Method Validation and Determination of Polycyclic Aromatic Hydrocarbons in Vegetable Oils by HPLC-FLD

Abstract

The aim of this work was to determine the level of contamination of different groups of vegetable oils available on the Polish market with polycyclic aromatic hydrocarbons, i.e. benzo(a)pyrene and sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene and chrysene, the content of which in foodstuffs is limited by Commission Regulation (EU) 2015/1125 of 10 July 2015. The research materials were refined rapeseed oils, sunflower oils, olive pomace oil, rapeseed oils with olive oil and unrefined soybean and coconut oils. The research methods included process of saponification of the vegetable oils, extraction of the polycyclic aromatic hydrocarbons fraction, clean-up by use a column packed with aluminum oxide and elution by petroleum ether, and then quantitative and qualitative determination of by high performance liquid chromatography with fluorescence detection method. Values of limit of detection and limit of quantification obtained during validation of the method were 0.18 and 0.25 μg/kg, respectively, and were significantly lower than the respective maximum values given in Commission Regulation (EU) 836/2011. The highest polyaromatic hydrocarbons content was found in unrefined coconut and soybean oils. The benzo(a)pyrene content and sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene and chrysene in all the tested sample did not exceed the maximum levels given in Commission Regulation (EU) 2015/1125.

Full text

Method Validation and Determination of Polycyclic Aromatic
Hydrocarbons in Vegetable Oils by HPLC-FLD
Alicja Zachara
1,2
&Dorota Gałkowska
1
&Lesław Juszczak
1
Received: 15 April 2016 /Accepted: 22 September 2016 / Published online: 5 October 2016
#The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract The aim of this work was to determine the level of
contamination of different groups of vegetable oils available
on the Polish market with polycyclic aromatic hydrocarbons,
i.e. benzo(a)pyrene and sum of benzo(a)pyrene,
benz(a)anthracene, benzo(b)fluoranthene and chrysene, the
content of which in foodstuffs is limited by Commission
Regulation (EU) 2015/1125 of 10 July 2015. The research
materials were refined rapeseed oils, sunflower oils, olive
pomace oil, rapeseed oils with olive oil and unrefined soybean
and coconut oils. The research methods included process of
saponification of the vegetable oils, extraction of the polycy-
clic aromatic hydrocarbons fraction, clean-up by use a column
packed with aluminum oxide and elution by petroleum ether,
and then quantitative and qualitative determination of by high
performance liquid chromatography with fluorescence detec-
tion method. Values of limit of detection and limit of quanti-
fication obtained during validation of the method were 0.18
and 0.25 μg/kg, respectively, and were significantly lower
than the respective maximum values given in Commission
Regulation (EU) 836/2011. The highest polyaromatic hydro-
carbons content was found in unrefined coconut and soybean
oils. The benzo(a)pyrene content and sum of benzo(a)pyrene,
benz(a)anthracene, benzo(b)fluoranthene and chrysene in all
the tested sample did not exceed the maximum levels given
in Commission Regulation (EU) 2015/1125.
Keywords Polycyclic aromatic hydrocarbons .
Benzo(a)pyrene .HPLC-FLD .Vegetable oils
Introduction
Vegetable fats are recognized as important components of the
human diet. They are a source of energy and essential fatty
acids as well as carriers of fat soluble vitamins. Fats are natural
multi-mixture of different lipids, including triacylglycerols
(97-99%) and accompanying substances in proportions de-
pending on the type of raw material and methods of extraction
and purification of the resulting product (Dubois et al. 2007;
Zachara and Juszczak 2016). In addition to the unsaturated
fatty acids content in oilseeds, vegetable oils and fatty prod-
ucts another important factor is the content of compounds with
strong antioxidant properties, which large amounts determine
the appropriate nutritional value and the storage stability of
these products (Krygier et al. 2011). Foods high in fat are
particularly susceptible to contamination with polycyclic aro-
matic hydrocarbons (PAHs). Due to lipophilic character they
accumulate in the fat cells of plants and animals (Moret and
Conte 2000; Wenzl et al. 2006). Vegetable oils may be con-
taminated with PAHs as a result of processing, especially dry-
ing of oil plants and due to use of contaminated extraction
solvent. To a lesser extent a contamination of these oils may
be associated with environmental pollution (Guillen et al.
2004; Lage Yusty and Cortizo Daviña, 2005; Moret et al.
