902 KONINGS ET AL. : JOURNAL OF AOAC INTERNATIONAL VOL. 79,
4,1996
FOOD COMPOSITION AND ADDITIVES
Liquid Chromatographic Determination of Tocopherols and
Tocotrienols in Margarine, Infant Foods, and Vegetables
ERIK J.M. KONINGS, HARRY H.S. ROOMANS, and PAUL R. BELJAARS
Inspectorate for Health Protection, Food Inspection Service, PO Box 2516, 6201 GA Maastricht, The Netherlands
A liquid chromatographic (LC) method was devel­
oped for determination of tocopherols and to­
cotrienols in foods. Tocopherols and tocotrienols
were released after saponification of test portions
for 40 min at 80°C, followed by extraction with hex-
ane-ethyl acetate. After evaporation of the organic
solvents, the extract was injected into the LC sys­
tem. Separation of individual tocopherols and to­
cotrienols was satisfactory. Some interferences
were encountered from tocomoneols, tocodienols,
2-terf-butyl-4-hydroxyanisole (BHA), 2,6-di-fert-bu-
tyl-4-methylphenol, (BHT), and plastochromanol-8.
The response of the LC system was linear over a
range of 0-10 jig/mL for tocopherols and to­
cotrienols. The detection limit for these com­
pounds was 0.1 ng/mL. Repeatabilty relative stand­
ard deviation values for tocopherols and to­
cotrienols in margarine, infant formula, and
broccoli (concentration range, 0.11-22 mg/100 g)
varied from 1.3 to 6.4%. Recoveries ranged from 91
to 105%.
ancer, heart disease, and vascular disease occur fre­
C quently in industrialized countries. Recent investiga­
tions show the protective effects of antioxidants from
foods against these diseases (1). In particular, vitamin E, vita­
min C, (3-carotene, and selenium exert a positive influence on
the prevention of these diseases as was found in various
epidemiological studies. Results from intervention studies are
now needed to confirm these findings.
Vitamin E consists of a group of 8 vitamers, tocopherols (T),
and tocotrienols (T3), all of which have biological activity.
McLaughlin and Weigrauch (2) derived activities for the vari­
ous forms relative to the activity of a-tocopherol, which is
fixed at 1.0 and defined as 1 tocopherol equivalent (TE). TE
values for the other vitamers are ^-tocopherol, 0.4; y-toco-
pherol, 0.1; 8-tocopherol, 0.01; cc-tocotrienol, 0.3; (3-to-
cotrienol, 0.05; and y-tocopherol, 0.01. A complete analysis of
a-tocopherol and its vitamers is needed to establish the total
biological activity of vitamin E.
Received August 30,1995. Accepted by JL January 17, 1996.
No.
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The main sources of vitamin E are vegetable oils, fats, grain,
nuts, and seeds. Recently published values (3-8) are based on
liquid chromatographic (LC) analyses. The purpose of this
study was to develop and validate a routine LC method for the
determination of all tocopherols and tocotrienols in various
foods.
The proposed method is based on saponification of test por­
tions to release tocopherols and tocotrienols, followed by ex­
traction of these vitamers. Extracts are analyzed by LC as de­
scribed by Balz et al. (9, 10). Results for analyses of margarine,
infant formula, and broccoli are presented.
METHOD
Apparatus
Trade names and sources are for user information only.
(a) Homogenizer.—Any food processor is suitable.
(b) Freeze dryer.—Cenco Instruments (Breda, The Nether­
lands), or equivalent.
(c) Water bath.—At80°C.
(d) Rotary evaporator.—Biichi (Flawil, Switzerland), or
equivalent.
(e) LC system.—Equipped with high-pressure pump,
30 jxL injection loop, autosampler, column oven adjustable to
23 °C, fluorescence detector capable of excitation at 295 nm
and emission at 330 nm, electronic integrator and recorder
(Waters Chromatography, Milford, MA). LC mobile phase
flow rate, l.OmL/min.
(f) Spectrometer.—Adjustable between 285 and 300 nm
(Perkin-Elmer Corp., Norwalk, CT, or equivalent).
(g) LC column.—(i) Guard column.—10 x 4.6 mm id,
stainless steel, packed with diol phase (5 jxm particle size,
100 A pore size). Lichrosorb Diol available from E. Merck,
Darmstadt, Germany, is suitable. (2) Separator column.—250
x 4.6 mm id, stainless stell, packed with diol phase (5 \\m par­
ticle size, 100 A pore size) Lichrosorb Diol from E. Merck is
suitable.
Reagents
All reagents should be of analytical purity, unless otherwise
stated. Water used should be Milli-Q grade or equivalent.
(a) Solvents and reagents.—Ethanol (96%, v/v), n-hexane
(LC grade),tert-butylmethyl ether, 2,6-di-terf-butyl-4-methyl-
phenol (BHT), D,L-a-tocopherol, D,L-P-tocopherol, D,L-y-toco-
 KONINGS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 4,1996 903

