
jug
ated
die
ne
hyd
rop
ero
xid
es
(uM
)
1a
2a
3a
Control
a-tocopherol
0
400
800
1200
1600
0 30 60 90 120
Time (min)
Co
nju
gat
ed d
ien
e h
ydro
per
oxi
des
(u
M)
3a
3p
3f
3s
3c
Control
a-tocopherol
0
100
200
300
400
500
600
2c 2p 2f
Co
nce
ntr
atio
n (
pea
k ar
ea/g
)
s
r
Avenanthramides in crown rust Avenanthramides in crown rust susceptible and resistant cultivarssusceptible and resistant cultivars
Radical scavenging activity of Radical scavenging activity of different avenanthramidesdifferent avenanthramides
a p f s c
12
30
10
20
30
40
50
60
70
Ra
dic
al
sc
av
en
gin
g
ac
tiv
ity
(d
elt
a A
(1 m
in) A
U)
a p f s c
12
30
10
20
30
40
50
60
70
Ra
dic
al
sc
av
en
gin
g
ac
tiv
ity
(d
elt
a A
(5 m
in) A
U)
1 min
5 min
Radical scavenging activity of Radical scavenging activity of different avenanthramidesdifferent avenanthramides
a p f s c
12
30
10
20
30
40
50
60
70
Ra
dic
al
sc
av
en
gin
g
ac
tiv
ity
(d
elt
a A
(1 m
in) A
U)
a p f s c
12
30
10
20
30
40
50
60
70
Ra
dic
al
sc
av
en
gin
g
ac
tiv
ity
(d
elt
a A
(5 m
in) A
U)
1 min
5 min
HPLC-chromatogram HPLC-chromatogram (UV-detection 340 nm) (UV-detection 340 nm)
--oat extract and reference compounds-oat extract and reference compounds-
0
10
20
30
40
50
15 20 25 30 35 40
Time (min)
Ab
so
rba
nc
e (
mA
U)
a
2c
2p2f
2fd
3f
2pd
0
40
80
120
160
15 20 25 30 35 40
Time (min)
Ab
so
rba
nc
e (
mA
U)
b
2c3c
2p
p
2f
3p
3f1c 2fd
1p1f
Tran