Food Chemistry 141 (2013) 11751180

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Validation of a fast and accurate chromatographic method for detailed

quantication of vitamin E in green leafy vegetables
Rebeca Cruz, Susana Casal
REQUIMTE, Laboratrio de Bromatologia e Hidrologia, Faculdade de Farmcia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

a r t i c l e

i n f o

Article history:
Received 23 May 2012
Received in revised form 17 March 2013
Accepted 19 March 2013
Available online 16 April 2013
Keywords:
Method validation
Tocopherols
Lettuce
Green leafy vegetables
NP-HPLC

a b s t r a c t
Vitamin E analysis in green vegetables is performed by an array of different methods, making it difcult
to compare published data or choosing the adequate one for a particular sample. Aiming to achieve a consistent method with wide applicability, the current study reports the development and validation of a fast
micro-method for quantication of vitamin E in green leafy vegetables. The methodology uses solid
liquid extraction based on the Folch method, with tocol as internal standard, and normal-phase HPLC
with uorescence detection. A large linear working range was conrmed, being highly reproducible, with
inter-day precisions below 5% (RSD). Method sensitivity was established (below 0.02 lg/g fresh weight),
and accuracy was assessed by recovery tests (>96%). The method was tested in different green leafy vegetables, evidencing diverse tocochromanol proles, with variable ratios and amounts of a- and c-tocopherol, and other minor compounds. The methodology is adequate for routine analyses, with a reduced
chromatographic run (<7 min) and organic solvent consumption, and requires only standard chromatographic equipment available in most laboratories.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Vitamin E is an essential nutrient of human diet, which ubiquitously occurs in eight main chemical forms of related structure, tocopherols (a-, b-, c-, and d-) and tocotrienols (a-, b-, c-, and d-).
Among the tocochromanols family, a-tocopherol is believed to present the most biological antioxidant activity, mainly attributed to
inhibition of membrane lipid peroxidation (Schneider, 2005) and
maintenance of membrane stability (Munn-Bosch & Falk, 2004).
These lipophilic compounds are synthesized by photosynthetic
organisms, occurring mainly in leaves and seeds (Munn-Bosch &
Falk, 2004). Green leafy vegetables, like lettuce, represent a good
source of these compounds (Colombo, 2010) but literature data
are still scarce, particularly regarding leafy vegetables other than
lettuce, increasingly consumed in fresh salads. Moreover, the reported amounts are highly variable among cultivars, growth conditions and particularly analytical methods where diverse extraction
and chromatographic approaches are used.
Indeed, choosing a method for quantication of vitamin E is complicated by an array of variations including food matrix, relatively
analytical complexity and, more recently, available technologies.
As regards the solvents used, single solvent extraction has been
described including direct extraction with ethanol for Brassica vegetables (Guzman, Yousef, & Brown, 2012), methanol for rapini
Corresponding author. Tel.: +351 2220428638.
E-mail address: sucasal@ff.up.pt (S. Casal).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.03.099

(Annunziata et al., 2012) and hexane for baby leaf lettuce (Samuoliene, Sirtautas, Brazaityte, & Duchovskis, 2012) and spinach
(Gleize, Steib, & Reboul, 2012) as well as acetone with sonication
for bay leaves (Gmez-Coronado, Ibnez, Ruprez, & Barbas, 2004).
All these techniques are usually fast to perform but lack in
extraction efciency, and thus report lower vitamin E concentrations than actual levels (Ruprez, Martn, Herrera, & Barbas,
2001). Recent methods such as supercritical uid extraction or
SPME are usually expensive and the highly selective molecular
imprinted solid phase extractions are not only expensive but also
laborious to prepare (Puoci et al. 2007). The use of solvent mixtures
of sufcient polarity to ensure full extraction while ensuring
minimal interference for chromatographic analysis seems to be
the best approach. It has already been used for total lipid quantication by several authors (Ferraz, Fiza, dos Santos, Pontes de
Carvalho, & Soares, 2004; Araujo et al., 2013), particularly with
the classic Folch method (Folch, Lees, & Sloane-Stanley, 1957),
which is among the most frequently used methods worldwide in
food and biological samples.
Chromatographic analyses of extracts are usually performed by
RP-HPLC (reversed-phase), which requires solvents compatible
with the lipophilic characteristics of vitamin E, and the chromatographic system does not completely resolve b- and c-isomers
(Irakli, Samanidou, & Papadoyannis, 2011). Moreover, co-extracted
lipids interfere with the chromatographic resolution, requiring
prior saponication (Barba, Esteve, & Frgola, 2011; Annunziata
et al., 2012), which is a complex and time-consuming process that

