ARTICLE IN PRESS

JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 17 (2004) 749–765
www.elsevier.com/locate/jfca

Original Article

A small-scale sample preparation method with HPLC analysis

for determination of tocopherols and tocotrienols in cereals
M. Ryyn.anen, A.-M. Lampi*, P. Salo-V.aa. n.anen, V. Ollilainen, V. Piironen
Department of Applied Chemistry and Microbiology, Food Chemistry, Latokartanonkaari 11, P. O. Box 27,
University of Helsinki, FIN-00014 Helsinki, Finland
Received 21 February 2003; received in revised form 8 August 2003; accepted 22 September 2003

Abstract

A small-scale sample preparation method was developed for reliable and economic analysis of
tocopherols and tocotrienols in rye and other cereals. The saponification parameters were optimized using
two experimental design protocols with statistical analyses. Three critical factors were optimized for hot
saponification: time, temperature and amount of potassium hydroxide (KOH). The amount of KOH had
the greatest effect. Two direct solvent extraction methods without saponification were tested and found to
yield in comparable values than with saponification. Finally, three solvent mixtures for extracting
tocopherols and tocotrienols after the optimized saponification step were studied in order to examine the
effect of solvent polarity on their recovery. Adding a polar modifier to the solvent, tocopherol and
tocotrienol values of rye were significantly increased. The optimized sample preparation method included
saponification with 0.5 mL KOH at 100
C for 25 min followed by extraction with n-hexane:ethyl acetate
(8:2). The repeatability of the recommended analytical procedure was good and the recoveries of added
tocopherols from rye flour samples ranged from 90.3% to 94.3%. The procedure developed was applied to
examine the amounts and distribution of tocopherols and tocotrienols in ten rye varieties. The average total
tocopherol and tocotrienol content of rye grains was 48.8 mg/g with a 9.3% standard deviation between
varieties.
r 2003 Elsevier Inc. All rights reserved.

Keywords: Tocopherols; Tocotrienols; HPLC; Rye; Cereals

*Corresponding author. Tel.: +358-919158412; fax: +358-919158475.

E-mail address: anna-maija.lampi@helsinki.fi (A.-M. Lampi).

0889-1575/$ - see front matter r 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2003.09.014
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1. Introduction

