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, EMD Biosciences,
Inc. (USA) and stored at 18
C until use.
The mobile phases were prepared using HPLC grade reagents:
hexaneandisopropanol (Mallinckrodt, USA) andacetic acid(Merck,
Brazil).
Filtration of the samples included use of lter paper n
JP41
(J. Prolab, Brazil), 3mL sterile syringes (Rymco, Colombia), and
polyethylene lter units Millex HV (Millipore, 0.45m, Brazil).
2.3. Equipment
The following equipment were used for extraction of vitamin
E in the samples: microgrinder, model MA 102 (Marconi, Brazil),
vacuum pump model CA (FANEN, Brazil) and rotative evaporator,
Q-344.1 (Quimis, Brazil). The mobile phases were degassed in an
ultrasonic vibrator T-14 (Odontobras, Brazil). A spectrophotome-
ter UV 1601 (Shimadzu, Japan) was used to scan the absorption
spectra of vitamin standards. Analyses were performed by a high
performance liquid chromatography system (Shimadzu, Japan)
equipped with a high-pressure pump, model LC-10AD VP; auto
injector SIL-10AF withloopof 50L; columnLiChrosorb 5m, Si60
Phenomenex, 250mm4mm; and uorescence detector model
RF-10A XL. The systemwas controlled by the software Multi Sys-
tem Class VP 6.1.
2.4. Methods
2.4.1. Collection and sample preparation
The vegetable and egg samples, purchased locally, were brought
to the laboratory where they were stored in refrigerator for a
maximumperiod of 5 days at temperatures between 0
C and +5
C
until extraction and analysis. Vegetable oils were purchased from
local markets. For a better representation of the analysis, care was
taken to purchase oil products that were of the same brand, but
fromdifferent lots.
Flaxseed was bought locally. Sorghumwas developed and sup-
plied by Embrapa Maize and Sorghum (Sete Lagoas, Minas Gerais,
Brazil). The sorghum seeds were manually selected and subjected
to sieving to remove dirt and impurities.
To obtain axseed our, the seeds were crushed in a blender at
mediumspeed for 5min and sieved to obtain a maximumparticle
size of 20 mesh.
For the production of our from sorghum and soybean, the
seeds were ground with the pericarp in a knife grinder (Braben-
der, Germany), with sieve-limit number 0 (zero) for 10min or until
all material passed through the sieve.
2.4.2. Preparation of vitamin standards and analytical curves
Stock solutions were prepared with 5mg of each standard dis-
solved in hexane containing BHT 0.01% and the volume completed
to 100mL, resulting in concentrations of 50ng/mL.
Real concentrations of the vitamin E isomers were veried by
spectrophotometry and corrected using the LambertBeer equa-
tion. The coefcients of molar absorptivity (in E
1%
) were: 70.8 for
-tocopherol, 86.4for -tocopherol, 92.8for -tocopherol and91.2
for -tocopherol in a 96% ethanol solution. Since the specic values
of the molar absorptivity coefcients of tocotrienols are not found
in literature, we used in this study the maximum wavelength of
each tocopherol isomer corresponding to the real concentration of
-, -, - and -tocotrienols, as indicated by Piironen et al. [9]. In
additionto spectrophotometric analysis, standardsolutions of each
of the isomers of vitamin E were individually analyzed by HPLC in
order to conrmtheir purity. As we detected only one peak for each
of the standards, we concluded that they had a high level of purity
and did not co-elute with any other component on the same wave-
length. The percentage of purity of the standards can be seen in
Table 1. Although it was observed that all eight standards showed
high levels of purity, no correction of the gravimetric concentra-
tions could result in errors in the quantication of compounds in
samples.
Analytical curves of the vitamin E isomers were constructed
using ve increasing concentrations of each isomer, with injections
of each concentration in triplicate. A linear correlation between
peak areas and concentrations of each injected isomer was deter-
mined. The linear regressionequationobtainedfor eachisomer was
used to calculate the content of isomers in food samples.
2.5. Optimization of the extraction method
All tests were performed in the Laboratory of Vitamin Analysis
of the Department of Nutrition and Health, Federal University of
Vic osa, Minas Gerais, Brazil.
For the extraction of vitamin E isomers, different methods
described in literature were tested and evaluated in accordance
with the response in resolution and quantication of compounds
by HPLC.