2005;WHO2010; Bojanowska and Czerwiński 2010); how-
ever, in a case of high air pollution with PAHs and due to
atmospheric precipitation, a superficial contamination of
plants during growing season may occur. This contamination
may be then transferred to the final product (Rodríguez-Acuna
et al. 2008). Both storage of seeds in silos and the processes of
deodorization and cleaning affect the reduction of the level of
*Lesław Juszczak
rrjuszcz@cyf-kr.edu.pl
1
Department of Food Analysis and Evaluation of Food Quality,
Faculty of Food Technology, University of Agriculture in Krakow,
Balicka Street 122, 30-149 Krakow, Poland
2
Laboratory of Food Hygiene and Nutrition, Voivodeship
Sanitary-Epidemiological Station in Rzeszow, Wierzbowa Street 16,
35-959 Rzeszow, Poland
Food Anal. Methods (2017) 10:1078–1086
DOI 10.1007/s12161-016-0673-5
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contamination with PAHs. On the other hand, direct impact
of exhaust gases during drying of the seeds and/or impact of
high temperature during extraction of oil from seeds result
in increased amount of polyaromatic hydrocarbons in a final
product. Many researchers observed significant differences
in the level of PAHs in different production batches of the
same type of oil (Cejpek et al. 1998; Tys et al. 2003;Guillen
et al. 2004).
Polycyclic aromatic hydrocarbons were evaluated by the
International Programme on Chemical Safety (IPCS) of the
World Health Organization (WHO) in 1998, the Scientific
Committee on Food of the European Commission in 2002
and the Joint FAO/WHO Expert Committee on Food
Additives in 2005. The International Agency for Research
on Cancer has classified benzo(a)pyrene to the Group 1 of
carcinogens, i.e. compounds with proven carcinogenic effect
to humans, while benz(a)anthracene, benzo(b)fluoranthene
and chrysene to Group 2B (compounds possibly carcinogenic
to humans) (WHO 2010). The research results confirming the
carcinogenic effect of PAHs on humans and the available data
on food contamination with these compounds resulted in the
adoption of the Commission Regulation (EC) 208/2005 of 4
February 2005 and then of the Commission Regulation (EC)
1881/2006 of 19 December 2006, in which maximum levels
for benzo(a)pyrene in foodstuffs have been settled. Further
regulations on maximum levels for polycyclic aromatic hy-
drocarbons in foodstuffs have been given in Commission
Regulation (EU) 835/2011, where benzo(a)pyrene –previous
marker for the occurrence of polycyclic aromatic hydrocar-
bons in food has been replaced with four specific substances:
benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene
and chrysene. Moreover, maximum levels of sum of these four
PAHs in different foodstuffs have been established. It was
found that these compounds are present in foods in varying
amounts, but their sum covers approx. 60% of fifteen carcino-
genic PAHs listed in Commission Regulation (EU) 835/2011.
According to the above mentioned Regulation, oils and fats
(excluding cocoa butter and coconut oil) intended for direct
human consumption or use as ingredients in food can contain
maximum 2 μg/kg of benzo(a)pyrene, while sum of
benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene
and chrysene is limited to the level of 10 μg/kg. Maximum
level of sum of the four PAHs for coconut oil intended for
direct human consumption or use as an ingredient in foodis 20
μg/kg. This is due to proportionally higher benzo(a)pyrene
and chrysene contents, which cannot be easily removed from
coconut oil during refining process with the current technical
possibilities of producing countries (Commission Regulation
(EU) 2015/1125).
ThePAHsinfoodaremainlydeterminedwithuseof
high performance liquid chromatography with fluores-
cence detection or gas chromatography-mass spectrometry
(Sadowska-Rociek et al. 2015). The methods of liquid
chromatography are more often used in food analysis
due to the lower cost of equipment and lower cost of
routine determination of the PAHs limited by law regula-
tion. Moreover, they are sufficient to official food control.
More expensive GC-MS technique is used in scientific
works due to the possibility of determination and identi-
fication of a number of polycyclic aromatic hydrocarbons
which affect food safety (Zachara and Juszczak 2016).
The European Union is the world’s largest producer of
rape –it produces about 19-20 million tonnes per year,
representing 34% of global production. Compared to oth-
er European Union countries, Poland ranks third in terms
of production volume, after Germany and France
(Dmochowska 2012). In Poland, rapeseed oil is the most
popular fat and it is consumed by approx. 50% of con-
sumers (Krygier et al. 2009). Much less people consume
other types of vegetable oils: sunflower or soybean oils –
approx. 4%, while olive oil –approx. 1.5% of consumers
(Zachara and Juszczak 2016). In recent years, increased
consumer interest in vegetable fats is observed and var-
ious types of rapeseed, sunflower, soybean and olive oils
as well as their mixtures appeared on the market.