Tocopherols and tocotrienols are a group of dietary plant constituents considered to have
beneficial health effects. There are four tocopherol and four tocotrienol vitamers. All of them
consist of a chromanol ring and a hydrophobic side chain, which is a phytyl in tocopherols
and an isoprenyl with three double bonds in tocotrienols. The vitamers can be distinguished by
the number and location of methyl groups on their chromanol ring. Generally, when the
vitamin E activity of a food item is evaluated, a sum of the different vitamers is calculated
taking into account their relative activities. For healthy people it is relatively easy to
obtain sufficient tocopherols and tocotrienols from the diet to prevent well-defined vitamin E
deficiency symptoms. However, higher intakes of E vitamers may also decrease the risk of
several chronic diseases related to oxidative damage, e.g., coronary heart diseases and
cancer. Most of the effects in the human body are due to their action as lipid-soluble anti-
oxidants (e.g., Stampfer and Rimm, 1995; Stone and Papas, 1997; Theriault et al., 1999;
Schwenke, 2002).
Despite the fact that the latest North American guidelines consider a-tocopherol to be the only
biologically active form of vitamin E and discount other vitamers (Anon, 2000), the biological
importance of other E vitamers has gained attention. Recent studies have shown that tocotrienols
have a variety of novel beneficial functions. For example, they may have a protective effect by
lowering LDL cholesterol by inhibiting cholesterol biosynthesis (Qureshi et al., 1995; Hood, 1998;
Theriault et al., 1999). There is growing evidence that the greatest advantage of a tocopherol- and
tocotrienol-rich diet is achieved when various E vitamers are administered concurrently. It has
been shown that a-tocopherol supplementation alone has little effect on mammary tumors,
and evidently, other forms of vitamin E-like substances, such as a-, g- and d-tocotrienols, reduce
the risk of breast cancer (Schwenke, 2002). Thus, it has been stated that the ratios of the
individual tocopherols and tocotrienols play an important role in determining the hypocholester-
olemic, antioxidant and antitumor properties of palm oil and rice bran (Qureshi et al., 2000,
2002). For example, a-tocopherol and a-tocotrienol had opposite effects on the cholesterol
metabolism of chicks, a higher ratio of tocotrienols to tocopherols being optimal (Qureshi et al.,
1989). Recently, the vitamers have also been shown to have non-antioxidant roles (Azzi and
Stocker, 2000).
The main sources of vitamin E-active compounds in the human diet are vegetable fats and oils
and products derived from them, but cereals also contribute significantly to the dietary intake in
the United States (14.6%, Murphy et al., 1990) and in Finland (18%, Heinonen and Piironen,
1991). In terms of amounts of tocopherols and tocotrienols, cereals are more important than in
terms of vitamin E activity, since they are rich sources of the less vitamin E-active tocotrienols.
For example, barley contains all four tocopherols and four tocotrienols. Other cereals such as
wheat, oats and rye also contain more tocotrienols than tocopherols (Piironen et al., 1986; Balz
et al., 1992; Peterson and Qureshi, 1993) and thus have a potentially beneficial distribution of the
vitamers. Since cereals contain a number of E vitamers and each of them has a diverse biological
and chemical character, they are a challenge for a food analyst, because it is essential to be able to
analyse each vitamer separately.
It is possible to accurately analyse all forms of tocopherols and tocotrienols with modern
chromatographic methods (Eitenmiller and Landen, 1999; Abidi, 2000; Piironen, 2000; Rupe! rez
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et al., 2001; Lampi et al., 2002). There are three major sample preparation approaches for food
samples: simple dilution of oil samples in organic solvent, direct extraction of the vitamers with
organic solvents, and extraction of the vitamers after saponification. In cereals, tocopherols and
tocotrienols are extracted by either a direct solvent method or by saponification followed by
extraction. Extraction with organic solvents disrupts the structures to which the vitamers are
attached and solubilizes them. n-Hexane is the commonly used solvent, but more polar
solvent mixtures are also used, e.g., ethyl acetate in n-hexane or acetone (Ueda and Igarashi,
1987a, 1990; Eitenmiller and Landen, 1999; Rupe! rez et al., 2001). Saponification further assists in
releasing the vitamers by degrading the food matrix, and removes the bulk of fat, which improves
their chromatographic separation (Piironen, 2000; Rupe! rez et al., 2001). Tocopherols and
tocotrienols are relatively unstable in alkaline conditions, and care must be taken to avoid
their destruction. They are protected by using antioxidants, flushing the saponification vessel
with nitrogen and working under subdued light (Eitenmiller and Landen, 1999). E vitamer
analysis is most commonly conducted with normal-phase high-performance liquid chromato-
graphy with fluorescence detection (NP-HPLC-FLD). A fluorescence detector is preferred
over an ultraviolet detector in analysing tocopherols and tocotrienols in complex food
matrices because of its specificity and sensitivity (Piironen et al., 1986; Eitenmiller and
Landen, 1999; Abidi, 2000). Other means of separation include reversed-phase HPLC, gas
chromatography and, most recently, capillary electrochromatography (Abidi et al., 2002).
Electrochemical detection is also applied as a sensitive detector for E vitamers after HPLC
separation (Ueda and Igarashi, 1987b; Yamauchi et al., 2002), while mass spectrometry (MS) in
combination with HPLC assists in identification of compounds (Strohschein et al., 1999; Stoggl .
et al., 2001). As the sensitivity of the HPLC analysis has improved, it has become possible to scale
down the food sample size, as has been done, e.g., for animal products (Salo-V.aa. n.anen et al.,
2000). At the same time, a need for shorter sample preparation times and decreased amounts of
chemicals has arisen.
Since there are multiple factors to be optimized in the sample preparation method for E vitamer
analysis, we chose an experimental design approach to develop a fast and reliable saponification
method for cereal samples. Earlier, Lee et al. (2000) used response surface methodology to
optimize an extraction procedure of tocopherols from tomato and broccoli. They used 70
C as the
saponification temperature and varied the amounts of alkali and ethanol in the saponification
mixture and the saponification time. In our study, a wider range of analytical conditions during
saponification and extraction of E vitamers was investigated. Our hypothesis was that
saponification is an efficient means to liberate tocopherols and tocotrienols from cereal matrix
prior to lipid extraction, but the saponification conditions should be carefully examined and
controlled to avoid decomposition of the analytes.
In order to reliably and economically investigate the distributions of tocopherols and
tocotrienols in rye and other cereals, a small-scale and rapid sample preparation method was
developed. The saponification conditions were optimized using experimental design and solvent
mixtures for extraction of tocopherols and tocotrienols were evaluated. Applicability of the
recommended sample preparation method was tested by examining the amounts and distribution
of tocopherols and tocotrienols in ten rye varieties grown in Finland. This was also performed in
order to expose the most promising varieties with regard to the optimal ratio of tocopherols and
tocotrienols.
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2. Materials and methods

2.1. Sample materials, standards, and reagents

The material used for method development was rye flour (Myllyn paras; Helsingin mylly,
Hyvink.aa. , Finland). Rapeseed oil was used as an in-house reference material for HPLC analysis
(Kultasula; Raisio Group, Raisio, Finland). Samples of ten rye varieties were cultivated in the
Agricultural Research Centre, Jokioinen, Finland, in 1999 (Boreal Plant Breeding Ltd, Jokioinen,
Finland). Sample grains were milled with a hammer mill at the Department of Food Technology,
University of Helsinki. All sample materials were stored in small portions in the dark at 20
C
until analysed.
HPLC-grade n-hexane, ethyl acetate and 1,4-dioxane were purchased from Rathburn
(Walkerburn, UK). Milli Q water was of HPLC-grade. Ethanol was of spectrophotometric
grade (AAS, 99.5%, Primalco, Finland). Ascorbic acid, p.a., was purchased from Merck
(Darmstadt, Germany) and potassium hydroxide (KOH) pellets from EKA Nobel (Bohus,
Sweden). Saponification solution was prepared by dissolving 50 g of KOH pellets in 100 mL Milli-
Q water. Sample extracts were filtered (GHP Acrodisc, 0.45 mm, Pall Corp., Anna Arbor, MI)
before injecting to HPLC.
a-, b-, g- and d-tocopherols (fur. biochemische Zwecke, Art no. 15496) were purchased from
Merck. The standard stock solutions of the tocopherols were prepared to a concentration of
approximately 500 mg/mL (in ethanol, AAS). The stock solutions were stored at 20
C. The
concentrations were confirmed spectrophotometrically using the known absorption coefficients of
each vitamer in ethanol Eð1%
1 cm Þ: 75.8 for a-tocopherol, 89.4 for b-tocopherol, 91.4 for g-tocopherol
and 87.3 for d-tocopherol at 292, 296, 298 and 298 nm, respectively (Podda et al., 1996). The
combined working solution was prepared by pooling suitable amounts of each tocopherol and
diluting with n-hexane to obtain concentrations ranging from 1–100 ng/injection. Each tocotrienol
vitamer was quantified with the respective tocopherol (e.g., AOCS, 1990; Kramer et al., 1997).
Tokovid-palmoil extract (Hovid Sdn. Bhd., Malaysia) was used as a qualitative standard for
tocotrienols.

