Food Anal.
Methods
DOI 10.1007/s12161-017-0943-x
Simultaneous Determination of Alpha-Tocopherol
and Alpha-Tocopheryl Acetate in Dairy Products, Plant Milks
and Health Supplements by Using SPE and HPLC Method
Slavica Sunarić 1 & Jelena Lalić 2 & Ana Spasić 2
Received: 15 November 2016 / Accepted: 21 May 2017
# Springer Science+Business Media New York 2017
Abstract A solid phase extraction technique for sample
cleanup and extraction of α-tocopherol and α-tocopheryl ac-
etate is described and applied to their simultaneous HPLC
analysis in dairy products, plant-based milks, infant formulas,
and pharmaceutical colostrum. The proposed method includes
sample pretreatment by using ethanol, followed by solid-
phase extraction of the organic supernatant on C18 cartridges.
The identification and quantification were done by reversed-
phase HPLC with fluorescence detection (ex 295 nm, em 330
nm) for tocopherol and with UV detector set at 220 nm for
tocopheryl acetate. The mobile phase consisted of 100% ace-
tonitrile. The linear ranges were from 0.02–0.40 μg/mL (dairy
products) and 0.50–5.00 μg/mL (plant milks, infant formulas,
powdered products) for α-tocopherol and those of α-
tocopheryl acetate were from 0.20–2.00 μg/mL (dairy prod-
ucts) and 1.00–10.0 μg/mL (plant milks, infant formulas,
powdered products). The LOD and LOQ of α-tocopherol
were 0.1 and 0.4 μg/mL, while those of the α-tocopheryl
acetate were 2.0 μg/mL for LOD and 6.0 μg/mL for LOQ.
The method gave extraction recoveries from 73–94% with
RSDs values being 5–10%. This method is simple, SPE pro-
cedure is fast and HPLC analysis time is less than 10 min. The
total time of performing analysis per sample is less than 1 h.
The important advantage of the proposed method is the capa-
bility of determining and reporting α-tocopherol and α-
tocopheryl acetate separately. Moreover, the developed
* Slavica Sunarić
ssunaric@medfak.ni.ac.rs
1
Department of Chemistry, Faculty of Medicine, University of Niš,
Niš, Serbia
2
Department of Pharmacy, Faculty of Medicine, University of Niš,
Niš, Serbia
method is suitable for fast screening of the synthetic form of
vitamin E in dairy products, plant-based milks, infant formu-
las, and pharmaceutical colostrum.
Keywords Synthetic vitamin E . Solid-phase extraction .
Liquid chromatography . Food
Introduction
Vitamin E is the major lipid-soluble antioxidant in mammalian
and plant cells located in the cell membranes. It protects poly-
unsaturated lipids from oxidation and also has some functions
in cell signaling and gene expression (Eitenmiller and Lee
2004). Humans and animals cannot synthesize vitamin E;
therefore, fat products of vegetal origin as well as dairy prod-
ucts are the main sources of this vitamin in the human diet.
The term vitamin E is used for α-, β-, γ- and δ-tocopherols
and α-, β-, γ- and δ-tocotrienols, a family of eight molecules
of a related structure named tocols. Tocols are a natural con-
stituent of many foods. All the tocols possess biological activ-
ity; however, α-tocopherol is the most bioactive form. Initial
estimations of the biological activity of the tocopherols and
some of the tocotrienols were established by the rat fetal re-
sorption assay. Compared to the natural RRR-α-tocopherol
form, β-, γ- and δ-tocopherols, α- and β-tocotrienols were
found to be 50%, 10%, 1–3%, 30–50% and 5% of the α-
tocopherol activity, respectively. Bioactivity data of γ- and
δ-tocotrienols are still unknown (Eitenmiller and Lee 2004).
There are natural and synthetic forms of α-tocopherol. The
natural α-tocopherol is the most potent form of vitamin E and
it is found in vegetal and dairy foods of natural origin, with no
extra vitamin E addition. Synthetic α-tocopherol is a racemic
mixture known as all-rac-α-tocopherol (or dl-α-tocopherol)
showing lower biological activity (≈74% of the RRR-α-