Methods involving saponication using different combinations
of time, temperature and potassium hydroxide (KOH) concentra-
tion, as well as a direct extraction technique with solvents were
evaluated. The procedures tested for extraction prior to chromato-
graphic analysis are presented in Table 2.
Because the method involving direct extraction with solvents
presented the best results in this study, it was used for the extrac-
tion of vitamin E isomers, as described below.
The extraction procedure (based on [11]) involved the extrac-
tion of the isomers, evaporation of solvents and dissolution in a
8498 H.M. Pinheiro-SantAna et al. / J. Chromatogr. A 1218 (2011) 84968502
Table 1
Maximumwavelength, absorbance and purity of standards of tocopherols and tocotrienols in solution of ethanol 96%.
Standard max (nm) Absorbance (A) Gravimetric concentration
a
(g/mL) Spectrophotometric concentration
b
(g/mL) Purity (%)
-T 292 0.380 55.00 53.67 97.58
-T 295.5296.5 0.477 57.00 55.21 96.86
-T 297298 0.475 57.00 52.08 91.37
-T 297298 0.468 56.00 50.23 90.05
-T3 289.5291 0.343 54.00 48.45 89.72
-T3 296 0.447 56.00 51.74 92.39
-T3 298.6299.5 0.497 57.00 53.56 93.86
-T3 297298.5 0.484 56.00 53.07 94.77
a
Uncorrected for reference standard purity.
b
Purity corrections made through HPLC with uorescence detection (excitation at 290nmand emission at 330nm) and spectrophotometer analysis.
known volume. For this, the following steps were taken. Roughly
10g of vegetable and egg and 4g of samples of sorghum, axseed
(our andseeds) andsoy(our) were weightedina tube. Next, 4mL
of heated water (about 80
Ctoconcentrate the
volume of the extract and re-dissolve it later in a known volume.
Subsequently, theextracts werequantitativelytransferredto25mL
volumetric asks and the volume completed witha mixture of hex-
ane and ethyl acetate (85:15, v/v). After complete homogenization
in the ask, an aliquot of 5mL was removed and evaporated under
nitrogen gas, stored in hermetically sealed amber glass asks at
temperatures of 5
Cto0
C
for 50min
Extraction with ethanol
and hexane
Determination of - and
-tocopherol and tocotrienol in
tomato and broccoli
[6]
Saponication at room
temperature
Use of KOH 50%, at room
temperature, overnight
Extraction with ethanol
and hexane
Determination of tocopherols and
tocotrienols in vegetables, fruits
and diets
[9,10]
Direct extraction with
solvents
Use of isopropanol and
mixture of solvents
compound by hexane and
ethyl acetate (85:15, v/v)
Determination of tocopherols in
infant cereals extrudates
[11]
2.5.1. Optimization of HPLC conditions for analysis
The efciency of chromatographic separation of eight vitamin
E isomers was evaluated from tests with different proportions of
components inthemobilephase, owrateandwavelengthof great-
est sensitivity of the detector, according to conditions suggested in
theliteratureandreportedinTable3. For this, weusedtheHPLCand
a normal phase system, with greater efciency for the separation
of isomers, especially -tocopherol and -tocotrienol.
The choice of mobile phase components and the excitation and
emission wavelengths was made considering the use of a normal
phase systemand the conditions described in literature for studies
with similar analyses [7,12]. In most cases, the separation of -
tocopherol and -tocotrienol isomers was the decisive factor in
assessing the conditions tested.
Identication of compounds was performed by comparison
of retention times obtained for standards and samples, analyzed
under the same conditions, by co-chromatography andcomparison
of quantitative results observed in literature to verify consistency
of data.
Analyses of the vitamin E isomers were conducted using
the chromatographic condition that presented the best results.
Thus, the selected conditions included: a normal phase column
LiChrosorb Si60, 5m; 250mm 4mm, uorescence detec-
tion (Ex 290nm and Em 330nm), mobile phase consisting of
hexane:isopropanol:acetic acid (98.9:0.6:0.5), and ow rate of
1mL/min.
After extraction, aliquots (5mL) were dried in nitrogen, redis-
solved in hexane (2mL) and ltered through 0.45m lter units.