1%
pherol, D,L-6-tocopherol, D,L-a-tocotrienol, D,L-pVtocotrienol, Table 1. Specific absorption coefficients (A-\ cm) and
D,L-Y-tocotrienol, D,L-5-tocotrienol, ethyl acetate, L(+)-ascorbic maximum wavelengths (kmax) for tocopherols and
acid, phenolphthalein, and potassium hydroxide (all available tocotrienols in 96% (v/v) ethanol solutions3
from E. Merck). nm A1%
Vitamer V.ax> " 1 cm
(b) Phenolphthalein solution.—1 g/L in ethanol.
(c) Hexane-BHT solution (20 mg/L).—Dissolve 20 mg
a-Tocopherol 292 70.8
BHT in 1 L n-hexane. (3-Tocopherol 296 89.4
(d) Potassium hydroxide solution (600 g/L).—Dissolve y-Tocopherol 298 91.4
600 g KOH in 500 mL water. Cool to room temperature and 5-Tocopherol 298 87.3
transfer to 1 L volumetric flask. Dilute to volume with water a-Tocotrienol 292.5 91.0
and mix. p-Tocotrienol 294 87.3

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(e) Tocopherol and tocotrienol standard solutions.—(7)
Stock solutions (50 mg/100 mL).—Dissolve ca 50 mg D,L-a-
tocopherol in ethanol. Transfer to 100 mL volumetric flask and
dilute to volume with ethanol. This solution is stable for
>6 months if stored at -20°C and the air above the solution is
replaced with nitrogen. Similarly, prepare stock solutions for
D,L-|3-tocopherol, D,L-y-tocopherol, D,L-8-tocopherol, D,L-a-to-
cotrienol, D,L-p-tocotrienol, D,L-y-tocotrienol, and D,L-5-to-
cotrienol. (2) Standard working solutions (I; 5 mg/100 mL).—
Pipet 5.0 mL tocopherol stock solution (e, 1) into 50 mL
volumetric flask. Dilute to volume with ethanol and mix. This
solution is stable for >6 months if stored at -20°C under nitro­
gen. Similarly prepare working solutions for the other stand­
ards. Determine absorbance difference (A - A0) for each stand­
ard working solution with spectrometer at suitable
wavelengths, using settings given in Table 1. A is absorbance
of the standard solution and A0 is absorbance of the blank (etha­
nol). Calculate concentration of each standard working solu­
tion. (3) Standard intermediate solution (5 mg/100 mL).—
Prepare mixed standard solution containing 4 tocopherols and
4 tocotrienols. Pipet 5.0 mL of each stock solution (e, 1) into
round-bottom flask. Evaporate to dryness under reduced pres­
sure at 40°C. Dissolve residue in hexane-BHT solution. Trans­
fer to 50 mL volumetric flask and dilute to volume with hex­
ane-BHT solution. Prepare solution weekly and store in
refrigerator. (4) Standard working solutions (II; containing 1,
2, 4, 6, and 10 \ig tocopherol or tocotrienol/mL).—Pipet 1,1,
2, 3, and 2 mL intermediate solution (e, 3) into 50, 25, 25, 25,
and 10 mL volumetric flasks, respectively, and dilute to volume
with hexane-BHT solution. Prepare fresh on day of use.
(f) LC mobile phase.—n-Hexane-te/?-butyl methyl ether
(94 + 6). Dilute 940 mL n-hexane with 60 mL te/t-butyl
methyl ether and degas.
(g) Phase-separatingfilter.—S&S597 HY (Schleicher and
Schuell, Dassel, Germany).
(h) Extraction solvent.—n-Hexane-ethyl acetate (900 +
100). Dilute 900 mL n-hexane with 100 mL ethyl acetate and
dissolve 20 mg BHT in this solution.
Sample Preparation
The described procedure is suitable for assays of vitamin E
in margarine, powdered infant formula, and broccoli.
Gently melt solid margarines at ± 40°C before analysis and
homogenize melted margarine. Suspend powdered infant for­
mula products by mixing 20 g powder with 20 g water at 50°C.