tocopherol) based on resorption–gestation test in rats (IOM
2000; Ruperez et al. 2001).
Nowadays, it is a common practice to add different forms of
synthetic vitamin E to food products. The main reasons for that
are particular nutritional purposes and prevention of lipid oxida-
tion, rancidity and deterioration and thus, maintaining food qual-
ity for the extended shelf life. Exogenous vitamin E in milk
powders supplemented with this nutrient appears to be stable
for long storage periods. It is considered that the addition is
necessary to ensure that vitamin is present in sufficient amounts,
as it tends to be destroyed by light, air and food processing (Fox
and Mc Sweeney 1998). For this purpose not only synthetic α-
tocopherol but also the ester forms of α-tocopherol are used. The
synthetic compounds are widely used owing to their cost, avail-
ability and stability. Addition of all-rac-α-tocopherol, tocopheryl
acetate and tocopheryl succinate is allowed by the EC Regulation
(Regulation (EC) No 1925/2006 of the European Parliament and
of the Council 2006). In processed food, dietary supplements,
pharmaceutical and health products tocopheryl acetate is the
most commonly used, because the ester form of α-tocopherol
is more stable to oxidation compared to the corresponding alco-
hol form.
Contents of different forms of vitamin E in foods have been
presented in mg, μmol, α-tocopherol equivalents (α-TEs) or
in international units (IU). For dietary purposes, vitamin E
activity is expressed as α-tocopherol equivalents (α-TEs),
which were defined as 1.0 mg α-tocopherol, 0.5 mg
β-tocopherol, 0.1 mg γ-tocopherol, 0.03 mg δ-tocopherol,
0.3 mg α-tocotrienol, 0.05 mg β-tocotrienol, while the activ-
ities of γ- and δ-tocotrienols were considered to be below the
limit of detection (IOM 2000; WHO/FAO 2004). On the other
hand, one IU was defined as equivalent to 1 mg of all-rac-α-
tocopheryl acetate and 0.91 mg of all-rac-α-tocopherol. α-TE
unit has been the accepted way of reporting vitamin E con-
centration in foods, while the recommended daily allowance
(RDA) for vitamin E is expressed both in IU and α-TE.
There is some controversy regarding the use of these syn-
thetic forms of vitamin E, especially tocopheryl acetate, as a
supplement or food additive. Animal and human studies have
shown that biological activity of tocopherols is dependent on
their chemical form and particular stereochemistry (Brigelius-
Flohe and Traber 1999; Stone et al. 2003; Yang et al. 2009;
Han et al. 2010; Vagni et al. 2011; Kuchan et al. 2016). The
esterified form possesses less bioactivity than natural vitamin
E. Also, the biochemical behavior of this ester is still not
suficiently clear. Natural and synthetic forms of the tocoph-
erols are equally absorbed from the intestinal lumen in the
form of mixed micelles, but the esters need to be further hy-
drolyzed by pancreatic esterases in the gut to the free alcohol
form, which possesses activity in vivo (Mathias et al. 1981;
Schneider 2005; El Hassan et al. 2006). Thus, all-rac α-
tocopheryl acetate is hydrolyzed in the gut to all eight α-to-
copherol stereoisomers and consequently has lower
Food Anal. Methods
bioactivity, approximately 67% of the natural RRR α-tocoph-
erol (Weiser et al. 1996; Kiyose et al. 1997). According to
Food and Drug Administration, tocopherols and α-
tocopherol acetate are generally recognized as safe (GRAS)
substances (U.S. Food & Drug Administration 2015). The
tocopherols used in foods as antioxidants are readily absorbed
and metabolized. Although they are relatively non-toxic, with
LD50 values of 2000 mg per kg (U.S. Food & Drug
Administration 2015), high vitamin E intakes could impact
absorption of the other fat-soluble vitamins, such as vitamin
A, D and K. Moreover, high intakes of vitamin E could have a
pro-oxidant effect (Loughrill et al. 2016).
To determine α-tocopherol in foods, different analytical pro-
cedures were proposed. Nowadays, the most frequently
employed analytical tools are HPLC techniques with UV or
fluorescence detection. Both reversed-phase and normal-phase
high-performance liquid chromatography were used in the vi-
tamin E analysis. The HPLC methods differ with respect to the
sample preparation procedures, the polarity of the stationary
phase and mobile phase composition (Albalá-Hurtado et al.
1997; Ruperez et al. 2001; Escriva et al. 2002; DeVries and
Silvera 2002; Korchazhkina et al. 2006). Ruperez et al.
(2001) and Luque-García and Luque de Castro (2001) reviewed
most common sample pretreatment procedures for
α-tocopherol chromatographic analysis in various matrices.
The traditional methods of α-tocopherol assay in milk and dairy
products usually include a complex and time consuming sam-
ple preparation procedure. The samples are prepared by alkaline
saponification of lipid residues followed by liquid extraction
with organic solvents (diethyl ether or n-hexane). The alkaline
hydrolysis is recommended to remove lipids when analyzing by
RP-HPLC and to release α-tocopherol from the complex sample
matrix. Many authors have concluded that alkaline saponification
is a destructive method resulting in α-tocopherol degradation, as
most procedures use high concentrations of potassium hydroxide
(Blanco et al. 2000; Czauderna and Kowalczyk 2007). Also,
under such conditions, tocopherol esters are completely hydro-
lyzed. In our previous paper, we confirmed that treating spiked
milk with 30–50% KOH resulted in the hydrolysis of the ester
form. This leads to transformation of the tocopheryl acetate into
tocopherol and determination only the total α-tocopherol in milk.
On the other hand, when 10% KOH was used, unsufficiently
purified sample was obtained and a large interfering peak of milk
lipids was observed (Sunarić et al. 2012).
Many authors have suggested different, less destructive,
sample preparation methods for individual or simultaneous