2.2. Optimization of the sample preparation method

The sample preparation method was based on the room temperature saponification method
with n-hexane as an extraction solvent (Piironen et al., 1986). In this study a small scale and rapid
procedure using hot saponification was developed for determining tocopherols and tocotrienols in
cereals, using rye flour. To avoid destruction of labile vitamers, all work was carried out under
subdued light, ascorbic acid was added as an antioxidant and reaction vessels were flushed with
nitrogen before adding KOH.
At first, sample amounts and solvent volumes were reduced from a previous method (Piironen
et al., 1986) and hot saponification was conducted. Total tocopherol and tocotrienol values from
rye flour obtained with the room temperature saponification and the hot saponification methods
were comparable (24.571.0 mg/g and 23.872.2 mg/g) as were the recoveries of added tocopherols
(range 58–129%). Thereafter we proceeded to optimize the hot saponification conditions. Three
critical experimental factors of the hot saponification method were evaluated: saponification time,
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temperature and amount of KOH. The saponification method was developed and optimized
using two experimental design protocols (Haaland, 1989). The first experiments included three
critical factors with a wide range of analytical conditions, i.e., levels of factors, and the second
included only two factors with a narrow range (Tables 1 and 2). All other sample preparation
parameters remained constant. The results were analysed by using multiple linear regression
analysis and visualized by response surface plots (Statgraphics Plus, 1997). The output factor for
the trials was the total tocopherol and tocotrienol value and the target was to obtain as high a
value as possible. Each trial was performed in duplicate and the mean value was used as the
output factor.
On the basis of the results of the experiments described above, it was further investigated
whether saponification could be omitted in sample preparation. Thus two direct solvent extraction
methods without saponification were tested. Solvent extraction method 1 included hot extraction
with 2-propanol and n-hexane as extraction solvents (Koivu et al., 1997) and method 2 was the
optimized hot saponification method excluding the addition of KOH. Optimization of the sample
preparation method was finally fulfilled by investigating the effect of solvent polarity on
extracting tocopherols and tocotrienols from the saponified mixture. Three extraction solvents
were compared: n-hexane, n-hexane:ethyl acetate (9:1) and n-hexane:ethyl acetate (8:2). In order
to evaluate the steps other than saponification in the sample preparation method as described
above, all analyses were performed in triplicate at minimum. Comparisons of means or pairs were
conducted with t-tests, and of three groups with one-way analysis of variances (ANOVA)
followed by multiple range tests. All analyses were carried out using the same software as for
multiple linear regression analyses (Statgraphics Plus, 1997).

Table 1
Analytical conditions as the input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor
in a Box-Behnken experiment to study the effects of saponification time (min), temperature (
C) and amount of
KOH (mL) on the output factor (Haaland, 1989) (mean values from duplicate analyses)
Trial Temperature Time KOH Total tocopherols
and tocotrienols
1 60 20 2 21.7
2 60 60 2 18.5
3 100 20 2 21.5
4 100 60 2 18.6
5 60 40 1 21.2
6 60 40 3 14.9
7 100 40 1 23.6
8 100 40 3 16.5
9 80 20 1 22.6
10 80 20 3 17.1
11 80 60 1 20.1
12 80 60 3 18.1
13 80 40 2 22.1
14 80 40 2 18.6
15 80 40 2 19.0
16 80 40 2 21.4
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Table 2
Analytical conditions as input factors and total values of tocopherols and tocotrienols (mg/g) as the output factor in a
Central Composite experiment to study the effects of saponification time (min) and the amount of KOH (mL) on the
output factor (Haaland, 1989) (mean values from duplicate analyses)
Trial Time KOH Total tocopherols
and tocotrienols
1 5 0.25 22.5
2 5 1 24.6
3 20 0.25 27.6
4 20 1 24.4
5 1.9 0.625 23.6
6 23.1 0.625 26.9
7 12.5 0.096 25.2
8 12.5 1.154 24.7
9 12.5 0.625 26.0
10 12.5 0.625 24.6
11 12.5 0.625 24.5

2.3. Recommended analytical procedure

After method optimization, a recommended analytical procedure was established. The main
steps are illustrated in Fig. 1.