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R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180

induces vitamin E loss (Ruprez et al., 2001). Recent developments

in chromatographic separation and detection systems (e.g. LCMS)
are typically expensive particularly for routine control methods.
NP-HPLC (normal-phase) has the advantage of allowing direct
use of a polar solvents and, thus, achieving better lipid solubility
and loading capacity (Kamal-Eldin, Gorgen, Peterson, & Lampi,
2000), which is essential for the analysis of minor compounds such
as vitamin E without the need for prior hydrolysis. As regards separation efciency, silica columns provide higher chromatographic
resolution for tocochromanols, and the use of chemically related
compounds as internal standards (e.g. tocol) minimizes potential
losses during sample preparation, while maintaining or improving
reproducibility.
The present work reports the development of a fast and accurate method for quantication of tocopherols in fresh green leafy
vegetables samples based on the Folch extraction method and
NP-HPLC chromatography. It aims to provide a consistent tool
responding to increased interest in tocochromanols characterization, which are nutritionally important, while making use of standard laboratorial chromatographic equipment.

Butylated hydroxytoluene (BHT) was obtained from SigmaAldrich (Germany), ascorbic acid was purchased from SigmaAldrich
(China), n-hexane was HPLC grade from SigmaAldrich (Germany),
and 1,4-dioxane was obtained from SigmaAldrich (p.a., USA).
Methanol (SigmaAldrich, Germany) and dichloromethane (SigmaAldrich, Germany) were of analytical grade. Sodium chloride
was purchased from Merck (Germany) and anhydrous sodium sulfate was obtained from Merck (Germany).
2.2. Standards preparation
Individual stock solutions (5 mg mL 1) of each tocopherol and
tocotrienol were prepared in n-hexane and stored at 20 C, protected from light, as well as a stock solution of tocol at
100 lg mL 1. Tocopherol standards purity (a- and c-) was monitored by spectrophotometry (UV-1800, Shimadzu, Japan), based
on their E1% 1 cm values (Nesaretnam, Yew, & Wahid, 2007). A
mixture of each tocochromanol standard and tocol was prepared,
thus obtaining the chromatogram seen in Fig. 1.
2.3. Sampling and extract preparation procedure

2. Experimental
2.1. Standards and reagents
Tocopherols (a-, b-, c-, and d-) and tocotrienols (a-, b-, and c-)
were acquired from Supelco (USA) and Calbiochem (USA). 2Methyl-2-(4,8,12-trimethyltridecyl)-chroman-6-ol (tocol) (Matreya
Inc., USA) was used as internal standard (IS).

Three different lettuce cultivars (Lactuca sativa L. cvs. Butterhead, Green Leaf, and Red Leaf), watercress (Nasturtium ofcinale
L.), chard (Beta vulgaris L. cv. Rhubarb Chard) and corn salad (Valerianella locusta L.) were analysed. Butterhead lettuce was used during method development and validation procedures.
Fresh samples were bought in local supermarkets from Porto,
Portugal. On the arrival at the laboratory, the vegetables were

Fig. 1. NP-HPLC-FLD chromatogram of a standard solution. (1) a-Tocopherol, RT = 1.74 min; (2) a-tocotrienol, RT = 2.04 min (3) b-tocopherol, RT = 2.79 min;
(4) c-tocopherol, RT = 3.14 min; (5) b-tocotrienol, RT = 3.45 min; (6) c-tocotrienol, RT = 3.92 min; (7) d-tocopherol, RT = 4.77 min; and (8) tocol, RT = 5.88 min.