tocopherol and tocopheryl acetate determination in various
matrices. The chromatographic systems applied were either
reversed-phase or normal-phase. The procedures for individual
determination are based on a solid-liquid or liquid-liquid ex-
traction using a large number of organic solvents, or by using
an ultrasonic bath, a supercritical fluid extraction, a small-scale
extraction and a matrix solid-phase dispersion technique (Indyk
Food Anal. Methods
1988; Chase and Long 1998; Turner and Mathiasson 2000;
Salo-Vaananen et al. 2000; Blake 2007; Brabcová et al. 2013;
Tsochatzis et al. 2012; Irakli et al. 2016). Woollard et al. (2016)
described vitamin E ester extraction from the infant formulas
by using the protease. The determination of the ester included a
normal-phase high-performance liquid chromatography. The
authors used α-tocopheryl propionate as the internal standard;
therefore, the fluorescence was measured by a selective dual-
channel detector. In most of the procedures of simultaneous
RP-HPLC determination, organic solvent extraction was per-
formed (Balz et al. 1993; Huo et al. 1999; Ruperez et al. 1999;
Rodas Mendoza et al. 2003; Castan et al. 2005). Also, liquid
extraction with iso-octane followed by simultaneous NP-
HPLC determination of the total vitamin E (dl-α-tocopherol
and dl-α-tocopheryl acetate), vitamin A palmitate and vitamin
A acetate in infant formula was proposed by McMahon et al.
(2013).
Solid-phase extraction (SPE) has also been employed for
the extraction of different forms of vitamin E in food matrices.
SPE is a non-destructive technique which enables selective
extraction avoiding any chemical changes of the sample and
analytes. Several SPE stationary phases (silica cartridges,
coarse-grained kieselguhr, C18, C8, C4, C6H11, HLB) as well
as different extraction protocols have been used depending on
the sample matrix (Braunstein 2006). Among them, C18 car-
tridges provide the highest efficiency being the most popular
sorbent for fat-soluble vitamin analysis.
Although the methods for individual solid-phase extraction
of tocopherol and tocopheryl acetate prevail (Iwase, 2000;
Fedder and Plöger 2005; Braunstein 2006; Blake 2007;
Grigoriadou et al. 2007), several methods for their simulta-
neous extraction and HPLC determination in human foods
and animal feeds have also been developed (Papadoyannis
et al. 1997; Blanco et al. 2000; Terada and Tamura 2004;
Braunstein 2006).
While numerous food products claim to contain Bvitamin
E^, these products may actually contain very different con-
centrations and forms including the most active vitamin E, its
several esters and other derivatives (Eitenmiller and Lee
2004). The levels of tocopheryl acetate in foods with addition
are usually high (5–500 μg/g) (Eitenmiller and Lee 2004). On
the other hand, in most of the food products, the content of T
and TA was not separately labeled or it is unclear which form
of vitamin E a producer declared. In many cases, the amount
of α-tocopherol is labeled and declared by the manufacturer,
while the presence and the content of α-tocopheryl acetate is
not. Also, producers often declare only the Bvitamin E^.
Therefore, the consumers have no information about dietary
intake of the synthetic vitamin E form. For that reason, it is
important to measure vitamin E acetate added to different food
products. Moreover, there is an increasing interest in the anal-
ysis of both α-tocopherol and α-tocopheryl acetate in com-
mercial food and health products.
In this work, we developed a non-destructive method for
the simultaneous isolation of both α-tocopherol and α-
tocopheryl acetate by using solid-phase extraction, followed
by their simultaneous HPLC determination. The method was
applied to dairy products, dairy substitutes, plant milks and
health products. In addition to this, simultaneous α-tocopherol
and α-tocopheryl acetate determination in commercial al-
mond, hazelnut and soy milks was described for the first time.
Materials and Methods
Materials and Reagents
The dl-α-tocopherol (1.1 units/mg) and dl-α-tocopheryl ace-
tate (1.00 units/mg) of analytical grade were purchased from
Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol
and acetonitrile used in this work were supplied by J.T.Baker
(Deventer, Netherlands). Ethanol, (95–96%, Ph.Eur.V) was
purchased from Zorka-Pharma (Šabac, Serbia). L-(+)-ascorbic
acid (99%) was from Sigma-Aldrich (St. Louis, MO, USA).
Apparatus and Chromatographic Conditions
Chromatographic analysis was performed on the Agilent
Technologies 1200 Series apparatus with PDA-UV and FL
detection. For the monitoring of chromatographic system
and data acquisition, the Agilent ChemStation program was
used. HPLC determination was performed at 40 °C using
Restek Ultra IBD column (150 × 3 mm, 3 μm) (Restek,
Bellefonte, PA) with the mobile phase consisted of 100% ace-
tonitrile. Fluorescence detection for α-tocopherol (λex
295 nm, λem 330 nm) and UV detection at 220 nm for α-
tocopheryl acetate were employed. The flow rate was kept at
0.45 mL/min.
Food and Pharmaceutical Products
The food and health products of representative producers were
purchased in local markets. Milk and dairy products were
from the Serbian manufacturers. Starter infant formulas (for
0 to 6 months old) were infant food products with prebiotics.
Commercial pharmaceutical colostrum was Ekolostrum®
capsule. The producer has claimed that this product contains
480 mg of 100% natural bovine colostrum and was made from
the fresh bovine colostrum obtained in the first 16 h after
calving. Colostrum is natural food that has been used in tradi-
tional medicine for hundreds of years. Commercial pharma-
ceutical colostrum is a health supplement and its regular intake
is recommended for slowing the aging process and healing the
body.
Almond milk (1.1% fat) and hazelnut milk (1.6% fat) were
sterilized beverages from the same manufacturer. Both