2.3.1. Saponification
Rye flour (0.5 g) was accurately weighed into a 30 mL Pyrex glass tube with a Teflon screw cap.
Ascorbic acid (0.1 g), ethanol (5 mL) and water (2 mL) were added. After mixing the tube with a
Vortex-mixer, the tube was flushed with nitrogen and KOH (0.5 mL) was added. The tube was
capped and transferred to a boiling water bath for 25 min. The tube was mixed with the Vortex-
mixer after 10 min of boiling. The tubes were cooled in an ice-water bath.

2.3.2. Extraction
Water (2.5 mL) and ethanol (2.5 mL) were added to the cooled tubes. Unsaponified lipids were
extracted by using three portions (each 10 mL) of n-hexane:ethyl acetate (8:2). Tubes were shaken
for 10 min with 500 strokes/min, and after separation of the phases, the organic layers were
collected in a separation funnel. Organic extracts were washed three times with water and with
addition of NaCl to avoid emulsion formation. The washed extract was transferred to a round-
bottomed flask, and the organic phase was evaporated. Ethanol (2 mL) and n-hexane (2 mL) were
added and evaporated to dryness. The residue was dissolved in n-hexane and transferred
quantitatively to a 5 mL volumetric flask. Sample extracts were stored in Kimax test tubes in the
dark at 70
C. Prior to HPLC analysis, extracts were filtered through a 0.45 mm filter.

2.3.3. Analytical HPLC

Normal phase HPLC with fluorescence detection (excitation 292 nm, emission 325 nm) was
used to analyse tocopherols and tocotrienols (Kamal-Eldin et al., 2000). The HPLC system
consisted of a pump (Waters 510; Waters Corp., Milford, MA), an autosampler with a cooling
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Preparing the sample for saponification:

0.5 g sample
0.1 g ascorbic acid, 5 mL ethanol, 2 mL water
flushing with nitrogen

Saponification:
0.5 mL KOH solution
100˚C, 25 min

Extraction and purification of non-saponifiable lipids:

2.5 mL ethanol, 2.5 mL water
3 x 10 mL n-hexane:ethyl acetate (8:2)
washing the extract 3 x with water and evaporating to dryness

Preparing the sample for HPLC analysis:

2 mL ethanol and 2 mL n-hexane
evaporating to dryness
dissolving the residue in 5 mL n-hexane

NP-HPLC analysis with fluorescence detector

Fig. 1. Recommended analytical procedure.

module (Waters 712), a scanning fluorescence detector (Waters 474), and an Inertsil silica column
(5 mm, 250 mm4.6 mm; Varian Chromapack, Middelburg, Netherlands) with a silica guard
column (Guard-Pak Silica, Waters). The temperature of the column oven was 30
C. Separation of
the vitamers was based on isocratic elution. The mobile phase contained 3% 1,4-dioxane and 97%
n-hexane. The flow rate of the mobile phase was 2 mL/min.
Tocopherols and tocotrienols were quantified with an external standard method in which
quantification was based on peak areas. Calibration curves with six points were obtained daily.
The method was validated by determining the following parameters: detection and determination
limits, range of linearity and repeatability. Detection limits were defined as a signal three times the
height of the noise. Determination limits were defined as three times the detection limit. The day-
to-day repeatability of the HPLC method was also confirmed with in-house reference material
(rapeseed oil) which was analysed daily during the 2-month period.

2.4. Evaluation and application of the recommended analytical procedure

Verification of the vitamer identification was based on the HPLC-FLD data and HPLC-MS
data. Full scan mass spectra and selected ion monitoring tracings derived from an atmospheric
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pressure chemical ionization (APCI) in positive mode were recorded using a quadrupole ion trap
mass spectrometry (Esquire LC-MS, Bremen, Germany). The liquid chromatographic method
used was the same as in analytical HPLC except that the eluent flow (2 mL/min) was split (1:10)
prior ionization interface with a splitter (Acurate Post-Column Splitter, LC Packings, USA). The
temperature of APCI interface and drying gas (nitrogen) was set at 300
C, and the potential of
discharge corona needle was 4.5 kV. Mass spectra were recorded at the scan range of 100–700 m/z
and the protonated molecular ions ([M+H]+) were measured.
The reliability of the recommended analytical procedure was verified by recovery and
repeatability tests with rye flour. Accuracy was verified by recovery tests, in which samples were
spiked with tocopherols (10 mg/g of rye flour) before saponification. Spiked samples were prepared
in duplicate on 3 days. Repeatability of the developed method was verified by analysing the
tocopherol and tocotrienol contents of rye flour in duplicate over 3 days.
The applicability of the recommended procedure to, e.g., whole grain samples was examined by
analysing the tocopherol and tocotrienol contents of ten rye varieties, and by finding any variation
in E vitamer composition. The varieties studied represent winter classes of rye cultivars grown in
Finland and are thus a relevant set of samples to be analysed by the current method.