R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180

1177

Fig. 2. Spectra obtained for a-tocopherol and c-tocopherol in a Butterhead lettuce sample with the DAD detector.

cleaned and washed with deionized water, dried, and the inedible
portion was discarded. Afterward, samples were homogenised in
a food processor (Silvercrest, Germany), and immediately sampled
for the chemical analyses. Small portions of nely ground fresh
sample (<1.5 g) was weighed accurately into amber glass vials containing ascorbic acid (50 mg), BHT (1 mg) and IS (1 lg). Samples
were homogenised with methanol (2 mL) by vortex mixing for
1 min. Then, dichloromethane (4 mL) was added and vortex mixed,
for 1 min. Subsequently, 0.9% (w/v) NaCl (1 mL) was added, the
mixture was homogenised (1 min), centrifuged (3 min, 5000 rpm)
and the clear lower layer was transferred to an amber ask. Extraction was repeated twice with dichloromethane. The extracts were
combined and vacuum-dried in a rotary evaporator (Bchi RE
111, Switzerland) at 25 C. The extract was recovered with 1 mL
of n-hexane and anhydrous sodium sulfate was added (around
100 mg). After a further centrifugation (5 min, 8000 rpm), the
supernatant was analysed immediately.
2.4. HPLC equipment and chromatographic separation
Analyses were carried out using an integrated system with a data
transmitter (Jasco LC NetII/ADC, Japan), pump (Jasco PU-980,
Japan), a refrigerated auto-sampler (Jasco AS 2057 Plus, Japan)
and a uorescence detector (Jasco FP-2020 Plus, Japan) programmed for excitation at 290 nm and emission at 330 nm, gain
10. Data were analysed using ChromNAV Control centre JASCO
Chromatography Data Station.
The chromatographic separation was achieved with a normalphase column (Supelcosil LC-SI; 7.5 cm  3 mm; 3 lm)
(SigmaAldrich, Germany). The isocratic system comprised a
mobile phase mixture of 1,4-dioxane in n-hexane (2.5%, v/v) at a
ow rate of 0.70 mL min 1, operating at constant room temperature (22 2 C), and the injection volume was 20 lL. The compounds were identied by chromatographic comparisons with
authentic standards and against UV spectra comparison (Fig. 2)
using a PDA detector (Jasco MD-2015 Plus, Japan) connected in series. Quantication was based on the uorescence signal response,
using the internal standard method.
3. Results and discussion
3.1. Extraction performance and chromatographic conditions
Single-solvent extractions have already proven to be inefcient
for the extraction of lipophilic compounds in several matrices
(Ferraz et al., 2004; Araujo et al., 2013). Based on published methods for vitamin E analysis in other food matrices (Annunziata et al.,
2012; Gleize et al., 2012), we used methanol and hexane to extract
vitamin E and analysed it using NP-HPLC. Lower extraction efciencies were obtained in both cases compared with literature data
(data not shown). Acetone extraction was not tested based on
Ruprez et al. (2001).

Such observations inevitably lead to complex extraction methods to ensure the solvent interacts adequately with the analyte
(Ruprez et al., 2001; Araujo et al., 2013). The Folch method uses
chloroform:methanol (2:1) (Folch et al., 1957), although increasingly chloroform is replaced by dichloromethane because of its
reduced toxicity.
We tested the efciency of the Folch method to extract vitamin
E and its impact on chromatographic analysis as detailed in
Section 2.3. In terms of extraction time and solvent consumption,
and in comparison with other recent extraction protocols (Barba
et al., 2011; Annunziata et al., 2012; Slavin & Yu, 2012), our
proposed technique was faster, did not include saponication,
and thus preserved vitamin E from oxidation, and used less organic
solvents per sample (14 mL).
Additionally, chromatographic analysis by NP-HPLC in a short
column provided separation of all tested molecules in a seven minute run (Fig. 1), consuming only 5 mL of eluent. This method is
more suitable for the processing of large numbers of samples than
most published examples (Barba et al., 2011; Gleize et al., 2012;
Guzman et al., 2012; Slavin & Yu, 2012). Furthermore, the addition
of a PDA detector in series allowed real-time conrmation of tocochromanols spectral characteristics, as shown in Fig. 2 for a Butterhead lettuce.
These chromatographic conditions are used in our laboratory on
a routine basis, for several other food matrices, with positive
outcomes (Alves, Casal, & Oliveira, 2009; Malheiro, Casal, Lamas,
Bento, & Pereira, 2012). The analysis of more complex samples
with more tocopherols and tocotrienols benets from the use of
a longer silica column (25 cm) for better peak resolution (Barreira
et al., 2009) but a mobile phase gradient is unnecessary, avoiding
stabilization and therefore reducing run time and solvent
consumption.
3.2. Method validation
The proposed analytical method was validated in terms of
linearity, precision, accuracy and stability for a- and c-tocopherol
only. For each compound, calibration curves were achieved with
six standard concentrations across the working range (Table 1)
and subjected to the entire extraction and analysis procedures.
The high correlation coefcients obtained after extraction (Table 1),
always greater than 0.999, conrm method reliability.
The limits of detection (LOD) and quantitation (LOQ) to determine sensitivity were dened as the lowest concentration of each
compound, over ve replicate injections with real sample extracts,
which gave an average signal-to-noise ratio greater than 3 or 10,
respectively. Only a- and c-tocopherol were detected in signicant
amounts, despite the tentative identication of d-tocopherol and
a-tocotrienol in some samples, which were clearly below quantiable amounts (Fig. 3). The limits of detection and quantication
ranged from 6 ng mL 1 to 9 ng mL 1 and from 21 ng mL 1 to
29 ng mL 1, respectively (Table 1). These values are below