products have similar composition, except that almond milk
contains 2% almonds, and hazelnut milk contains 2.5%
hazelnuts. Soy milk (1.9% fat) with and without calcium were
claimed to be organic products containing 8% soya, sugar
cane and 0.4% red seaweed. Two different commercial coco-
nut water brands were also included in this study. All plant
milks were imported products.
Whey powder and a substitute for powdered milk were
from Serbian producer. According to the manufacturer’s dec-
laration, a substitute for powdered milk (Palmlat 26/10 EF)
contains whey, milk proteins, as well as vegetable fats.
Analytical Procedures
Preparation of Standards and Working Solutions
Stock standard solutions of dl-α-tocopherol (T), concentration
of 1.0 mg/mL, and dl-α-tocopheryl acetate (TA), concentra-
tion of 1.0 mg/mL, were prepared by dissolving standard sub-
stances in absolute ethanol. For the calibration curves, work-
ing standard solutions were prepared by appropriate dilution
of the stocks aliquots. The solutions were stored at 4 °C in
light-resistant glass bottles and were stable for 1 week. For the
sample preparation, 0.5% (m/m) ascorbic acid solution (AA)
was used.
Sample Preparation
For the liquid food samples preparation the procedure was as
follows: a volume of 1 mL of the sample was measured in a
50-ml centrifuge tube and mixed by vortex with 0.5 mL of
0.5% (m/m) ascorbic acid and 5 mL of ethanol. The sample
was then centrifuged at 4000 rpm and 20 °C for 10 min. The
obtained ethanolic supernatant was purified by solid-phase
extraction. For the powdered food and pharmaceutical sam-
ples (infant formulas, pharmaceutical colostrum, whey pow-
der and a substitute for powdered milk), a quantity of 5 g of
the homogenized sample was dissolved in 50 mL of warm
deionized water (50 °C). After complete dissolving, 1 mL of
the sample was treated in the same way as the liquid foods.
Solid-Phase Extraction
Cartridges Oasis HLB (1 cm3, Waters Corporation, Milford,
Mass., USA), Bond Elut C18 (1 mL/100 mg, Agilent
Technologies, Santa Clara, CA, USA), Chromabond C18ec
(1 mL/100 mg, Macherey-Nagel GmbH, Düren, Germany)
and Chromabond HR-X (1 mL/100 mg, Macherey-Nagel
GmbH, Düren, Germany) were initially tested for the solid-
phase extraction of both compounds of interest. Cartridges
conditioning was done with 1 mL of water followed by
1 mL of methanol and 1 mL of acetonitrile.
Food Anal. Methods
After the cartridge selection and SPE method optimization
and validation, the procedure adopted for extraction was as
follows: 3 mL of the obtained ethanolic supernatant was
passed through the SPE column at a flow rate lower than
1 ml/min. After the sample aspiration, elution with 2 × 1 mL
of methanol was performed at a flow rate about 1 ml/min.
Depending on the examined product and target analyte con-
centrations, the aliquots of 10 to 50 μL of the eluate were
injected into HPLC column.
Method Validation
Validation of the developed method included evaluation of
selectivity, sensitivity, linearity, system suitability, stability,
accuracy, intra- and inter-day precision. The system suitability
tests were performed by replicate injections of the standard
solutions. The examination of the system suitability parame-
ters was conducted in terms of retention time, peak area, the
number of theoretical plates, capacity factor (k′), peak sym-
metry, resolution and selectivity. Mean values and standard
deviations of retention time and peak area were also calculat-
ed. Variation of the chromatographic method was assessed by
intra- and inter-day precision of retention times and peak
areas. Intra-day precision was evaluated by triplicate analysis
of the mixed standard solutions of T (0.5 or 0.17 μg/mL) and
TA (1.0 or 0.33 μg/mL) on the same day and calculated as
RSD. Inter-day variation was checked by injecting the same
standard mixtures on three different days. The number of the-
oretical plates, peak symmetry, capacity factor, resolution and
selectivity were obtained from chromatograms by using
Agilent ChemStation software.
Linearity was determined by constructing the calibration
curves in two concentration ranges (0.02–0.40 μg/mL and
0.50–5.00 μg/mL for T; 0.20–2.00 μg/mL and 1.00–
10.0 μg/mL for TA). The curves were constructed by using
peak area and the external standard method at eight calibration
points of each compound. Calibration standards were ana-
lyzed in triplicate. Linear regression analysis was conducted
to calculate the intercept, slope and linear regression coeffi-
cient (R 2 ) with OriginPro 6.1. Software (OriginLab
Northampton, Massachusetts, USA).
To estimate the chromatographic method sensitivity LOD
and LOQ were calculated based on the analytical parameters
of the calibration curves. The limits of detection (LOD), limits
of quantification (LOQ) and lower limits of quantification
(LLOQ) for α-tocopherol and α-tocopheryl acetate were cal-
culated by using the equations: LOD = 3.3 SEa/b, LOQ = 10
SEa/b and LLOQ = 5 SEa/b, where SEa is the standard devi-
ation of the intercept, and b is the slope. In addition, real LOD,
LOQ and LLOQ were experimentally estimated by injecting
different volumes of the post-extracted spiked fruit yogurt
sample until the signal-to-noise ratio reached ten for LOQ,
five for LLOQ and three for LOD.
Food Anal. Methods
The selectivity of the method was evaluated for different
food samples by checking the peak purity and peak resolution
onto the chromatograms.
Stabilities of T and TA working standard solutions, as well
as of the extracted food samples were tested at +4 °C for 24 h,
48 h and 1 week. Triplicate measurements of the standard
solutions (low and high concentrations) and extracted samples
(milk, almond milk and infant formula) were performed im-
mediately after preparation. The analysis was repeated after
24 h, 48 h and 1 week. The stability of the analytes in the
samples was estimated by comparing the peak area after stor-
age with that of freshly prepared solutions.
The recovery of the extraction procedure was evaluated in
two ways: by applying SPE method to standard mixture of T
and TA, and by the standard addition method. In the first
procedure aliquots of T and TA standards at three concentra-
tion levels (0.17, 0.33, 4.0 μg/mL T and 0.33, 0.67, 4.0 μg/mL
TA) were mixed with 0.5 ml 0.5% (m/m) ascorbic acid and
5 ml of ethanol (in order to simulate the full extractive proce-
dure), followed by centrifugation and solid-phase extraction.
Recoveries were calculated as the ratio of the found and nom-
inal values. In the second procedure, milk samples (1.5% fat)
were spiked with 0.3 or 2.2 μg/mL T and 0.6 or 2.2 μg/mL TA
before and after SPE procedure. The samples spiked after and
before extraction were compared for the recovery test.
The accuracy of the developed method was evaluated by
comparing the results obtained by the proposed SPE method
and results obtained by applying the saponification method.
Two samples were used in the recovery experiments and each
assay was performed in five replicates.
Statistical Analysis
The data obtained from the saponification method and the
developed solid-phase extraction method were subjected to
statistical analysis. All the samples were analyzed in five rep-
licates. The results are presented as the mean value ± standard
deviation. The statistical analysis was performed using F test
and Student’s t test at 95% confidence level.
Results and Discussion
Method Development
Optimization of the Chromatographic Conditions
The chromatographic conditions regarding composition of the
mobile phase, working temperature and flow rate, which were
optimized in our previous work for the α-tocopherol determi-
nation in milk (Sunarić et al. 2012), were initially tried for the
simultaneous determination of α-tocopherol and α-tocopheryl
acetate standard solutions. Recording of the chromatograms
and the system suitability data were analyzed for three differ-
ent columns: Restek Ultra IBD (150 × 3 mm, 3 μm), Zorbax
Eclipse Plus C8 (150 × 3 mm, 3.5 μm) and Zorbax Eclipse
Plus C18 (150 × 3 mm, 3.5 μm). The best resolution, peaks
shapes, sensitivity and peaks areas, limits of detection and
quantification for both α-tocopherol and α-tocopheryl acetate
were obtained on Restek Ultra IBD column. Figure 1 shows
the chromatograms corresponding to T and TA standards.
Also, the retention times were optimal on this column for both
analytes (under 7 min), while on the Zorbax Eclipse Plus C18
the retention times were higher than 17 min. Simultaneous
detection with fluorescence (ex 295 nm, em 330 nm) and
UV (220 and 295 nm) detectors was employed in order to find
an optimal detection mode. On all of the three chromatograph-
ic columns α-tocopherol had a higher peak area with fluores-
cence than UV detection, while acetate ester showed very
weak fluorescence. Although literature data give tocopheryl
acetate absorption maximum at 286 nm and α-tocopherol ab-
sorption maximum between 292 and 298 nm, both substances
had minor signals when detection at 295 nm was used (Fig. 1).
As a result of these observations, and in order to achieve lower
limits of detection, fluorescence detection for T and UV de-
tection at 220 nm for TA were chosen. Any interference from
the solvent at wavelength of 220 nm was avoided by using
100% acetonitrile as the mobile phase. The purity of the chro-
matographic peaks of both analytes at 220 and 295 nm were
proved by PDA detector.
Validation of the Chromatographic Method
α-Tocopherol and α-tocopheryl acetate peaks in the extracted
food samples were confirmed by spiking milk with T and TA
standards before SPE procedure (Fig. 2). Peaks at 220 and
295 nm were monitored by a photodiode array detector and
were identified by retention times and their UV spectra. Under
the conditions adopted, T and TA were fully separated for less
than 8 min with symmetrical and good shaped peaks. α-
Tocopheryl acetate elutes at about 4.42 ± 0.04 min, followed
by α-tocopherol at about 6.92 ± 0.03 min. For the α-
tocopherol peak, no interferences were observed. The resolu-
tion of the tocopheryl acetate peak depended on the sample
matrix. Thus, in spiked milk sample given in Fig. 2, the peak
of TA (Rt 4.38 min) was not completely separated from close-
ly eluting substance, while for the other extracted food sam-
ples (Figs. 3, 4, and 5) no interferences were found.
The chromatographic system suitability parameters, as well
as repeatability of the retention time and peak areas are sum-
marized in Table 1. Intra- and inter-day repeatability was
expressed as a relative standard deviation (RSD). Retention
time of both analytes showed good repeatability and
reproductivity with RSDs being <1%, while for the peak area
RSDs values were higher (1.7–6.8%). As expected, peak area
RSDs were lower for higher T and TA concentrations.
 Food Anal. Methods