Cut broccoli into small lumps and mix in homogenizer. Freeze-
y-Tocotrienol 296 90.5
8-Tocotrienol 297 89.1
a
Fromref. 11.
dry for 48 h at -40°C, and grind until fine powder is obtained.
Make a homogeneous suspension of 5 g freeze-dried broccoli
in 30 mL water.
Accurately weigh approximate portions of 5.00 g marga­
rine, 20.00 g infant formula, and 35.00 g broccoli suspensions
in 500 mL conical flasks and proceed as described below.
Saponification and Extraction
In the case of margarine, for each gram of fat add 5 mL KOH
solution to test portion; in the case of suspensions, add solid
KOH until KOH concentration is 60% (w/v). For each 5 mL
60% KOH, add 15 mL ethanol and shake flask until all KOH
is dissolved. Add 0.25 g ascorbic acid for each gram of test
portion. Mix, flush with nitrogen, and connect flasks to air
cooler. Saponify contents in water bath at ca 80°C for 40 min.
After saponification, immediately cool to room temperature.
Bring ethanol/water ratio in each flask to 0.3 by adding water;
add 100 mL extraction solvent, stopper flask, and shake con­
tents. Quantitatively transfer mixture to separatory funnel,
shake funnel, and let layers separate. Drain aqueous phase and
extract 2 more times with 100 mL extraction solvent as de­
scribed before. Combine organic phases and wash them with
100 mL portions of water until reaction of washes to phenol­
phthalein is neutral. Dry organic phase by filtration through
phase-separating filter. Evaporate filtrate to dryness under re­
duced pressure (40°C). Dissolve residue in appropriate volume
of hexane-BHT solution.
Determination
Inject 30 jiL sample extract and 30 ^iL standard working so­
lutions II into LC system. Determine peak areas of vitamin E
vitamers in samples and standards. Establish peak heights for
p-tocotrienol and 6-tocotrienol. Construct linear regression
plot of standard curve for each vitamer and calculate concen­
tration of each vitamer in sample.
Results and Discussion
The method is suitable for determination of tocopherols and
tocotrienols in various foods and food products. LC separation
of individual tocopherols and tocotrienols is satisfactory. Fig-
904 KONINGS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No.
ure 1 illustrates separation of 4 tocopherols and 4 tocotrienols.
Stabilities of the stock and the standard working solutions were
checked on each day of use over 6 months by absorbance read­
ings at the wavelengths of maximum absorption. Concentra­
tions were calculated by using the specific absorption coeffi­
cients in Table 1. If the stock and the standard working
solutions are not stored at -20°C under nitrogen or if they are
older than 6 months, concentrations should be determined on
the day of use.
The small shoulders near the p-tocotrienol and 5-tocotrienol
peaks in the Figure 1 chromatogram are caused by cis isomers.
According to the supplier's specifications, the individual stand­
ards contain about 20% cis isomers. A satisfactory approach for
calculating p-tocotrienol and 5-tocotrienol concentrations is
measurement of peak heights instead of peak areas because
peak shoulders have less influence on the final result. Identifi­
cation of peak shoulders as cis isomers in sample chromato-
grams was not possible. The interference of other compounds
such as tocomoneols, tocodienols, 2-tert-butyl-4-hydroxyan-