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Table 1
Method analytical parameters for a- and c-tocopherol content in Butterhead lettuce cultivar.
Compounds

Retention time

a-Tocopherol
c-Tocopherol
Tocol

Min

CV (%, n = 6)

1.84
3.40
6.15

1.0
0.8
1.5

Correlation
coefcient (r2)

Working
range (lg mL

0.9994
0.9995

0.0518.0
0.0518.5

Limits
1

LOD (ng mL
8.8
6.4

LOQ (ng mL

29.4
21.2

CV, coefcient of variation; LOD, limit of detection; LOQ, limit of quantication.

Fig. 3. Examples of NP-HPLC-FLD chromatographic prole of several green leafy vegetables analysed. Peak identication according to Fig. 1.

0.02 lg/g fresh weight and comparatively lower (i.e. more sensitive) than published examples (Gmez-Coronado et al., 2004; Irakli
et al., 2011; Annunziata et al., 2012; Slavin & Yu, 2012).
In order to evaluate the assay precision, six sample extracts
were prepared on non-consecutive days. Intra-day and inter-day
precisions below 5% (Table 2) were achieved. These values are

comparable with data found in the literature including those of

Annunziata et al. (2012) (intra-day precision <2.1% and 2.2%;
inter-day precisions <2.7% and 2.6%, for a- and c-tocopherol,
respectively) and Gleize et al. (2012) (intra-day precision <1.9%
and inter-day precisions <3.1%, for a-tocopherol). However, our
method exhibited better repeatability and reproducibility than

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R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180

Table 2
Method validation features of a- and c-tocopherol content in Butterhead lettuce cultivar.
Compounds

Instrumental precision
CV (%, n = 6)

Precision
Intra-day CV (%, n = 6)

Inter-day CV (%, n = 6  2)

a-Tocopherol
c-Tocopherol

2.7
2.7
4.4

2.4
4.1

2.9
4.8

Tocol

Accuracy (%, n = 5)

96.2 3.3
99.8 5.9

CV, coefcient of variation.

Table 3

a-Tocopherol and c-tocopherol content in green leafy vegetables (lg 100 g

Compounds

a-Tocopherol
c-Tocopherol

fresh weight; n = 4).

Lettuce cultivar
Butterhead

Green leaf

Red leaf

340 6
625 21

147 4
511 24

364 5
493 14

Puoci et al. (2007) (intra-day precision <3.3% and inter-day precisions <6.5%, for a-tocopherol), and Slavin and Yu (2012) (intraday precision <5.3% and inter-day precisions <6.9%, for c-tocopherol). Instrumental precision was also estimated for both retention
time (Table 1) and area counts variation (Table 2) by injecting the
same extract six times consecutively. Accurate temperature control was also a determinant for retention time precision, but the
area counts ratios were not inuenced by slight retention time
variations.
The accuracy of the method was assessed by the standard addition procedure with two addition levels (25% and 50% of the
expected values, each one executed ve times), which revealed
notable recovery outcomes (>96%) for both tocopherols (Table 2).
The present method provided higher extraction yields than those
reported by Annunziata et al. (2012) (93.2 1.5% for both a- and
c-tocopherol in rapini), Barba et al. (2011) (89.1% for c-tocopherol
in vegetable beverages), Kim, Giraud, and Driskell (2007) (9093%
for both a- and c-tocopherol in vegetables including lettuce) and
Puoci et al. (2007) (60% for a-tocopherol in bay leaves).
Extract stability was evaluated daily over a period of a week
during storage at 4 C, with losses of less than 10%.