3. Results and discussion

3.1. Optimization of saponification conditions

Three critical experimental factors were optimized in the small-scale hot saponification method
for analysis of tocopherols and tocotrienols in cereals: saponification time, temperature and
amount of KOH. Using the Box-Behnken design the total tocopherol and tocotrienol value of rye
flour measured as the output factor ranged from 14.9 to 23.6 mg/g (Table 1). Two factors were
shown to have a significant impact on the output factor, namely the saponification time
(P ¼ 0:0877) and the amount of KOH (P ¼ 0:0002). Saponification temperature, however, was
not an important factor (P ¼ 0:3634) and was excluded from the linear regression model. The
influences of the two important factors are illustrated in Fig. 2. The coefficient of determination
indicating the model’s ability to explain the relationships between the factors was 64.7%. The
figure shows that the smaller the amount of KOH solution, the greater was the total tocopherol

26.8363 - 2.615*KOH - 0.047312*Time

24
T + T3, ug/g

22
20
18
60
50
16 40
1 1.4 30
1.8 2.2 2.6 20
3
Time, min
KOH, mL

Fig. 2. Response surface plot of effects of saponification time and amount of KOH on total value of tocopherols and
tocotrienols.
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and tocotrienol value. The effect of saponification time on the output factor was less than that of
the amount of KOH. For example, when the time increased from 20 to 60 min, the total
tocopherol and tocotrienol value increased by 1.9 mg/g, but when the amount of KOH decreased
from 3 to 1 mL, the output factor increased by 5.2 mg/g. Since the saponification temperature had
an insignificant effect on tocopherol and tocotrienol values, the highest setting of 100
C was
chosen for further analyses, e.g., to enhance decomposing of cereal matrix.
In order to examine the effect of even lower levels of KOH and shorter saponification times,
another experiment using the Central Composite design was carried out at a temperature
stabilized to 100
C (Table 2). The total tocopherol and tocotrienol values were higher and the
range smaller than in the first experiment, ranging from 22.5 to 26.9 mg/g. Here both the amount
of KOH and the time had a significant effect on the output factor with P ¼ 0:0038 and P ¼
0:0002; respectively. Moreover, there was an evident interaction between these two factors
(P ¼ 0:0014) in the linear regression model (Fig. 3). The coefficient of determination of this model
was even higher, 87.1%, than that of the first model. Again, the smaller the amount of KOH, the
greater was the total tocopherol and tocotrienol value. However, at low levels of KOH, o1 mL, a
longer saponification time was needed than at high KOH levels. The interaction between these
two factors was so strong that at 0.096 mL of KOH, the total tocopherol and tocotrienol value
was 19.8 mg/g higher at the longest saponification time (23.1 min) than at the shortest time
(1.9 min) whereas at 0.625 mL of KOH, the difference was only 3.4 mg/g. When more than 1 mL of
KOH was used, changes of saponification time within the range of the experiment had only minor
effects on the output factor. Thus under these hot saponification conditions a compromise can be
made between the amount of KOH and saponification time.
Our finding that a small amount of KOH produces higher total tocopherol and tocotrienol
values is supported by the results of Piironen et al. (1984), who investigated the effects of different
amounts of KOH on E vitamer contents in a diet sample. They showed that treating samples with
higher amounts of alkaline than used in this study partly destroyed tocopherols. In addition, high
levels of soap might decrease the extractability of tocopherols and tocotrienols, because the soap
produced during saponification might solubilize them into the alkaline media (Ueda and Igarashi,
1987b). However, saponification degrades the food matrix and purifies the lipid extract, which
improves the efficacy of the sample preparation method. We also found that emulsions were
formed less frequently when at least 0.25 mL of KOH was used than with less alkaline. With low
levels of KOH, saponification times should be long enough as indicated in Fig. 3. Thus we

19.7418 + 5.19666*KOH + 0.444822*Time - 0.460444*KOH*Time

31
T + T3, ug/g

29
27
25
23
21 24
19 1620
0 8 12
0.2 0.4 0.6 0.8 1 1.2 0 4
Time, min
KOH, mL

Fig. 3. Response surface plot of effects of time and potassium hydroxide on the total amount of tocopherols and
tocotrienols.
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concluded that the recommended saponification method should consist of a reasonable amount of
KOH, 0.5 mL, with a saponification time of 25 min at 100
C.
3.2. Comparisons of direct solvent extraction and saponification