The current state of knowledge on genotoxic, mutagenic
and carcinogenic properties of polycyclic aromatic hydro-
carbons contributes to the fact that these compounds are of
interest in a broad spectrum of sciences. According to the
assessment of the European Commission it is necessary to
constantly monitor the PAHs content in food, including
fats of vegetable origin, for the continuous monitoring of
risks.
The aim of this work was to determine, using validated
chromatographic method, the level of contamination of differ-
ent groups of vegetable oils available on the Polish market
with polycyclic aromatic hydrocarbons, i.e. benzo(a)pyrene
(BaP) and sum of benzo(a)pyrene (BaP), benz(a)anthracene
(BaA), benzo(b)fluoranthene (BbFA) and chrysene (CHR),
the content of which in foodstuffs is limited by Commission
Regulation (EU) 2015/1125.
Materials and Methods
Determination of polycyclic aromatic hydrocarbons in the
samples was performed by validated method that meets the
criteria set out in the Commission Regulation (EU) 836/2011.
The Laboratory is accredited in this analysis by Polish
Accreditation Centre. The Laboratory participated in the
inter-laboratory study conducted by the National Institute of
Public Health –National Institute of Hygiene, which is the
national reference laboratory and satisfactory results of deter-
mination of four PAHs in oil samples enriched with PAHs at
two levels were obtained.
Food Anal. Methods (2017) 10:1078–1086 1079
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Materials and reagents
For the chromatographic analysis the following reagents
were used: acetonitrile, cyclohexane, methanol (all of
HPLC purity) (Merck, Darmstadt, Germany), sodium
chloride and potassium hydroxide (all of analytical grade)
(POCH, Gliwice, Poland). Water of HPLC purity was
from LiChrosolv (Merck, Darmstadt, Germany), while ni-
trogen of analytical grade was from Air Liquide
(Rzeszow, Poland). For the clean-up procedure, neutral
aluminum oxide of activity stage I for column chromatog-
raphy (Merck, Darmstadt, Germany), petroleum ether of
HPLC purity (Merck, Darmstadt, Germany) and anhy-
drous sodium sulphite of analytical grade (POCH,
Gliwice, Poland) were used. The certified standard PAH
Solution Mix from AccuStandard (New Haven, USA)
consisted of PAHs solution in methanol-dichloromethane
(MeOH-DCM), with 200.6 μg/ml of BaP, 197.8 μg/ml of
BaA, 198.8 μg/ml of BbFA and 199.0 μg/ml of CHR. In
order to validate the method, certified reference material
FAPAS 0618 Olive oil from Central Science Laboratory
(York, UK) was used.
Apparatus
HPLC was used for the determination and quantitation of
each of the PAHs. The UltiMate 3000 (Dionex, Sunnyvale,
U.S.A.) chromatographic system was used, which
consisted of WPS-3000TSL auto-sampler, DGP-3600A
pump, TCC-3200 2×2P-10P thermostated column com-
partment, RF 2000 fluorescence detector, connected to
Chromeleon software (version 6.80 SP2 Build 2284).
The Hypersil Green PAH (tailored alkyl-bonded silica with
high carbon loading; Thermo Scientific, Waltham, U.S.A.)
column (250×4.6 mm, I.D., 5 μm) and guard column
(10×4.0 mm, I.D., 5 μm) were used.
Samples
Samples of edible oils: 30 samples of rapeseed oil, 10
samples of sunflower oil and 12 samples of other oils:
olive pomace oil, rapeseed oil with olive oil, soybean oil
and coconut oil were commercially available. Samples of
rapeseed oil included universal refined oils, virgin rape-
seed oils and virgin cold-filtered refined rapeseed oil and
they were both of domestic origin (12 samples) and
imported (18 samples). Before analysis the samples were
stored according to the manufacturer’s recommendations.
Liquid samples were conditioned at room temperature
within 2 hrs, while solid samples were melted in a water
bath at 60 °C (PN-EN ISO 661:2006).