isole (BHA), di-tert-butyl-4-methylphenol (BHT), and plasto-
chromanol-8, which would result in overestimation of the vi­
tamers, was small. Tocomoneols and tocodienols are related to
tocotrienols but contain fewer double bounds. BHA and BHT
are antioxidants frequently used in foods. Plastochromanol-8 is
structurally related to y-tocotrienol and is found in vegetable
oils (12). Figure 2 shows the LC separation of these com­
pounds and tocopherols and tocotrienols.
BHT elutes immediately after injection (to). Rammell and
Hoagenboom (13) defined peak 2 as a-tocomoneol in palm oil
but had no separation between peak 3 and peak 4, a-to-
cotrienol. Peak 3 is considered to be oc-tocodienol. With the
method described here, it is not possible to discriminate be-
1
IO
T»
7
cu
J 1
!■ ■
l Ul
■1
11
11) I
f extraction.
Ritinttan tins in ukwfeM
Figure 1 . Chromatogram of standard mixture of
tocopherols (± 0.5 ug/mL) and tocotrienols (± 0.4 ug/mL)
in hexane. 1 = oc-tocopherol, 4 = a-tocotrienol, 5 =
{^-tocopherol, 6 = ^-tocopherol, 8 = p-tocotrienol, 10 =
Y-tocotrienol, 11 = S-tocopherol, and 12 = 5-tocotrienol.
4,1996
r 2
•*
ID
«-
cu
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/JL
0.00 10J00
fVivj
i
U
20.00 301)0
Rstntkn tint n ninufcM
40.00
Lj
Figure 2. Chromatogram of mixture of palm oil,
lineseed oil, BHA, and BHT in hexane. 1 = a-tocopherol,
2 = a-tocomoneol, 3 = a-tocodienoi, 4 = a-tocotrienol, 6 =
^tocopherol, 7 = BHA, 8 = p-tocotrienol, 9 =
plastochromanol-8,10 = y-tocotrienol, and 12 =
5-tocotrienol.
tween different stereomers or cis and trans isomers of toco­
pherols and tocotrienols.
Tocopherol and tocotrienol recoveries were improved by
bringing the ethanol/water ratio to 0.3 after saponification and
by using 10% ethyl acetate in the extraction solvent. The toco­
pherol and tocotrienol recoveries from margarine were in­
creased by approximately 25% for a-tocopherol and a-to­
cotrienol, 50% for ^-tocopherol and p-tocotrienol, 55% for
y-tocopherol and y-tocotrienol, and 85% for 5-tocopherol and
5-tocotrienol. Ueda and Igarashi (14,15) reported similar re­
sults for vitamin E determinations in blood and tissue samples.
The response of the LC system was linear over the range
0-10 fig/mL for tocopherols and tocotrienols, with correlation
coefficients >0.999. The detection limit for these compounds
was 0.1 |Xg/mL.
The method was validated for 3 matrixes: margarine, pow­
dered milk-based infant formula, and broccoli (Table 2; 16).
Recoveries were determined by spiking samples with various
concentrations of different standards before saponification and
A typical chromatogram for an infant formula sample is
shown in Figure 3. The chromatogram for soy-based infant for­
mula is similar to that for milk-based formula. Peak 8 is com­
parable in chromatograms for different brands of milk and soy-
based infant formulas. In chromatograms for certain
milk-based infant formulas, peak 9 is smaller and may be as
high as peak 8. This may be explained by the type of fat used.
The mean standard recoveries for tocopherols and to­
cotrienols in margarine, infant formula, and broccoli ranged
from 91 to 105%. Peak purities of tocopherols and tocotrienols
were established in 2 ways: with a method by Haroonetal. (17)
 KONINGS ET AL. : JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 4,1996 905