Fig. 1 Chromatograms of the standard mixture of α-tocopherol (T) and (ex 295 nm, em 330 nm) and UV (220 and 295 nm) detection. Rt (T)
α-tocopheryl acetate (TA), concentrations of 1 and 2 μg/mL, respectively, 6.8 min; Rt (TA) 4.4 min
obtained on Restek Ultra IBD analytical column by using fluorescence
Food Anal. Methods

Fig. 2 Chromatograms of milk spiked with mixture of α-tocopherol (T) and α-tocopheryl acetate (TA) standards before solid-phase extraction. Spiked
concentrations of T and TA in milk were 4.0 μg/mL. Rt (T) 6.89 min; Rt (TA) 4.38 min

The capacity factor (k′) of both analytes had optimal values
between 2 and 4. The values of k′ larger than 5 lead to longer
retention and analysis time, while values less than 1 are unre-
liable and cause low resolution. In our work, good separation
was obtained with the resolutions in the range of 6.2–9.8. Peak
symmetries of both analytes were between 0.98 and 1.17, and
for the vitamin E acetate these values were better than 1.456
obtained on monolithic HPLC column (Brabcova et al. 2013).
The theoretical plate number (N) was higher than 3000 which
was similar to the value found by Brabcova on monolithic

Fig. 3 Chromatograms of the almond milk 1.1% fat. Rt (T) 6.86 min; Rt (TA) 4.42 min
column (N = 3986). It can be concluded that a good peak
symmetry, resolution and selectivity were achieved with N
between 3080 and 3500 which indicates satisfactory overall
column efficiency.
The calibration curves in two concentration ranges for α-
tocopherol and α-tocopheryl acetate were constructed by mea-
suring peak area of the T and TA working standard solutions.
Under the chromatographic conditions tested, linear relationships
of the standard solutions were obtained in the selected concen-
tration ranges with the linear regression coefficients (R2) higher
than 0.99. Analytical parameters of the calibration curves are
given in Table 2. The results of LOD and LOQ are expressed
as μg/mL of the analytical standards in the chromatographed
solution. The method showed good sensitivity with the low
LOD, LLOQ and LOQ values.
The real limits of detection and quantification depended on a
sample matrix. Matrix interferences were examined by spiking
extracted yogurt which had minor T and no TA content. The fruit
yogurt with strawberries served as a blank, because it had T
concentration about LOD. Signal-to-noise ratios (S/N) were ex-
perimentally obtained as the lowest T and TA concentrations in
Food Anal. Methods
the post-extracted spiked fruit yogurt which met the criteria for
the reliable identification of analyte and for the analyte response
at least 3, 5 and 10 times the response of the blank. The results of
S/N in μg/mL are given in Table 2. Based on S/N values, it can
be concluded that no matrix interferences were observed for α-
tocopherol (S/N values were lower than theoretical LOD, LOQ
and LLOQ), but they were found for α-tocopheryl acetate (S/N
values were two to three times higher than theoretical LOD,
LOQ and LLOQ). This finding confirmed higher sensitivity of
the fluorescence compared to UV detection. Both theoretical and
real detection and quantification limits of vitamin E acetate were
significantly lower (three to six times) than those found by
Brabcova et al. (2013) on monolithic HPLC column (LOD
0.58 μg/mL, LOQ 1.94 μg/mL), which additionally confirmed
column and chromatographic efficiency.
LOD, LLOQ and LOQ of the method were sufficiently low to
determine Tcontent which was expected in most of the processed
foods and dairy products. Regarding TA determination, sensitiv-
ity of the method was acceptable for higher TA concentrations
which is expected to be found in plant milks, milk substitute,
infant formulas and pharmaceutical colostrum.
Food Anal. Methods
Fig. 4 Chromatograms of the hazelnut milk 1.6% fat. Rt (T) 6.86 min; Rt (TA) 4.42 min
Stability of the Samples
As it is expected that the samples will be analyzed on the same
or next day, the short-term stability of α-tocopherol and α-
tocopheryl acetate in the stock solutions and extracted samples
was studied. The stock solutions were stored in a glass vial,
while the extracted samples were in plastic containers or
Eppendorf tubes. All solution were refrigerated at +4 °C.
The stability study showed that α-tocopherol and α-
tocopheryl acetate concentrations were not significantly
changed in stock solutions stored at +4 °C for 24 h, 48 h
and 1 week (deviation is within ±7%). On the other hand,
the concentration of T and TA in the extracted samples stored
at +4 °C for 24 h was decreased (deviation of 15% for T and
10% for TA). After 1 week of being refrigerated at +4 °C the
concentrations of T and TA in processed samples were signif-
icantly changed (deviation higher than 30% for T and 15% for
TA). The results demonstrated that α-tocopherol and α-
tocopheryl acetate had a good short-term stability in stock
solutions refrigerated at +4 °C for 1 week, while extracted
samples were stable for 24 h under the same conditions.
Sample Preparation Optimization
When avoiding the saponification step, Ruperez et al. (2001)
recommended sample treatment with ethanol or methanol pri-
or to the extraction, in order to disrupt the lipoproteins and fat
droplets structures where vitamin E is associated. As many of
our examined samples have high protein and fat content, the
release of vitamin E from lipids and elimination of interfer-
ences from large-scale protein molecules must be done before
SPE treatment. Therefore, optimization of the sample prepa-
ration prior to the solid-phase extraction was a very important
step determining an overall accuracy of the developed meth-
od. The optimization of the sample preparation included se-
lection of the low-toxicity organic solvent capable of protein
precipitation and vitamin E release from the sample matrix
with high recovery. For that purpose, different volumes of
methanol and ethanol were tested. The solutions of strong
acids (such as perchloric acid) were not tried because of an
undesirable aqueous medium in which vitamin E is not solu-
ble and low pH which may affect the stability of the vitamin.
Ethanol, as less polar solvent, was finally selected for the

Fig. 5 Chromatograms of the infant formula. Rt (T) 6.86 min; Rt (TA) 4.43 min
protein precipitation giving higher recoveries (over 80%) for
T compared to the recoveries obtained when using methanol.
An optimal sample to ethanol volume ratio was found to be
1:5 (v/v). To prevent tocopherol oxidation during the sample
preparation, it is recommended to add some of the antioxi-
dants (Ruperez et al. 2001). Higher concentrations of AA
solution would significantly change pH of the sample and
decrease the recovery; therefore, 0.5 mL of 0.5% (m/m) ascor-
bic acid was added in the sample solution.
SPE Procedure Optimization and Validation
A comparative analysis was conducted on four commercially
available SPE sorbents in order to select a proper sorbent for
isolation and retention of both T and TA. Tocopherol and
tocopheryl acetate are slightly polar compounds, therefore,
only less polar sorbents were taken into account. The
octadecyl bonded phases of choice were Chromabond
C18ec (Macherey-Nagel), Bond Elut C18 (Agilent
Technologies) and Oasis HLB (Waters). Additionally, a hy-
drophobic polystyrene-divinylbenzene copolymer phase
Food Anal. Methods
Chromabond HR-X (Macherey-Nagel) was also tested. All
cartridges were conditioned with water, methanol and aceto-
nitrile. In the next step, evaluation of the optimal elution sol-
vents (ethanol, methanol and acetonitrile), their volume
(1 mL, 2 mL and 3 mL) and the washing step with water
was conducted.
Solid-phase extraction protocol was firstly evaluated on
mixture of T and TA standards treated in the same way as
the real food samples. The verification, that α-tocopherol
and α-tocopheryl acetate were retained by the selected sorbent
and then eluted by the appropriate solvent, was obtained by
analyzing retention parameters and UV spectra of the chro-
matographic peaks, as well as by calculating the extraction
recoveries. The best chromatographic peak areas for both
compounds of interest were obtained on Chromabond C18ec
cartridges by using methanol as an elution solvent and by
avoiding the washing step. Extraction recoveries ranged 73–
94% for the mixture of T and TA standards subjected to the
full SPE procedure are given in Table 3.
A solid-phase extraction of commercial milk sample (1.5%
of milk fat) at the selected cartridges was also carried out.
Food Anal. Methods