Analytical procedure
Isolation of the hydrocarbon fraction
In order to get PAH extracts clean enough for chromatograph-
ic analysis the purification step with use of glass chromato-
graphic column packed with alumina was performed. Mixture
of aluminum oxide and water (9:1, w/w) was poured into a
glass column equipped with fritted glass and the column was
tapped to pack it. A anhydrous sodium sulphite as a drying
agent was made on the alumina packing. The column was
conditioning by passing petroleum ether.
A 0.4 g aliquot of oil was diluted with 10 ml of petroleum
ether and the sample was loaded onto the alumina column.
Then, 60 ml of petroleum ether was poured onto the column
with flow rate for approx. 1 ml/min. The first 20 ml eluted
fraction was discarded. The exact fraction (PAH compounds)
was collected. The solution was evaporated in Laborota 4000
rotary evaporator (Heidolph, Schwabach, Germany) at 35 °C
under vacuum to the final volume of less than 1 ml and then
the sample was quantitatively transferred to vials and evapo-
rated under a gentle stream of nitrogen.
In the case of coconut oil samples the liquid-liquid extrac-
tion was preceded by a saponification step. For this purpose, a
2 g aliquot of sample was hydrolyzed with 1.5 M potassium
hydroxide in methanol for 90 min at reflux. The hydrolyzed
sample was then filtered, extracted three times with 50 ml of
cyclohexane and washed three times with 50 ml of water. The
resulting extract was dried by addition of anhydrous sodium
sulphate and the supernatant was concentrated in a vacuum
evaporator at 50 °C. The concentrated solution was quantita-
tively transferred to the 25 ml flask and made up to volume
with petroleum ether. The resulting sample was purified on the
alumina column according to the above described procedure.
Each sample was prepared in duplicate.
HPLC- FLD analysis
An aliquot of 100 μl was injected into HPLC using an auto-
sampler. The temperature of the column was maintained con-
stant at 18 °C. The mobile phase was constituted of acetoni-
trile and water. The elution conditions applied were: 0 –3min,
60%ofacetonitrileisocratic;3–15 min, 60-100% of aceto-
nitrile gradient, 15 –46 min, 100% of acetonitrile isocratic, 46
–53 min, 100-60% of acetonitrile, gradient. The flow rate was
1.0 ml/min. The effluents were monitored using the following
excitation and emission (Ex/Em) wavelengths: 260/420 nm
for BaA and CHR and 290/430 nm for BbFA and BaP.
Preparation of calibration curve
Calibration curves were performed by external standard meth-
od. The working standard solution at a concentration of 10 ng/
1080 Food Anal. Methods (2017) 10:1078–1086
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ml was made with the certified standard solution of PAHs and
it was used to make the following calibration solutions: 0.20,
0.80, 4.00, 8.00 and 10.00 ng/ml. The calibration curves were
plotted as linear dependence (y=a∙x) of the measured signal
(y) in function of the peak area of the standard substance (x).
Measurement of samples
The measurement procedure consisted of dosing the following
samples: blind samples, oil samples, two calibration solutions
(1.00 and 9.00 ng/ml) and oil samples enriched with certified
standard sample or with certified reference material.
Statistical analysis
Determination of the working range and linearity
of the method
For each of PAH calibration curves the coefficients of varia-
tion for concentration limits were calculated and then the F-
Snedecor test was used for testing homogeneity of coefficients
of variation at a 0.05 significance level. Correlation coefficient
(r) was also calculated. The limit of detection (LOD) and the
limit of quantification (LOQ) were calculated according to the
following formulas: C
m
+3SD and C
m
+6SD, respectively,
where C
m
is mean concentration of PAH in a sample of very
low PAH concentration and SD is standard deviation. The
sensitivity of the method was measured as the slope of the
calibration curve.
Statistical analysis of results
Results obtained in the procedure of validation of the method
were tested by Dixon Qtest for identification and rejection of
outliers. Then, for each of PAH concentrations the following
statistical parameters were calculated: recovery factor (cor-
rectness), variance, standard deviation, coefficient of
variation, standard and expanded uncertainties, confidence in-
terval and relative standard deviation of repeatability. The
evaluation of the significance of differences between mean
values of PAHs was made using Tukey multiple comparison
test at a 0.05 significance level. All statistical procedures were
computed using Statistica version 10.0 (StatSoft, Poland).