3.3. Method application to green leafy vegetables

In order to extend the method applicability to green leafy vegetables other than lettuce, six different foodstuffs were evaluated for
vitamin E content (Table 3). Low standard deviations were obtained
among the four extracts prepared for each sample (RSD < 5%)
(Table 3) supporting method reliability and applicability. Representative chromatograms for each are presented in Fig. 3. In lettuce
samples, c-tocopherol was the major tocochromanol with amounts
ranging from 479 lg 100 g 1 to 645 lg 100 g 1 (fresh weight). In
contrast, a-tocopherol in chard, watercress, and corn salad was
the most abundant, especially in the former. Some vestigial
amounts of d-tocopherol, a- and c-tocotrienol were also present
in some samples (Fig. 3). Comparing a- and c-tocopherol levels in
Butterhead and Leaf lettuce samples, we obtained higher concentrations for both compounds than those reported by Chun, Lee,
Ye, Exler, and Eitenmiller (2006). This may be because of the aggressive extraction used by those authors (high temperature saponication and sonication), which destroyed the tocochromanols, as
described by Ruprez et al. (2001). Kim et al. (2007) also obtained
lower a- and c-tocopherol concentrations in lettuce samples, probably because they used only hexane for extraction as suggested by
Samuoliene et al. (2012); we proved hexane alone to be insufcient
for full extraction of tocochromanols.

Watercress

Chard

Corn salad

522 19
14 1

716 11
129 3

458 14
31 1

4. Conclusions
This study focused on developing a micro-method for detailed
and accurate determination of the main tocopherols in green leafy
vegetables with adequate sensitivity, precision and accuracy while
being simultaneously rapid, economic and requiring only standard
chromatographic equipment available in most analytical laboratories. In comparison with other published methods, the proposed
method is much faster to execute, and offers better accuracy, with
comparable precision, and increased sensitivity while enabling
analysis of more samples in a short period as well as reduced consumption of organic solvents. Being based on the classical Folch
method, the protocol could be used for other analyses.
This method is, thus, suitable for routine analyses, and can be
used for green vegetables quality control or other related studies.
Acknowledgments
We acknowledge FCT (Fundao para a Cincia e a Tecnologia)
and FEDER through the COMPETE program under the projects
FCOMP-01-0124-FEDER-008703
(FCT/PTDC/AGR-AAM/102447/
2008) and PEst-C/EQB/LA0006/2011. Rebeca Cruz acknowledges
the research scholarship within the same project.
References
Alves, R. C., Casal, S., & Oliveira, M. B. P. P. (2009). Tocopherols in espresso coffee:
Analytical method development and validation. Food Chemistry, 115,
15491555.
Annunziata, M. G., Attico, A., Woodrow, P., Oliva, M. A., Fuggi, A., & Carillo, A. (2012).
An improved uorimetric HPLC method for quantifying tocopherols in Brassica
rapa L. subsp. sylvestris after harvest. Journal of Food Composition and Analysis,
27, 145150.
Araujo, G. S., Matos, L. J. B. L., Fernandes, J. O., Cartaxo, S. J. M., Gonalves, L. R. B.,
Fernandes, F. A. N., & Farias, W. R. L. (2013). Extraction of lipids from microalgae
by ultrasound application: Prospection of the optimal extraction method.
Ultrasonics Sonochemistry, 20, 9598.
Barba, F. J., Esteve, M. J., & Frgola, A. (2011). Determination of vitamins E (a-, c- and
d-tocopherol) and D (cholecalciferol and ergocalciferol) by liquid
chromatography in milk, fruit juice and vegetable beverage. European Food
Research and Technology, 232, 829836.
Barreira, J. C. M., Alves, R. C., Casal, S., Ferreira, I. C. F. R., Oliveira, M. B. P. P., &
Pereira, J. A. (2009). Vitamin E prole as a reliable authenticity discrimination
factor between chestnut (Castanea sativa Mill.) cultivars. Journal of Agricultural
and Food Chemistry, 57, 55245528.
Chun, J., Lee, J., Ye, L., Exler, J., & Eitenmiller, R. R. (2006). Tocopherol and tocotrienol
contents of raw and processed fruits and vegetables in the United States diet.
Journal of Food Composition and Analysis, 19, 196204.
Colombo, M. L. (2010). An update on vitamin E, tocopherol and tocotrienol
Perspectives. Molecules, 15, 21032113.
Ferraz, T. P. L., Fiza, M. C., dos Santos, M. L. A., Pontes de Carvalho, L., & Soares, N. M.
(2004). Comparison of six methods for the extraction of lipids from serum in