Table 1 HPLC system suitability parameters made in three replicates at two concentration levels of T and TA

Mean Rt precision Mean area Area precision Number of Peak k′ Resolution Selectivity
Rt (min) (arbitrary theoretical symmetry
Intra-day Inter-day units) Intra-day Inter-day plates
RSD (%) RSD (%) RSD (%) RSD (%)

T 6.92 0.43 0.31 152.14 3.58 3.11 3079 0.98 3.92 7.72 7.42
0.5 μg/mL
T 7.08 0.55 0.32 35.49 5.20 3.50 3572 1.00 4.00 9.82 7.79
0.17 μg/mL
TA 4.42 0.91 0.62 26.56 1.78 3.36 3116 1.17 2.24 6.17 3.06
1.0 μg/mL
TA 4.49 0.45 0.30 9.64 6.80 5.40 3323 0.98 2.18 7.67 2.94
0.33 μg/mL

After the extraction on the C18 and HLB cartridges, tocoph- three repeated extractions of the same spiked milk sample.
erol chromatographic peak was clearly separated. On the other The mean precisions were satisfactory (all RSDs were <10%
hand, the extract obtained after aspiration on HR-X sorbent at two spiked concentration levels) having in mind that for the
did not show any chromatographic peak after the 3rd minute. complex matrices RSDs up to 15% are considered acceptable.
The best chromatographic peak area was obtained on The recoveries of the proposed solid-phase extraction
Chromabond C18ec; therefore, this cartridge was selected method applied to milk sample ranged between 76 and 87%
for the extraction of real samples. In order to estimate matrix depending on the spiked concentrations, which is similar to
interferences in the real sample, UHT milk (1.5% of milk fat) the recoveries of 74–116% in fresh milk and 80–104% in
was spiked with T and TA standards before and after the SPE fortified milk on C18 cartridges reported by Blanco et al.
procedure. Then, the extraction recovery was evaluated ac- (2000) and recoveries of 57.1–88.3% for the fatty foods (pea-
cording to Matuszewski et al. (2003). The results of accuracy nut butter and margarine) on C18 cartridges found by Terada
and precision of the extraction method applied to standards and Tamura (2004). Compared to the procedure given by
and spiked milk samples are summarized in Table 3. The intra- Terada and Tamura (mixed solvent of acetonitrile-2-propanol
day precision of the extraction procedure was assessed for and post-column photochemical reaction for UV and fluores-
cence detection), our SPE method used less toxic and cheaper
Table 2 Analytical characteristics of the HPLC method solvent ethanol for the sample preparation and there was no
additional reaction for analytes detection. Some of the authors
Parameters α-Tocopherol α-Tocopheryl acetate
found higher recoveries, but the sample matrices and
Number of points 8 8 extraction procedures were quite different from those which
Linearity range (μg/mL) 0.02–0.40a 0.20–2.00a we analyzed. Thus, Irakli et al. (2016) obtained recoveries of
0.50–5.00b 1.00–10.0b 93 to 102% for α-, β-, γ- and δ-tocopherols in tomato fruits
Slope ± S.E. 223.7 ± 12.2a 30.0 ± 0.6a on hydrophilic-lipophilic balance cartridges, and Brabcova
219.6 ± 10.9b 31.6 ± 0.8b et al. (2013) found recoveries in the range 96.4–103.6% for
Intercept ± S.E. 8.1 ± 2.4a −2.7 ± 0.8a vitamin E acetate in tablets and powdered dietary supplements
−6.3 ± 18.1b −5.2 ± 1.2b extracted with methanol and ultrasound bath.
Linear regression coefficient (R ) 0.991a
2
0.997a The comparison of the results obtained by SPE-HPLC
0.992b 0.997b
method with the classical saponification followed by HPLC
LOD (μg/mL) 0.04 0.09
method was carried out only for dairy products (UHT milk
LOQ (μg/mL) 0.11 0.26
and soured milk). As stated above, the saponification proce-
LLOQ (μg/mL) 0.06 0.13
dure destroys all of the tocopheryl acetate. Therefore, in the
S/N (1:3) (μg/mL) 0.01 0.2 plant milks, infant formulas and health products only total α-
S/N (1:10) (μg/mL) 0.04 0.6 tocopherol content could be determined. For that reason, com-
S/N (1:5) (μg/mL) 0.02 0.4 parison with the saponification method could not be made for
S.E. standard error the examined samples in Table 6. For UHT milk and soured
a
Calibration graph used for the analysis of the samples with low analyte
milk samples, the results of α-tocopherol determination ob-
concentration tained by using solid-phase extraction were 15–25% lower
b
Calibration graph used for the analysis of the samples with high analyte compared to those found by using the saponification method
concentration (Table 4). Although the SPE method gave lower values than
 Food Anal. Methods

Table 3 Accuracy and precision

of the extraction method Mixture of standards Spiked milk

Concentration T (μg/mL)a TA (μg/mL)a T (μg/mL)b TA (μg/mL)b

(μg/mL) 0.17 0.33 4.0 0.33 0.67 4.0 0.3 2.2 0.6 2.2
Recovery (%) 73.2 85.6 94.0 75.4 84.1 92.5 76.5 85.0 82.1 87.0
RSD (%) (n = 3) 8.3 7.5 5.5 9.5 8.2 8.0 9.2 7.1 10.1 8.5
a
Concentrations of T and TA in the standard mixture
b
Spiked concentrations of T and TA standards in milk