Results and discussion
The values of the limit of detection (LOD), the limit of quan-
tification (LOQ) and the recovery complied with the criteria
set out in the Commission Regulation (EU) 836/2011
(Table 1). In the concentration range of 0.20-10.00 ng/ml the
calibration curves for each of the standard substances were
linear, with values of correlation coefficient higher than
0.998. These curves were used to check the reproducibility
and precision of the method at two concentration levels of
0.80 and 8.00 ng/ml. Acceptable results were obtained, there-
fore this part of the study allowed for approval to use the
method for determining the sum of four PAHs in vegetable
oils.
Examples of chromatogram profiles of the determined
PAHs in standard solution and in refined rapeseed oils are
presented in Fig. 1and Fig. 2,respectively.
The determined limited benzo(a)pyrene, benz(a)anthracene,
benzo(b)fluoranthene and chrysene as well as sum of these
four PAHs contents of rapeseed oils are given in Table 2.
The PAHs concentration values were corrected for the recov-
eries obtained for each of the measurement series, in accor-
dance with the requirements of the Commission
Regulation (EU) 836/2011. The recovery values were
within the ranges of 80-103%, 87-109%, 87-108%
and 91-110% for benzo(a)pyrene, benz(a)anthracene,
chrysene and benzo(b)fluoranthene, respectively.
The refined rapeseed oils contained benzo(a)pyrene in the
amount not exceeding the permissible limit of 2.0 μg/kg
Tabl e 1 Validation parameters of the method and evaluation criteria
Parameter Kind of PAH Evaluation criteria
BaA CHR BbFA BaP
Linearity—correlation coefficient 0.9999 1.0000 0.9999 1.0000 ≥0.998
Sensitivity (slope) by regression equation 7.277 2.890 8.532 6.613 –
Limit of detection (LOD) (μg/kg) 0.18 0.18 0.18 0.18 ≤0.30
1
Limit of quantification (LOQ) (μg/kg) 0.25 0.25 0.25 0.25 ≤0.90
1
Recovery (%) Level I (0.80 ng/ml) 97.77 83.42 92.71 108.65 50–120
1
Level II (8.00 ng/ml) 102.30 102.89 100.16 102.02
Repeatability RSD
r
[%]
2
0.2 0.6 0.3 0.3 HORRAT
r
<2
1
1
Commission Regulation (EU) 836/2011
2
Repeatability relative standard deviation
Food Anal. Methods (2017) 10:1078–1086 1081
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(Commission Regulation (EU) 2015/1125). In two samples of
the refined rapeseed oils and in one sample of refined virgin
rapeseed oil the BaP content was higher than one (1.20 ± 0.29
μg/kg, 1.92 ± 0.29 μg/kg and 1.10 ± 0.17 μg/kg,
respectively).
Maximum amount of the analyzed PAHs in the rapeseed oil
samples ranged from 1.30 to 2.40 μg/kg, while in the refined
virgin rapeseed oil samples it was from 1.10 to 1.95 μg/kg.
Taking into account the value of uncertainty this is not a sig-
nificant difference. Significantly lower amounts of
benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene
and chrysene, being lower than 1 μg/kg, were found in the
cold-filtered refined virgin rapeseed oil samples (Table 2). For
these samples, also the sum of four PAHs (2.91 ± 0.58 μg/kg)
was significantly lower than in the rapeseed oils and in refined
virgin rapeseed oils.
Chrysene was the most quantitatively dominant compound
of all the PAHs determined in the rapeseed oils, with its con-
tent exceeding 1 μg/kg in three samples of the rapeseed oils
and in four samples of the refined virgin rapeseed oils. This
phenomenon is in agreement with that of Alomirah et al.
(2010), who determined chrysene in 83% of vegetable oil
samples, while benz(a)anthracene in 70% of them.