Table 2. Results and statistical findings of determinations (n -10) of vitamin E vitamers in margarine, powdered
infant formula, and broccoli9

Vitamer Mean, mg/100g sr, mg/100g RSDr, % r, mg/100g Mean recovery, %

Margarine

a-Tocopherol 8.41 0.12 1.4 0.34 92

P-Tocopherol 0.573 0.014 2.4 0.039 97
-^Tocopherol 22.3 0.51 2.3 1.43 93
5-Tocopherol 7.08 0.16 2.3 0.45 91
oc-Tocotrienol 1.85 0.08 4.3 0.22 96
p-Tocotrienol 0.74 0.03 3.6 0.07 105

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■fTocotrienol 3.23 0.09 2.8 0.25 92
8-Tocotrienol 0.85 0.03 3.7 0.08 96

Powdered infant formula

a-Tocopherol 15.71 0.21 1.3 0.59 98

P-Tocopherol 0.135 0.007 5.2 0.020 93
y-Tocopherol 2.40 0.09 3.5 1.43 97
5-Tocopherol 0.114 0.007 6.4 0.020 99
a-Tocotrienol 1.15 0.04 3.5 0.22 100
P-Tocotrienol 0.213 0.007 3.4 0.020 —
y-Tocotrienol 1.97 0.06 3.3 0.18 95
8-Tocotrienol 0.558 0.009 1.5 0.024 96

Broccoli

a-Tocopherol 6.32 0.16 2.5 0.44 98

sr = repeatability standard deviation; RSDr = repeatability relative standard deviation; r = repeatability (2.8 x sr)

and by diode array detection (DAD) for compounds detectable coeluter and therefore its value could not be included in the
in the UV region. All peaks consisted of single compounds ex­ total vitamin E content of broccoli. Peak purities were checked
cept for y-tocopherol in broccoli. The latter compound had a by DAD except for P-tocotrienol and 5-tocotrienol in marga­
rine and infant formula and for (3-tocopherol and 8-tocopherol
in infant formula.
Results were examined for outliers by the Grubbs test at the
p = 0.05 level of significance. One outlier was found in the
recovery series (n = 10) for y-tocopherol, oc-tocotrienol, and
y-tocotrienol in margarine, and another was found in the recov­
eries of 8-tocopherol in powdered infant formula.
Repeatability relative standard deviations (RSDr) for toco­
pherols and tocotrienols in margarine, infant formula, and broc­
coli varied from 1.3 to 6.4% (concentration range, 0.11—
22 mg/100 g). These values for the accompanying levels are
acceptable according to the IUPAC (1989) Harmonized Proto­
col (18), according to which the acceptable within-laboratory
method performance (RSDr) may range from one-half to two-
thirds of the predicted reproducibility relative standard devia­
tions (RSDR) for the levels of interest.
The described procedure determines tocopherols and to­
T r- cotrienols in foods with satisfactory, reliable, and reproducible
0.00 10.00 20.00 30.00 40.00 results. Caution is needed when interpretating results for y-to­
Retention timetominutes
copherol in broccoli because of an unkown impurity in the LC
Figure 3. Chromatogram of tocopherols and peak. Further research is required to check y-tocopherol peaks
tocotrienols in powdered milk-based infant formula. 1 - in chromatograms of other vegetables. The method is suitable
oc-T, 2 = cx-tocomoneol, 4 = a-T3, 5 = p-T, 6 = ^T, 8 = 0-T3, for surveying foods for vitamin E vitamers under routine con­
9 = plastochromanol-8,10 = y T 3 , 1 1 = 5-T, and 12 = 6-T3. ditions.
906 KONINGS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 4,1996
Acknowledgments
We thank E.M. J.P. Houben and K. A. Henriquez of the Food
Inspection Service, Maastricht, The Netherlands, for their help
with the experimental work.
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