saponification, the advantage of SPE is in simultaneous ex-
traction both α-tocopherol and tocopheryl acetate. By using
the saponification method, tocopheryl acetate is converted to
tocopherol; therefore, this method does not allow tocopheryl
acetate determination.
A statistical analysis by using F test and t test was carried
out in order to compare precision and accuracy of the two
extraction methods. For both UHT milk and soured milk F
(observed) < F (table), therefore, there is no significant differ-
ences between the precision of these two methods. On the
other hand, t (observed) > t (table) which indicates significant
differences in results of the analysis obtained by the two
methods.
Commercial Food and Health Product Analysis
α-Tocopherol and its acetate ester were determined in tripli-
cate by the proposed SPE-HPLC method in different dairy
foods, plant milks and health supplements. The retention
times of TA and T in real samples were 4.33 ± 0.097 min
(RSD 2.2%) and 6.87 ± 0.04 min (RSD 0.6%), respectively.
The corresponding peaks were analyzed regarding their UV
spectra and peak purity. The quantitation was done by using
calibration curves constructed for external standards. Samples
with high quantity of the tocopheryl acetate were assayed by
single point calibration comparing the chromatographic peak
area with that of the corresponding standard. The intra-day
precision, expressed as RSD was in the range of 3–11% for
all of the tested food samples.
In Tables 5 and 6, the results of the analysis of dairy and
health products, plant milks, infant formulas and pharmaceu-
tical colostrum are given. In Table 6, the ratio of tocopheryl
acetate to tocopherol for the products with the detected TA is
also given. Representative chromatograms of the samples
with T and TA content, purified by the solid-phase extraction,
are shown in Figs. 3, 4, and 5. Although it is likely that many
organic compounds from the complex matrices are being co-
eluted with vitamin E forms during the solid-phase extraction
on C18 cartridges, interferences from other co-eluted com-
pounds were additionally eliminated by selecting the appro-
priate detection wavelength. As can be seen in Figs. 3, 4, and
5, very Bclean^ chromatograms containing a few peaks were
obtained for the plant milks and one of the infant formulas.
These peaks were well separated and were not interfered with
the analytes. Also, in these samples, tocopheryl acetate peak
area was higher for UV than for FL detection. On the other
hand, in chromatograms of sour cream, substitute for pow-
dered milk and one of the infant formulas a strong fluores-
cence signal was obtained at retention time very closely to Rt
of tocopheryl acetate. This chromatographic peak had signif-
icantly higher peak area than that one obtained by UV detec-
tion at 220 nm, which indicated co-elution of some fluorescent
compound or fluorescent complex of tocopheryl acetate. The
occurrence of this interference at excitation 295 nm and emis-
sion 330 nm justified the selection of UV detection at 220 nm
instead of fluorescence detection for TA.
Dairy products should contain α-tocopherol, the content of
which is in positive correlation with milk fat and small
amounts of tocopheryl acetate which is added as antioxidant
(Fox and McSweeney 1998). The obtained results for UHT
milk, raw sheep milk, plain yogurt and fruit yogurt are in the
expected ranges for the same products given by Fox and
McSweeney (1998). In sour creams with 20% milk fat and
cream cheese with 19% milk fat a high concentration of α-
tocopherol was found, which was expected due to the high fat
content. Regarding tocopheryl acetate, we found that most of

Table 4 Comparison of the

developed SPE method with SPE method Saponification Recoverya (%) F valueb t valuec
saponification method Found T (μg/mL) ± SD Found T (μg/mL) ± SD

UHT milk 0.31 ± 0.04 0.40 ± 0.03 77.5 1.78 - 4.02

Soured milk 0.36 ± 0.03 0.43 ± 0.02 83.7 2.25 - 4.34
a
Recovery of the developed method calculated as ratio of the value found by SPE and that found by saponification
b
Theoretic F value was 6.39 for the 95% confidence level
c
Theoretic t value was 2.78 for the 95% confidence level
Food Anal. Methods

Table 5 α-Tocopherol and α-

tocopheryl acetate determination Dairy product, Pa Milk fat (%) Declared Found content and forms of RSD (%)
in dairy products content and vitamin E (n = 3)
forms of
vitamin E

Tb TAb Tb ± SD TAb ± SD

UHT milk P1 1.5% / / 0.031 ± 0.003 <LLOQ 9.7

P1 2.8% / / 0.055 ± 0.004 <LLOQ 7.3
A + D3 P1 3.2% / / 0.050 ± 0.002 <LLOQ 4.0
P2 2.8% / / 0.021 ± 0.002 ND 9.5
P3 2.8% / / 0.038 ± 0.003 <LLOQ 7.9
Plain yogurt P1 1.0% / / <LLOQ ND /
P1 2.8% / / 0.009 ± 0.001 ND 11.1
Fruit yogurt P1 0.0% / / <LLOQ ND /
P5 3.2% / / 0.028 ± 0.003 <LLOQ 10.7
Chocolate milk P1 1.0% / / 0.010 ± 0.001 ND 10.0
Kefir P3 2.8% / / 0.031 ± 0.003 <LLOQ 9.7
P4 0.9% / / 0.012 ± 0.001 ND 8.3
Soured milk P1 2.8% / / 0.030 ± 0.002 ND 6.7
P2 2.8% / / 0.032 ± 0.002 ND 6.3
P3 2.8% / / 0.036 ± 0.003 ND 8.3
Sour cream P2 20% / / 0.510 ± 0.015 1.55 ± 0.03 2.9 (1.9c)
P12 20% / / 0.084 ± 0.003 ND 3.6
P3 20% / / 0.085 ± 0.004 ND 4.7
P1 20% / / 0.077 ± 0.004 ND 5.2
Cream cheese P6 19% / / 0.150 ± 0.005 <LLOQ 3.3
Goat UHT milk P4 2.8% / / 0.044 ± 0.002 ND 4.6
Goat yogurt P11 3.2% / / 0.058 ± 0.004 <LLOQ 6.9
Goat sour cream P11 3.2% / / 0.076 ± 0.005 <LLOQ 6.6
Raw sheep milk / / / / 0.092 ± 0.004 / 4.3

ND not detected, SD standard deviation, n number of determination

a
Producer
b
Mean value expressed as mg/100 g
c
RSD for TA determination

our analyzed dairy products either contained TA lower than
LLOQ, or TA was not detected. Only one of the dairy prod-
ucts, sour cream with 20% milk fat from producer 2, contained
high amount of tocopheryl acetate.
Modern food industry promotes plant milks as healty prod-
ucts with high content of vitamin E. At best, the plant milks
should have only natural form of vitamin E. However, because
these products contain high quantities of vegetal oils, the ad-
dition of small amounts of tocopheryl acetate as a food anti-
oxidant is required. As our results showed, the tocopheryl
acetate concentrations in these products were almost four
times higher than α-tocopherol. There is a great interest in
tocopherol and tocopheryl acetate determination in commer-
cial plant milks; however, rapid and simple analytical
methods, as well as data about their concentrations in these
products were not found in the literature. Moreover, we found
only one paper dealing with α-tocopherol concentration in
commercial almond milk (Toro-Funes et al. 2014) and not a
single paper regarding hazelnut milk. Although almond milk
was produced according to the authors’ own production tech-
nology, we considered that comparing the results for α-
tocopherol content could be made. The concentration of α-
tocopherol in our sample is in the range also specified in the
work by Toro-Funes et al. (2014) (3.15–7.92 mg/L). On the
other hand, our examined processed almond milk also
contained a high concentration of added tocopheryl acetate.
Similar results for α-tocopherol and α-tocopheryl acetate
were obtained for the hazelnut milk from the same producer.
As can be seen in Table 6, the producer has only claimed
1.80 mg of d-α-tocopherol per 100 g in both products.
Regarding soya milk, it is interesting that milk with calcium
had three times higher α-tocopherol concentration than the
 Food Anal. Methods