The sum of four PAHs in all the samples did not exceed the
permissible value of 10 μg/kg of product (Table 2). Our results
of determination of PAHs in refined rapeseed oils, with rela-
tively the highest chrysene content, are in agreement with
these reported by Ciecierska and Obiedziński (2006). While,
Węgrzyn et al. (2006) determined lower PAHs content in re-
fined rapeseed oils (BaP –0.24 μg/kg; Σ4PAHs –1.49 μg/kg)
than in the present study. These authors also found significant
differences in benzo(a)pyrene, benz(a)anthracene,
Fig. 1 Enlargement of raw
HPLC-FLD chromatogram
profile of PAH Solution Mix
standard: benz(a)anthracene (1),
chrysene (2),
benzo(b)fluoranthene (3),
benzo(k)fluoranthene (4), and
benzo(a)pyrene (5)
Fig. 2 Enlargement of raw
HPLC-FLD chromatogram
profile of refined rapeseed oil:
benz(a)anthracene (1), chrysene
(2), benzo(b)fluoranthene (3), and
benzo(a)pyrene (5)
1082 Food Anal. Methods (2017) 10:1078–1086
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benzo(b)fluoranthene and chrysene contents between refined
rapeseed oils, cold-filtered refined virgin rapeseed oils and
rapeseeds. According to Ciecierska and Obiedziński (2006)
refining process play a key role in the oilseeds processing,
because it significantly reduces polyaromatic hydrocarbons
content in the final product. However, the amounts of
benzo(a)pyrene determined by Jędra et al. (2008)inrefined
rapeseed oils (3.74 μg/kg) and in refined virgin rapeseed oils
(0.92-1.03 μg/kg) were significantly higher than, and similar
to, respectively, these obtained in the present work. The levels
of contamination of rapeseed oils with benzo(a)pyrene,
benz(a)anthracene, benzo(b)fluoranthene and chrysene were
similar to the average levels of these compounds found in
refined vegetable oils tested in the EU countries (EFSA 2008).
On an industrial scale the oil from oilseeds is usually ob-
tained in two stages: first by hot mechanical pressing in screw
presses, and then by extracting the residual oil from pomace.
Oil is often subjected to a refining process in order to remove
some impurities such as metal ions, pesticides and polycyclic
aromatic hydrocarbons (PAHs). In the literature there is a lack
of data on levels of contamination of cold filtered extra virgin
rapeseed oils with polycyclic aromatic hydrocarbons. The
analysed rapeseed oils were advertised as 100% refined virgin
rapeseed oils, cold filtered; however, according to the produc-
er information given on the labels they were cold filtered at the
bottling stage. Filtration plays an important role in the
purification technology of vegetable oils. One of the most
popular filtration methods used for rapeseed oils is filtration
on vertical plate filters at 50°C. After pressing, the oil contains
a significant amount of pomace, a by-product of oil manufac-
ture. Pressed oil is of better quality than the extracted one
(Krygier et al. 2009,2011). Starski and Jędra (2011)reported
that there were differences in PAHs level of rapeseed oils
produced by different manufacturers and they concluded that
it could be due to regionalization of rape crops and differences
in filtration methods.
In the analysed refined sunflower oils the benzo(a)pyrene
content did not exceed the level of 2.0 μg/kg (Table 3). In 30%
of the samples the BaP content was lower than LOD (0.18 μg/
kg) determined during method validation (Table 1), while
50% of the samples contained from 0.18 to 0.25 μg/kg of
BaP (Table 3). In most refined sunflower oil samples the
BaP, CHR and BbFA content was lower than LOQ (in 80%,
70% and 80%, respectively). Similar results were reported by
other authors (Teixeira et al. 2007;Węgrzyn et al. 2006;
Alomirah et al. 2010;DostandIdeli2012). The highest
PAHs contents (1.86 ± 0.28 μg/kg; 0.92 ± 0.14 μg/kg; 1.42
±0.21μg/kg and 1.58 ± 0.24 μg/kg for BaP, BaA, CHR and
BbFA, respectively) were found in one sample of refined sun-
flower oil originating from UE (Table 3); however, in this case
the sum of four PAHs (5.78 ± 1.16 μg/kg) and BaP content did
not exceed the permissible limits given in Commission
Tabl e 2 The limited PAH content of rapeseed oils
Kind of PAH Number of products (%) with PAH content above limit Concentration (μg/kg)
>LOD >LOQ >1 (μg/kg) >2 (μg/kg) >5 (μg/kg) >10 (μg/kg) Min Max
Refined rapeseed oils (n=20)
BaP 55 10 10 ––– <LOD 1.92 ± 0.29
BaA 40 25 5 ––– <LOD 1.30 ± 0.20
CHR 30 45 10 5 –– <LOD 2.40 ± 0.36
BbFA 50 20 15 ––– <LOD 1.35 ± 0.20
Σ4PAHs2510203510–LOD–LOQ 5.80 ± 1.16
Refined virgin rapeseed oils (n=5)
BaP 60 20 20 ––– LOD–LOQ 1.10 ± 0.17
BaA 40 40 20 ––– LOD–LOQ 1.20 ± 0.18
CHR –20 80 ––– 0.98 ± 0.15 1.95 ± 0.29
BbFA 40 20 40 ––– LOD–LOQ 1.35 ± 0.20
Σ4PAHs –20 20 60 –– 0.98 ± 0.20 4.65 ± 0.93
Cold-filtered refined virgin rapeseed oil (n=5)
BaP 40 20 ––– <LOD 0.52 ± 0.08
BaA 60 20 ––– <LOD 0.81 ± 0.12
CHR 60 40 ––– LOD–LOQ 0.78 ± 0.12
BbFA 60 20 ––– <LOD 0.80 ± 0.12
Σ4PAHs –60 20 20 –– 0.58 ± 0.12 2.91 ± 0.58
Σ4PAHs sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene, and chrysene, LOD limit of detection, LOQ limit of quantification, nnumber
of samples
Food Anal. Methods (2017) 10:1078–1086 1083
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Regulation (EU) 2015/1125. The levels of contamination of
the refined vegetable oils with PAHs are comparable to these
found by other authors (Cejpek 1998; Guillen et al. 2004;
Teixeira et al. 2007). Most authors (Ciecierska and
Obiedziński 2006; Starski and Jędra 2011)reportedthatthe
PAHs content of edible oils was reduced as a result of refining
process, especially by bleaching with use of a mixture of ad-
sorbents (activated bleaching earth and activated charcoal)
and by deodorisation, while Teixeira et al. (2007)observeda
slight increase in PAHs content of soybean oil and olive oil
after the bleaching process.