1180

R. Cruz, S. Casal / Food Chemistry 141 (2013) 11751180

terms of effectiveness and protein preservation. Journal of Biochemical and

Biophysical Methods, 58, 187193.
Folch, J., Lees, M., & Sloane-Stanley, G. H. (1957). A simple method for the isolation
and purication of total lipids from animal tissues. The Journal of Biological
Chemistry, 226, 497509.
Gleize, B., Steib, M., & Reboul, E. (2012). Simple and fast HPLC method for
simultaneous determination of retinol, tocopherols, coenzyme Q10 and
carotenoids in complex samples. Food Chemistry, 134, 25602564.
Gmez-Coronado, D. J. M., Ibnez, E., Ruprez, F. J., & Barbas, C. (2004). Tocopherol
measurement in edible products of vegetable origin. Journal of Chromatography
A, 1054, 227233.
Guzman, I., Yousef, G. G., & Brown, A. F. (2012). Simultaneous extraction and
quantitation of carotenoids, chlorophylls, and tocopherols in brassica
vegetables. Journal of Agricultural and Food Chemistry, 60, 72387244.
Irakli, M. N., Samanidou, V. F., & Papadoyannis, I. N. (2011). Development and
validation of an HPLC method for the simultaneous determination of
tocopherols, tocotrienols and carotenoids in cereals after solid-phase
extraction. Journal of Separation Science, 34, 13751382.
Kamal-Eldin, A., Gorgen, S., Peterson, J., & Lampi, A. (2000). Normal-phase high
performance liquid chromatography of tocopherols and tocotrienols:
Comparison of different chromatographic columns. Journal of Chromatography
A, 881, 217227.
Kim, Y., Giraud, D. W., & Driskell, J. A. (2007). Tocopherol and carotenoid contents of
selected Korean fruits and vegetables. Journal of Food Composition and Analysis,
20, 458465.

Malheiro, R., Casal, S., Lamas, H., Bento, A., & Pereira, J. A. (2012). Can tea extracts
protect extra virgin olive oil from oxidation during microwave heating? Food
Research International, 48, 148154.
Munn-Bosch, S., & Falk, J. (2004). New insights into the function of tocopherols in
plants. Planta, 218, 323326.
Nesaretnam, K., Yew, W. W., & Wahid, M. B. (2007). Tocotrienols and cancer:
Beyond antioxidant activity. European Journal of Lipid Science and Technology,
109, 445452.
Puoci, F., Cirillo, G., Curcio, M., Iemma, F., Spizzirri, U. G., & Picci, N. (2007).
Molecularly imprinted solid phase extraction for the selective HPLC
determination of a-tocopherol in bay leaves. Analytica Chimica Acta, 593,
164170.
Ruprez, F. J., Martn, D., Herrera, E., & Barbas, C. (2001). Chromatographic analysis
of a-tocopherol and related compounds in various matrices. Journal of
Chromatography A, 935, 4569.
Samuoliene, G., Sirtautas, R., Brazaityte, A., & Duchovskis, P. (2012). LED lighting and
seasonality effects antioxidant properties of baby leaf lettuce. Food Chemistry,
134, 14941499.
Schneider, C. (2005). Chemistry and biology of vitamin E. Molecular Nutrition & Food
Research, 19, 730.
Slavin, M., & Yu, L. (2012). A single extraction and HPLC procedure for simultaneous
analysis of phytosterols, tocopherols and lutein in soybeans. Food Chemistry,
135, 27892795.