Table 6 α-Tocopherol and α-tocopheryl acetate in plant milks, health products, infant formulas, and pharmaceutical colostrum

Product, milk fat % P Declared content and forms of vitamin E Found content and forms of vitamin E (n = 3) TA/T RSD (%)

Ta TAa Ta ± SD TAa ± SD

Almond milk 1.1% P7 1.8 of d-α-T / 0.58 ± 0.02 2.25 ± 0.06 3.9 3.5 (2.7b)
Hazelnut milk 1.6% P7 1.8 of d-α-T / 0.59 ± 0.02 2.16 ± 0.08 3.7 3.4 (3.7b)
Soy milk 1.9% P14 / / 0.04 ± 0.004 ND / 10.0
Soy milk 1.9% with Ca P14 / / 0.12 ± 0.006 ND / 5.0
Substitute for powdered milk P8 / / 2.1 ± 0.1 3.37 ± 0.15 1.6 4.7 (4.5b)
Whey powder P8 / / < LLOQ ND / /
Infant formula P9 7.1 of vitamin E / 5.72 ± 0.2 6.67 ± 0.18 1.2 3.5 (2.7b)
Infant formula P10 5.7 of vitamin E / 1.29 ± 0.07 2.14 ± 0.09 1.7 5.4 (4.2b)
Pharmaceutical colostrum P16 / / 0.25 ± 0.018 7.64 ± 0.27 30.6 7.2 (3.5b)
Coconut water P13 / / ND ND / /
Coconut water P15 / / ND ND / /

ND not detected, P producer, SD standard deviation, n number of determination

a
Mean value expressed as mg/100 g
b
RSD for TA determination

product without Ca, although both products have the same fat
content. Tocopheryl acetate was not detected in processed
soya milks.
In powdered food products and substitutes, an expect-
edly large content of the tocopheryl acetate was found. It is
known that in these products high amount of the synthetic
vitamin E is usually added according to the Regulation
(EC) No 1925/2006. Pharmaceutical colostrum contained
a very high quantity of the synthetic form of vitamin E,
although the producer declared that a capsule contains
100% natural bovine colostrum powder with no additives
and flavors.
Although tocopheryl acetate in both products of soy
milk was not detected, another chromatographic peak at
7.97 min with significant area and UV spectra correspond-
ing to tocopherol was observed in these samples. Similarly,
in a substitute for powdered milk and sour cream 20% from
the producer 2, unidentified peaks with UV spectra of to-
copherol were registered at 3.8 min. The peaks at 3.8 and
7.97 min were probably other forms of natural or synthetic
vitamin E which we could not reliably identify without
analytical standards. Considering their large peak area,
we believe that there were other allowed synthetic tocoph-
erol esters.
Infant milk formulas had also high content of α-tocopheryl
acetate, since this is the form used for formulation. In many
cases, supplemented all-rac-α-tocopheryl acetate provided
higher than 80% of the vitamin E in these products
(Eitenmiller and Lee 2004). In the last 15 years, there have
been only few papers describing tocopheryl acetate determi-
nation in infant milks, so we compared the results with those
of these authors. Thus, Woollard et al. (2016) found vitamin E
acetate in manufactured formulations in the range 4.5–8.7 mg/
100 g, which is higher than in our examined samples. α-
Tocopherol levels in the examined infant formulas concur
with the values found by Rodas Mendoza et al. (2003) and
Chavez-Servin et al. (2006), while tocopheryl acetate was in a
significantly lower concentration. For the infant formula P9,
TA/T ratio was found to be 1.2 indicating closely the same
amounts of T and TA. For the infant formula P10 this ratio was
1.7, since this product contained TA almost twice as high as T.
Recent research by Loughrill et al. (2016) also indicated that
in infant’s nutrition the majority of vitamin E provided by the
infant formulas is in the form of α-tocopheryl acetate. The
concentrations of tocopheryl acetate in manufactured infant
milks are high and considerably exceed natural tocopherol
content in breast milk. On the other hand, infants have low
vitamin K levels and intake of excess vitamin E can lead to
blood clotting problems (Diplock et al. 1998; Loughrill et al.
2016). For that reason, it is important to control the vitamin E
content in infant foods.
Conclusions
There is a major interest to develop simple analytical methods
for simultaneous α-tocopherol and α-tocopheryl acetate de-
termination, because comon saponification methods could not
detect and quantify tocopheryl acetate. Considering that, now-
adays, addition of TA to processed foods is common, it is
important to control and to distinguish these two forms of
vitamin E.
A solid phase extraction method for α-tocopherol and α-
tocopheryl acetate simultaneous and selective HPLC
Food Anal. Methods
determination was developed and applied to dairy products,
plant milks and health supplements. The very clean chromato-
grams obtained for the most of the real samples justified the
use of the solid-phase extraction in order to remove lipids and
other smaller organic molecules. The results of the recoveries
were mostly satisfactory for both T and TA, having in mind
the complexity of the matrices. Although recoveries were low-
er, the advantage of this method is in avoiding both saponifi-
cation and liquid-liquid extraction. In contrast to the saponifi-
cation, the proposed SPE method is capable of reporting α-
tocopherol and α-tocopheryl acetate separately. Compared
with liquid extraction, the proposed method does not use large
volume of the harmful organic solvents and evaporation step.
In addition, the advantage of the developed method is in a
simple mobile phase (acetonitrile only), as well as simple
sample preparation and extraction by using one cartridge for
both analytes.
By the proposed method, the content of the added synthetic
α-tocopheryl acetate could be determined in food products. To
our knowledge, this is the first study reporting determination
of α-tocopherol and synthetic vitamin E ester in commercial
plant milks. A high content of α-tocopheryl acetate was found
in almond and hazelnut commercial milks. Powdered dairy
products, dairy substitute and a health supplement also
contained α-tocopheryl acetate in much higher quantities than
α-tocopherol form. By applying this method to different food
products, we demonstrated the potential of its use for various
food matrices with a wide range of α-tocopherol and α-
tocopheryl acetate concentrations. In that way, the analytical
range of the method was also demonstrated.
Acknowledgements This research was financially supported by the
Faculty of Medicine University of Niš internal scientific project No 2
(11-14629-4/2) and by the grant TR 31060 from the Ministry of
Education, Science and Technological Development of the Republic of
Serbia.
Compliance with Ethical Standards
Funding This study was funded by the Ministry of Education, Science
and Technological Development of the Republic of Serbia (grant TR
31060) and by the internal scientific project No 2 (11-14629-4/2) of
Faculty of Medicine, University of Niš, Serbia.
Conflict of Interest Slavica Sunarić declares that she has no conflict of
interest. Jelena Lalić declares that she has no conflict of interest. Ana
Spasić declares that she has no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed Consent Not applicable.
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