None of the tested samples of popular Polish olive pomace
oil and rapeseed oil with 5% of olive oil exceeded the maxi-
mum permissible PAHs levels and their minimum
benzo(a)pyrene content was below the limit of detection, while
the maximum BaP content amounted to 0.25 and 0.45 μg/kg
for olive pomace oil and rapeseed oil with olive oil, respectively
(Table 4). This result is consistent with this one reported by
Jędra et al. (2008), where BaP content of olive pomace oil
amounted to 0.31 μg/kg. No statistically significant difference
was found between the maximum sums of four polycyclic ar-
omatic hydrocarbons determined in olive pomace oil (3.15 ±
0.63 μg/kg) and rapeseed oil with olive oil (2.92 ± 0.58 μg/kg).
Rodríguez-Acuña and Pérez-Caminomdel (2008) found that
the PAHs content of olive oil and of olives is dependent on
environmental pollution and exposition of the fruits to fumes.
Polycyclic aromatic hydrocarbons are adsorbed on the surface
of the fruit and can be transferred to the oil during extraction
process. According to Ciecierska and Obiedziński (2006) olive
pomace oil can be a significant food source of PAHs. These
authors determined 61 μg/kg of benzo(a)pyrene and more than
96 μg/kg of sum of the four PAHs in this product, with the first
value exceeding 30 times the permissible limit. High amount of
polycyclic aromatic hydrocarbons in olive pomace oil results
from direct drying process of the pomace before oil extraction
(Ciecierska and Obiedziński 2006). Guillen et al. (2004)and
WuandYu(2012) found that the PAHs content can be a useful
factor in determining quality of different vegetable oils and in
optimizing refining process.
Benzo(a)pyrene and sum of four PAHs contents of un-
refined soybean coconut oils were significantly higher
than these of refined oils (Table 4). The amounts of each
of the determined PAHs in unrefined soybean oils were
higher than 0.7 μg/kg, while maximum sum of four
PAHs amounted to 9.10 ± 1.82 μg/kg. The latter did not
exceed the permissible level for vegetable oils
(Commission Regulation (EU) 2015/1125). Significantly
lower PAHs contents were determined by Yu et al. (2014)
in soybean and crude and refined soybean oils. These au-
thors found that benzo(a)pyrene, benz(a)anthracene,
Tabl e 3 The limited PAH content of sunflower oils
Kind of PAH Number of products (%) with PAH content above limit Concentration (μg/kg)
>LOD >LOQ >1 (μg/kg) >2 (μg/kg) >5 (μg/kg) >10 (μg/kg) Min Max
Refined sunflower oils (n=10)
BaP 501010 ––– <LOD 1.86 ± 0.28
BaA 50 20 –––– <LOD 0.92 ± 0.14
CHR 502010 ––– <LOD 1.42 ± 0.21
BbFA 50 10 10 ––– <LOD 1.58 ± 0.24
Σ4PAHs 501010 –10 –<LOD 5.78 ± 1.16
Σ4PAHs sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene, and chrysene, LOD limit of detection, LOQ limit of quantification, nnumber
of samples
Tabl e 4 The limited PAH
content (μg/kg) of the other
vegetable oils
Kind of oil Kind of PAH
BaP BaA CHR BbFA Σ4PAHs
Olive pomace oil (n=3) ND–0.25 ND–0.80 0.68–1.42 0.40–0.92 1.11–3.15
Rapeseed oil with 5 % olive oil (n=3) ND–0.45 0.27–0.85 0.64–1.10 0.43–0.97 1.34–2.92
Unrefined soybean oil (n=3) 0.84–1.79 0.70–1.05 1.39–3.95 0.91–1.35 3.84–9.10
Unrefined coconut oil (n=3) ND–2.20 2.40–9.60 4.10–10.8 ND–2.30 8.90–24.9
Σ4PAHs sum of benzo(a)pyrene, benz(a)anthracene, benzo(b)fluoranthene, and chrysene, nnumber of samples,
ND not detected
1084 Food Anal. Methods (2017) 10:1078–1086
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benzo(b)fluoranthene and chrysene contents were reduced
as a result of neutralization, bleaching and deodorization
of the oils.
