102 Acta Chim. Slov. 2007, 54, 102–105
Prýma et al.: Tocol Content in Barley
1. Introduction
Vitamin E is one of the most important antioxidants
in the organism. Unlike vitamin C, it is soluble in fats,
which is of special importance as free radicals in the orga-
nism may damage cell membranes and lipoproteins with a
lower density. The positive role of vitamin E was recently
proven in coronary artery disease.1 It belongs among the
most significant lipophilic antioxidants that protect unsa-
turated lipids in eukaryotic cells against free radicals that
damage DNA and cause e.g. age-related pigmentation and
skin ageing, cataract, neuritic plates of the Alzheimer’s di-
sease, arteriosclerosis, stroke, heart attack, tumors.2
Vegetable oils are the main source of vitamin E, but
of it is also contained in cereals in significant amounts. Vi-
tamin E occurs in alpha, beta, gamma, delta isomers – to-
copherols and tocotrienols.3 and barley is apparently the
only commonly grown cereal, the caryopsis of which con-
tains vitamin E in all eight forms of tocopherols and toco-
trienols.4,5 The determination of these substances was per-
formed using alkaline saponification and extraction of
nonsaponified portion with diethyl ether with subsequent
liquid-chromatographic determination.6,7 Green barley
plants are also commercially used as a source of natural
antioxidants.8 The content of vitamin E was determined in
selected varieties of malting barley caryopses and also in
different growth phases of green barley. 
2. Materials and Methods
2.1. Green Barley
We used spring two-row barley varieties Sebastian,
Malz and the line KM1910, grown in 2005 in plots of
MUAF in @ab~ice and Agrotest Krome∨r∨í`. Three growing
phases were selected for screening the content of active
components (according to a decadic scale – DC): I in DC
29, II in DC 31 and III in DC 32-33 in which plant green
matter was taken.
2.1. Barley Lines with Increased Vitamin E
Content
Seed samples used for analyses were from hand
sown plants grown in field trials in spacing 15 × 10 cm on
the field of the experimental station of MUAF in @ab~ice
near Brno. This station lies in the maize production area,
at the altitude of 184 m above the sea level. 
The varieties of American provenience Wabet (Wb),
Wanubet (Wnb), Washonubet (Wsnb) were used as donors
of vitamin E. Lines with higher vitamin E content and ot-
her antioxidants in comparison with malting-type varie-
ties were attained by their suitable combinations with the
malting varieties Kompakt (Ko), Krona (Kr).
Totally 12 varieties and lines of spring barley were
used for the analyses: standard hulled malting varieties re-
Abstract
Green barley and malting barley are sources of numerous compounds with positive effects on the human organism – es-
pecially substances with antioxidant effects, e.g. vitamin E. We determined tocol content in barley caryopses in depen-
dence of varietal and genetic properties of materials. Using chromatography, we studied vitamin E content in different
growth phases of green barley and in varietal lines of malting barley. The content of vitamin E was the highest in the first
growing phase (14.4–18.0 mg kg–1) in the variety Sebastian. Green plants contain significantly less tocotrienols than
barley caryopses.
Keywords: vitamin E, tocols, barley, green barley, HPLC analysis
Short communication
Tocol Content in Barley
Jaroslav Prýma,a,* Jaroslava Ehrenbergerová,b Natálie Belcrediová,c
Kater
∨
ina Vaculovác
aResearch Institute of Brewing and Malting, PLC, Malting Institute Brno, Mostecká 7, 614 00 Brno, Czech Republic. 
Tel.: +420 545244110, E-mail: pryma@brno.beerresearch.cz.
bMendel University of Agriculture and Forestry, Brno, Czech Republic
cAgricultural Research Institute, Krome∨r∨í`, Ltd., Czech Republic
Received: 26-09-2006
Paper based on a presentation at the 12th International Symposium on Separation Sciences, 
Lipica, Slovenia, September 27–29, 2006.