The results of the previous experiments indicated that the total tocopherol and tocotrienol value
from rye flour was greatest with the smallest amount of KOH. Thus it was further investigated
whether saponification could be omitted and, instead, direct solvent extraction would be used in
sample preparation where decomposition of matrix would be less efficient, but no losses due to
hot alkaline would occur. The total value of tocopherols and tocotrienols determined by two
direct extraction methods, i.e., hot extraction with 2-propanol and n-hexane, and optimized
saponification of the method excluding addition of KOH, and by the saponification method gave
statistically similar results (ANOVA, P > 0:05; n ¼ 3–5). The value was 24.270.8 mg/g for method
1, 23.970.9 mg/g for method 2, and 24.970.5 mg/g for the saponification method. Recoveries of
added tocopherols from rye flour were 73.7–90.5% and 86.1–90.4% for the saponification and the
direct extraction method 1, respectively. With saponification, recoveries of more polar
tocopherols were lower than that of a-tocopherol, decreasing in the order of their polarity,
while with solvent extraction with 2-propanol and n-hexane, no difference between the vitamers
was observed. The different recoveries of added tocopherols in the saponification method are
caused either by differences in their stabilities during saponification or extractabilities from the
saponification mixture, and not from liberation from the matrix, because tocopherols were added
as ethanol solutions. These findings illustrate one more important aspect of the sample
preparation procedure, i.e., the polarity of the extraction solvent after saponification. Similarly,
Ueda and Igarashi (1987b) found that soap formed from fat decreased the extractability of other
tocopherols more than that of a-tocopherol when an apolar n-hexane was used as the extraction
solvent. Hence, modification of extraction solvent following saponification was investigated to
improve the recoveries of more polar tocopherols.
Since the optimized saponification method was shown not to yield lower tocopherol and
tocotrienol values in rye flour than direct extraction methods, it was concluded that saponification
is beneficial for the efficient liberation of tocopherols and tocotrienols from cereals. Furthermore,
resolution of the vitamers analysed by HPLC was better with the saponification method.
Previously, comparisons of sample preparation methods consisting of saponification or direct
extraction have yielded inconsistent results. Piironen et al. (1984) preferred saponification due to
its higher total yield of tocopherols and tocotrienols, but Bonvehi et al. (2000) found ca 20%
higher tocopherol values of oils and biscuits when samples were prepared without saponification
than with saponification, and they therefore omitted saponification. One potential reason for the
different results may be that precautions during sample preparation, including addition of
antioxidants, working under subdued light and nitrogen atmosphere, are more important when
the sample is subjected to saponification, because the vitamers are easily oxidized in the presence
of alkali and the procedure must be carefully controlled.
3.3. Effect of solvent polarity on extraction of tocopherols and tocotrienols from saponified mixtures

Development of the saponification step was conducted using n-hexane as the extraction solvent.
A possible need for a more polar extraction solvent mixture to improve the extractability of
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tocopherols and tocotrienols was observed. The use of polarity modifying agents in extraction
solvents has also been suggested earlier (Ueda and Igarashi, 1987a; Eitenmiller and Landen, 1999;
Salo-V.aa. n.anen et al., 2000). Thus, after the saponification parameters had been obtained, three
different extraction solvents were compared: n-hexane, n-hexane:ethyl acetate (9:1) and n-
hexane:ethyl acetate (8:2). The total tocopherol and tocotrienol values obtained from rye flour
using these solvent mixtures were significantly different (ANOVA, Po0:001) giving values of
24.970.5, 28.770.6 and 29.570.3 mg/g (n ¼ 3–5), respectively. The multiple range tests showed
that the mean with n-hexane as solvent was lower than with others (Po0:05). Thus polar
modification of extraction solvent improved the extractability of tocopherols and tocotrienols
present in the hot saponified rye flour mixture. At the same time, minor peaks eluting at the
retention times of g-tocopherol, g-tocotrienol and d-tocotrienol also became detectable. When
Ueda and Igarashi (1987a) investigated the effect of ethyl acetate concentration in n-hexane as an
extraction solvent, they concluded that the addition of 10% ethyl acetate in n-hexane was optimal,
because as the proportion of ethyl acetate increased above this level, some of it partitioned in the
aqueous phase and decreased the volume of the organic layer. In our method, increasing the
concentration of ethyl acetate did not have adverse effects, because the extraction was repeated
three times and the organic layers were collected quantitatively to ensure the recovery of all
vitamers. Thus 20% ethyl acetate in n-hexane was preferred instead of 10%.

3.4. Repeatability and accuracy of the recommended analytical procedure

Finally, the recommended analytical procedure method was evaluated by repeating the analysis
of rye flour and by spiking rye flour samples with tocopherols (Table 3). All analyses were
performed in duplicate over 3 days. The total tocopherol and tocotrienol value was 28.770.1 mg/
g. b-tocotrienol was the major vitamer, followed by a-tocopherol, a-tocotrienol and b-tocopherol.
The repeatability of the method was excellent, since the coefficients of variation (%CV) of these
vitamers were 1.5–3.6%. Even the minor vitamers (g-tocopherol, g- and d-tocotrienols), that had
contents near to the determination limit, had a low %CV of o19%. It should be remembered that
this high repeatability was obtained with a highly homogenous 0.5 g sample of rye flour, and that
greater variations may occur with more uneven materials.
The average recovery of the added tocopherols at the 10 mg/g level was good. The highest
recovery was obtained for a- and b-tocopherols, 94.3% and 93.6%, and the lowest for g-
tocopherol, 90.3% (Table 3). Thus recoveries were all >90% and the variation between vitamers

Table 3
Tocopherol (T) and tocotrienol (T3) contents in mg/g expressed as mean7SD (n ¼ 6) of (1) rye flour and (2) rye flour
with added tocopherols. (3) Recoveries of added tocopherols determined by the recommended analytical procedurea
a-T a-T3 b-T g-T b-T3 g-T3 d-T d-T3 Total
(1) 8.270.1 8.170.3 2.970.1 0.270.02 9.370.3 0.170.01 0.070.0 0.170.01 28.770.1
(2) 17.570.7 8.070.4 13.070.5 9.970.4 9.270.4 0.170.02 10.470.4 0.170.02 68.170.4
(3) 94.3 93.6 90.3 91.0
a
Identification of T and T3 was confirmed by high performance liquid chromatographic mass spectrometric data
except for d-T3.
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was clearly lower than when n-hexane was used as the only solvent. This means that polar
modification improves the extractability of the more polar tocopherols. In the study of Piironen
et al. (1986), recoveries of a-, b-, g- and d-tocopherols were 94.3%, 95.2%, 96.5% and 95.7%,
respectively, with saponification at room temperature. Despite optimization of the saponification
method and careful carrying out of the procedure, hot saponification might destroy tocopherols
and tocotrienols, and recoveries as high as those obtained with room temperature saponification
might not be achieved.