Analyses were performed by injecting two different volumes (5
and 50L) for each sample in order to obtain the detection of all
compounds in quantities appropriate for identication and quan-
tication, since some vitamin E isomers can only be adequately
quantied when injected in volumes of 50L, due to their lowcon-
centrations in the samples. Fromthe start of sample processing to
extractiontoinjection, the total time required, for eachsample, was
one and a half hour.
2.5.2. Determination of linearity range
The linearity range was obtainedusing the previously optimized
chromatographic conditions fromthe injection, in triplicate, of six
increasing concentrations of the standard solution of each isomer.
The data obtainedfor the peak areas were usedfor linear regression
analysis. The correlation coefcient (R
2
) obtained in each case was
used to assess linearity [16].
2.5.3. Analysis of the standards recovery
Standards recovery tests were performed from the addition of
knownconcentrations of the different isomers insamples of water-
cress, peppers, parsley, arugula and axseed.
To evaluate recovery, samples were carefully weighed and
homogenized in two tubes in order to obtain two very similar
aliquots. In one sample, standards in concentrations sufcient to
H.M. Pinheiro-SantAna et al. / J. Chromatogr. A 1218 (2011) 84968502 8499
Table 3
Chromatographic conditions tested for determination of tocopherols and tocotrienols in foods by HPLC.
Mobile phase Flowrate (mL/min) excitation and emission (nm) Reference
HX 1.0 290 and 330 [7,12]
HX:IP (99:1) 1.0 285 and 325 [12,13]
HX:IP (99.1:0.9) 1.0 285 and 325 [11,12]
HX:IP (99.4:0.6) 1.0 290 and 330 [12]
HX:IP (99.4:0.6) 1.5 285 and 325 [7,12]
HX:IP (99.5:0.5) 1.0 290 and 330 [12,15]
HX:IP (99.7:0.3) 1.0 290 and 330 [12,14]
HX:IP (99.7:0.3) 1.5 285 and 325 [7,12]
HX:IP:HAC (99.35:0.6:0.05) 1.0 290 and 330 [12]
HX:IP:HAC (99.33:0.6:0.07) 1.0 290 and 330 [12]
HX:IP:HAC (99.33:0.6:0.07) 1.0 285 and 325 [12]
HX:IP:HAC (99.33:0.6:0.07) 1.5 290 and 330 [12]
HX:IP:HAC (99.33:0.6:0.07) 2.0 290 and 330 [12]
HX:IP (99.6:0.6), pH adjusted with HAC 2.0 290 and 330 [12]
HX, hexane; IP, isopropanol; HAC, acetic acid.
Table 4
Evaluation of extraction conditions for determination of tocopherols and
tocotrienols in foods by HPLC.
Extraction conditions Resolution of
compounds
a
Analytical
values
b
Saponication under heating +
Saponication at roomtemperature +
Direct extraction with solvents +++ +++
a
Rs: , Rs <0.6; , Rs =0.6; +, Rs =0.7 a 0.8; ++, Rs =0.9; +++, Rs =1.0 a 1.25.
Rs: 0.6, bad; 0.8, weak; 1.0, enough; 1.25, very good.
b
Values of the peak area observed: , lower values; , intermediate values;
+++, higher values.
represent about 50% of the original vitamin content of each sample
were added, and the other tube was used as control. The two tubes
of each sample were then subjected to the processes of extraction
and analysis. All procedures were performed in triplicate and the
samples injected in duplicate. The recovery values were obtained
from the percent difference between the contents analyzed and
added.
2.5.4. Determination of limits of detection and quantication
The limit of detection (LOD) was determined as the concentra-
tion where the ratio between the peak height of interest and the
baseline noise was 3. The limit of quantication (LOQ) was consid-
ered to be 5 times the detection limit [17].
2.5.5. Experimental design and statistical analysis of data
A randomized design was used with 3 repetitions for vegetable
oils, ours and axseed and sorghum; 5 repetitions for soybean
our, and 6 repetitions for vegetables and egg yolk. The contents
of tocopherols and tocotrienols were reported as meanstandard
deviation for each sample. The analysis of variance and Duncan test
at 5% probability were applied to detect differences between the
mean content of isomers of different food groups (vegetables and
herbs/spices, sorghum seedsorghum our, axseedaxseed
our; soybean oil olive oil canola oil). For the soybean our no
statistical analysis were used because there were no similar foods
to be compared with the specic sample. Statistical analysis was
conducted using SAS