103Acta Chim. Slov. 2007, 54, 102–105
Prýma et al.: Tocol Content in Barley
creating resolution problems. Therefore, reverse phase
chromatography was used for the analyses. Using this
procedure, we could not distinguish between the β and 
γ.-isomers, therefore the sum of these isomers is shown as
a combined value for both T and T3.9 Total contents of to-
cols and the vitamin E equivalent (VEeq) were calculated
for each cultivar as described by McLaughlin and Wei-
brauch.10
gistered in the CR Kompakt and Krona, experimental li-
nes were waxy types, Wabet, Wanubet and Washonubet,
obtained from the Montana Agricultural Experiment Sta-
tion, Bozeman MT USA and seven hulled lines, obtained
from crossing the waxy types and Kompakt and Krona.
Wabet is a hulled cultivar and Wanubet and Washonubet
are hull-less cultivars. 
The sampled grain from the manually harvested ears
of individual plants were cleaned, homogenized and che-
mically analyzed. The content of vitamin E and of its iso-
mer, α-, βγ-, δ-tocopherols and α-, βγ -, δ-tocotrienols we-
re determined.
2.2. Tocol Analysis
Total contents of tocols – tocopherol (T) and toco-
trienol (T3) isomers, α, β + γ and δ were determined as
follows: grain samples (2 g) were homogenized and then
100 mg of ascorbic acid, 50 mL of ethanol and 10 mL of
50% KOH were added. Saponification was performed at
room temperature (18–25 °C) in a dark room in a nitroge-
nous atmosphere overnight. Subsequently, the samples
were extracted with diethyl ether, washed with water,
dried with anhydrous sodium sulphate and evaporated.
The evaporated residue was dissolved in a defined volume
with reagent grade methanol. The extract was analyzed
using HPLC with fluorescence detection according EN
12822:2000. We used an HPLC SpectraSystem (Thermo
Separation Products, Inc., USA), Pump P2000, fluores-
cence detector FL 3000, column stationary phase Nucleo-
sil 120-5 C 18, 250 × 4 mm, mobile phase: methanol, 1.0
mL min–1, injection volume 20 µL, detection: λEX = 290
nm, λEM = 330 nm.
Normal phase chromatography (n-hexane eluent)
enabled the separation of all isomers and at the same time
the ratio between β and γ-isomers was determined. Howe-
ver, the column was extremely sensitive to humidity, thus
Figure 1. Chromatogram of tocopherols and tocotrienols in barley.
Table 1: Content of vitamin E in green barley.
Variety (Linie) α-T (β + γ)-T δ-T α-T3 (β + γ)-T3 δ -T3 
mg kg–1 mg kg–1 mg kg–1 mg kg–1 mg kg–1 mg kg–1
1st sampling
KM 1910 16.3 3.1 0.45 – – –
Sebastian 17.6 2.7 0.32 – – –
Malz 14.0 3.0 0.25 – – –
2nd sampling
KM 1910 6.8 2.0 0.36 – – –
Sebastian 8.5 2.1 0.29 – – –
Malz 8.7 1.2 0.34 – – –
3rd sampling
KM 1910 6.5 0.8 0.11 4.07 – – 
Sebastian 9.1 1.7 0.24 4.21 – – 
Malz 9.3 1.3 0.21 4.11 – – 
104 Acta Chim. Slov. 2007, 54, 102–105
Prýma et al.: Tocol Content in Barley
3. Results and Discussion
Contents of individual isomers of tocopherols and
tocotrienols in three growing phases of green barley were
determined and on the basis of these, the content of vita-
min E was determined (Table 1, Figure 1). It was the hig-
hest at the first sampling (14.4–18.0 mg kg–1) and in the
variety Sebastian. 
Dependence of vitamin E content on the variety
was confirmed. The varieties with the highest tocol con-
tent and the highest content of vitamin E (Wabet) can ser-
ve as donors when breeding new lines (Table 2, Figure 2).