3.5. Evaluation of the analytical HPLC method

Identification of tocopherols and tocotrienols was confirmed on HPLC-MS data (Fig. 4). The
m/z values of protonated molecular ions for a-, b- and g-tocopherols were 431.5, 417.4 and 417.4,
and for a-, b-, g- tocotrienols were 425.4, 411.4 and 411.4, respectively. Verification of d-
tocotrienol was performed with HPLC-FLD derived from the Tokovid-palmoil extract, because
the signal of protonated molecular ion of d-tocotrienol was too low for adequate identification
using LC-MS. This is apparently due to the low ionization efficiency of d-tocotrienol in APCI
process used in this study. However, the use of the mixture of n-hexane and 1,4-dioxane as a
mobile phase enables the more uniform ionization efficiency for individual vitamers compared to
.
the results of Stoggl et al. (2001) when the eluent consisted of isooctane and diisopropyl ether.
The quantitative HPLC method for analysis of tocopherols and tocotrienols was validated by
several detector parameters. Its repeatability was confirmed with an in-house reference material
and its stability by comparing the chromatograms during the 2-month period.
Detection limits varied between 0.10 and 0.18 ng/injection. Determination limits of the vitamers
ranged between 0.30 and 0.54 ng/injection. The detector response was linear in the tested ranges,
2–80 ng/injection (R2 ¼ 0:9998). The variation of the standard curve slopes varied from 0.2% to
0.3% (n ¼ 22), which showed that the stability of the detector response was excellent. The average
tocopherol contents (n ¼ 13) of the in-house reference (rapeseed oil) were: a-tocopherol:

α -T α -T3

30

25

β -T β -T3
20
FLD

15

10

γ -T γ -T3 δ -T3
5

0 2.5 5 7.5 10 12.5 15 17.5 20

Time [min]

Fig. 4. HPLC-chromatogram of rye flour (for details see Section 2).

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264716 mg/g, g-tocopherol 590728 mg/g and d-tocopherol 1977 mg/g, and the %CVs were 6.0%,
4.8% and 36.4%, respectively. These figures show that the HPLC method had good repeatability.
The high variation of d-tocopherol was due to its low concentration, which was near to the
determination limit.
The silica column used proved to be very stable, because the %CVs of the retention times of
tocopherols and tocotrienols over a two-month period were only approximately 0.5% (n ¼ 10).
The retention time of void volume (to ) was 1.96 min (measured with dodecane) and all tocopherols
and tocotrienols were well separated between retention times of 5.5 and 14.2 min. Resolution of
the column was good, being 1.98 (n ¼ 10) for b- and g-tocopherols which are usually difficult to
separate from each other completely.

3.6. Tocopherol and tocotrienol contents of rye

The total combined content of tocopherols and tocotrienols of the rye flour used in the method
development was 28.7 mg/g, of which the amounts of a- and b-tocopherols and -tocotrienols were
8.2, 8.1, 2.9 and 9.3 mg/g, respectively. In an earlier study, the corresponding amounts for whole
meal rye flour were 10.2, 14.4, 3.1 and 11.3 mg/g, and for rye flour 5.5, 4.3, 2.6 and 6.4 mg/g,
respectively (Piironen et al., 1986). Thus the values indicate that some parts of the kernel have
been removed during the milling process of the rye flour used in this study.
The average tocopherol and tocotrienol contents of the ten rye varieties commonly cultivated in
Finland were 48.874.5 mg/g as analysed by the recommended analytical method (Table 4). Total
E vitamer contents were higher than in the rye flour used, because whole grains were milled for
samples without removing outer layers of the kernel. The best source of tocopherols and
tocotrienols was the variety Akusti, 54.3 mg/g, and the poorest was Picasso, 39.9 mg/g. The overall
variation between the varieties was 9.3%, which is slightly more than the variation in folate and
plant sterol contents that were earlier shown to be 8% and 6%, respectively (Kariluoto et al.,
2001; Piironen et al., 2002). There was an apparent although not significant (P ¼ 0:051) positive

Table 4
Tocopherol (T) and tocotrienol (T3) contents of 10 rye varieties in mg/g. Mean of duplicate determinationsa
a-T a-T3 b-T g-T b-T3 g-T3 d-T3 Total T T3 T3/T-ratio
BOR9214 13.9 16.2 3.4 0.5 11.7 0.2 0.2 46.1 17.8 28.3 1.6
Akusti 14.3 19.0 3.2 0.6 16.8 0.3 0.2 54.3 18.0 36.3 2.0
Riihi 16.1 16.7 3.9 0.4 10.2 0.2 0.2 47.7 20.5 27.2 1.3
Picasso 10.0 14.7 2.3 0.4 12.2 0.2 0.1 39.9 12.7 27.2 2.1
BOR9414 14.8 17.5 4.1 0.6 13.2 0.3 0.2 50.8 19.6 31.2 1.6
Esprit 12.6 15.3 2.9 0.5 11.9 0.3 0.2 43.6 15.9 27.7 1.7
BOR7068 16.0 17.3 4.0 0.6 15.3 0.3 0.3 53.7 20.6 33.1 1.6
Elvi 14.5 17.8 3.6 0.6 14.2 0.3 0.2 51.3 18.8 32.6 1.7
Anna 15.8 16.5 4.2 0.6 11.8 0.3 0.2 49.4 20.6 28.8 1.4
Voima 16.3 17.6 3.8 0.6 12.2 0.3 0.2 50.9 20.6 30.3 1.5
Average 14.4 16.9 3.5 0.5 13.0 0.3 0.2 48.8 18.5 30.3 1.7
CV% 13.5 7.5 17.3 16.3 15.1 27.3 21.2 9.3 13.9 10.0 15.5
a
Identification of T and T3 was confirmed by high-performance liquid chromatographic, mass spectrometric data
except for d-T3.
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correlation between E vitamer and plant sterol contents. As in the case of rye flour, a- and b-
tocopherols and -tocotrienols were the major vitamers. Our values are in line with earlier studies,
in which total a- and b-tocopherol and tocotrienol contents ranged between 23 and 49 mg/g
(Morrison, 1978), between 32.4 and 54.7 mg/g (Barnes, 1982) and around 32.4 mg/g (Balz et al.,
1992). In Poland, a lower level of 27.7 mg/g of tocopherols and tocotrienols in rye was found
(Zielinski et al., 2001). The difference could be explained by variation between varieties as well as
by variation between the methods of analysis. The authors extracted tocopherols and tocotrienols
directly from rye with 80% methanol before NP-HPLC analysis.
In the ten rye variety samples, tocopherol and tocotrienol contents varied between 17.8 and
20.6 mg/g and between 27.2 and 36.3 mg/g, respectively, and the tocotrienols to tocopherols ratio
between 1.3 and 2.1 (Table 4). In earlier studies, the ratio was slightly lower at 1.6 (Balz et al.,
1992) and 1.1 (Zielinski et al., 2001). The tocotrienols to tocopherols ratio did not correlate with
the total E vitamer contents. For example, the highest ratio was measured for Picasso, which, on
the other hand, had the lowest vitamer level. The best source of tocotrienols was Akusti with a
content of 36.3 mg/g. Thus total vitamer contents and tocotrienols to tocopherol ratios could be
separately considered when new varieties and/or cultivation techniques are developed and tested.
Since other biological activities of tocopherols and tocotrienols have become evident in addition
to vitamin E activity, and the role of tocotrienols as bioactive compounds has emerged, the
tocotrienols to tocopherol ratio has become an important criterion for nutritional quality
(Qureshi et al., 1989; Hood, 1998). This study illustrated that rye grains have a beneficial ratio of
tocotrienols to tocopherols as well as a high level of tocopherols and tocotrienols, with evident
variation between varieties.

4. Conclusions

The optimized small-scale sample preparation method including hot saponification in

combination with a NP-HPLC-FLD procedure was shown to be reliable for the determination
of tocopherols and tocotrienols from cereals. Saponification under carefully controlled conditions
to avoid degradation of the vitamers was shown to be an effective and sufficiently sensitive
method for tocopherol and tocotrienol assay from cereals. Using hot saponification of 25 min, the
time needed for sample preparation could significantly be reduced, and by scaling down the
sample size to 0.5 g, the amounts of solvents needed were also reduced. Polar modification of n-
hexane with 20% ethyl acetate improved the extraction efficacy of the vitamers from the
saponification mixture. With the optimized method, total tocopherol and tocotrienol content of
rye flour was 27.870.1 mg/g, while those with cold saponification and direct extraction with hot 2-
propanol and hexane were lower being 24.571.0 and 24.17 0.8 mg/g, respectively. Thus with an
optimized hot saponification method, higher amounts of tocopherols and tocotrienols were
obtained than with the other methods studied. The NP-HPLC-FLD method was verified to be
sensitive and reliable for the analysis of eight vitamers of tocopherols and tocotrienols. Finally,
the repeatability and accuracy of the optimized procedure was confirmed by analysing rye flour
and its applicability by analysing ten rye varieties for tocopherols and tocotrienols. This study
showed that rye grains possess a beneficial ratio of tocotrienols to tocopherols as well as high
amounts of tocopherols and tocotrienols, although with evident variation between varieties.
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M. Ryynanen et al. / Journal of Food Composition and Analysis 17 (2004) 749–765 763

Acknowledgements

This study was financially supported by the Ministry of Agriculture and Forestry of Finland,
the National Technology Agency of Finland and some Finnish food companies. It was part of the
project ‘‘Bioactive compounds of rye’’ coordinated by Dr. Kirsi-Helena Liukkonen, VTT
Biotechnology. The authors thank Seppo Hovinen from Boreal Plant Breeding Ltd. for the rye
variety samples, Hanna Salminen for technical assistance as well as Dr. Afaf Kamal-Eldin (SLU,
Uppsala, Sweden) for personal consultation.

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