Vitamin E content in barley was determined in the range
of 16.2–23.8 mg kg–1, which is in agreement with litera-
ture data. Peterson and Quereshi11 and Holasová et al.12
found out that in some cases vitamin E content exceeds
even the amount detected in other cereals even four times
(wheat 15.6 , triticale 11.8 , rye 17.1 , oats 14.7 and bar-
ley 23.7 mg kg–1 VEeq). Holasová et al.13 found that the
content of vitamin E in barley caryopses was signifi-
cantly higher (21.9–25.5 mg kg–1) compared to triticale
(4.8–18.8 mg kg–1), wheat (13.5–17.6 mg kg–1), oats
Figure 2: Content of vitamin E in barley varieties.
Table 2. Content of vitamin E in barley grain.
Table 1: Content of Vitamin E in Barley Grain
Variety (Linie) α-T (β + γ )-T δ-T α-T3 (β + γ)-T3 δ-T3 
mg kg–1 mg kg–1 mg kg–1 mg kg–1 mg kg–1 mg kg–1
Wabet 9.8 5.3 1.3 43.0 8.2 0.5
Wb x Wsnb 9.5 5.0 1.1 40.9 7.8 0.6
Wanubet 8.6 5.4 1.2 36.6 7.5 0.8
Wb x Ko 8.8 4.8 1.4 33.9 9.4 0.3
Ko x Wb 8.2 6.9 1.5 33.9 8.4 0.5
Washonubet 6.9 4.4 1.1 39.6 8.4 0.5
Krona 8.5 7.4 1.5 31.9 8.0 0.5
Wnb x Kr 7.9 4.5 1.1 29.1 7.5 0.4
Ko x Kr 8.0 8.4 1.4 26.3 8.8 0.7
Kr x Ko 8.6 7.1 1.2 23.6 9.2 0.7
Kr x Wnb 7.9 5.8 0.9 26.2 7.7 0.6
Kompakt 8.2 7.0 1.0 22.5 6.7 0.4
(13.6–17.6 mg kg–1) and rye (15.5–18.7 mg.kg–1). Green
plants contain significantly less tocotrienols than barley
caryopses.
4. Conclusion 
Liquid chromatography with fluorimetric detection
was used for determination of vitamin E content in green
barley and malt barley. The highest content of vitamin E
was found in green barley after the first sampling. Consi-
derable variability in tocol content in barley varieties was
found, ranging from 16–24 mg kg–1.
5. Acknowledgement
The authors acknowledge the financial support of
the Ministry of Education, Youth and Sports of the Czech
Republic, Project RC No. MSM6019369701 and 1M0570
and the Grant Agency of the Czech Republic, Project 
No. 525/05/0781. 
105Acta Chim. Slov. 2007, 54, 102–105
Prýma et al.: Tocol Content in Barley
6. References
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2. K. Nesaretnam, N. Guthrie, A. F. Chambers, K. K. Carroll,
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3. D. M. Peterson, A. A. Qureshi, Cereal Chem. 1993, 70,
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4. A. Cavalero, A. Gianinetti, F. Finocchiaro, G. Delogu, A. M.
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6. EN 12 822:2000, Foodstuffs – Determination of vitamin E
by high performance liquid chromatography – Measurement
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Povzetek
Je~men je vir mnogih spojin s pozitivnim u~inkom na zdravje, {e posebej antioksidantov, kot je vitamin E. V zrnju smo
dolo~ili vsebnost tokola v kultivarjih razli~nega genetskega izvora. S pomo~jo kromatografske analize s fluorimetri~no
detekcijo smo preu~evali vsebnost vitamina E v razli~nih rastnih fazah je~mena. Najve~ja vsebnost je bila v prvi rast-
ni fazi (14,4–18,0 mg kg–1) pri kultivarju Sebastian. Zelene rastline vsebujejo bistveno manj tokotrienolov kot zrnje
je~mena.