The highest contamination with polycyclic aromatic hydro-
carbons was found for unrefined coconut oils (Table 4); how-
ever, due to much higher limits for the sum of the four PAHs
established for coconut oil used for direct human consumption
or as an ingredient in foods (20 μg/kg) the determined sum of
four PAHs do not exceed the permissible level. Coconut oil is
usually obtained by pressing a dry coconut meat yielding
crude protein oil, which is then filtered, washed and refined.
In the present study, a significant linear correlation (r=
0.9470) between benzo(a)pyrene content and sum of four
PAHs was found.
Our results of determination of the four limited poly-
cyclic aromatic hydrocarbons in different edible vegeta-
ble oils are consistent with these presented in the EFSA
report published in 2008 and involving 2100 vegetable
oil samples originating from 17 Member States, where
85.8% of the samples contained less than 2 μg/kg of
benzo(a)pyrene. Moreover, also chrysene was the most
quantitatively dominant PAH of all the determined poly-
cyclic aromatic hydrocarbons (EFSA 2008).
Conclusions
All the tested vegetable oil samples showed a benzo(a)pyrene
(BaP) and sum of benzo(a)pyrene, benz(a)anthracene,
benzo(b)fluoranthene and chrysene (sum of four PAHs) levels
below established limit values given in Commission
Regulation (EU) 2015/1125. The unrefined oils were charac-
terized by significantly higher contamination with PAHs as
compared to the refined oils. The unrefined coconut oil
contained the highest level of PAHs. No significant differ-
ences were found between the four PAHs contents of the re-
fined rapeseed oils and the refined virgin rapeseed oils, while
the maximum levels of each of the PAHs determined in the
cold-filtered refined virgin rapeseed oils were significantly
lower than these found in other rapeseed oils.
Acknowledgments The authors thank Monika Hoły for technical
assistance in carrying out the analyses.
Compliance with Ethical Standards
Conflict of Interest Alicja Zachara declares that she has no conflict of
interest. Dorota Gałkowska declares that she has no conflict of interest.
Lesław Juszczak declares that he 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.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
Alomirah H, Al-Zenki S, Husain A, Sawaya W, Ahmed N, Gevao B,
Kannan K (2010) Benzo[a]pyrene and total polycyclic aromatic
hydrocarbons (PAHs) levels in vegetable oils and fats do not reflect
the occurrence of the eight genotoxic PAHs. Food Addit Contam
27(6):869–878. doi:10.1080/19440040903493793
Bojanowska M, Czerwiński J (2010) Polycylic aromatic hydrocar-
bons in rape seeds with relation to their growing site and ther-
mal treatment. J Toxicol Env Heal A 73(17,8):1250–1259.
doi:10.1080/15287394.2010.492013
Cejpek K, Hajslova J, Kocourek V, Tomaniova M, Cmolik J (1998)
Changes in PAH levels during production of rapeseed oil. Food
Addit Contam 15(5):563–574. doi:10.1080/02652039809374682
Ciecierska M, Obiedziński M (2006) Vegetable oils contamination by
polycyclic aromatic hydrocarbons. Food. Science. Technology.
Quality 2(47):48–55
Commission Regulation (EC) No 208/2005 of 4 February 2005
amending Regulation (EC) No 466/2001 as regards polycyclic
aromatic hydrocarbons. Official Journal of the European Union
L 34, 8.2.2005.
Commission Regulation (EC) No 1881/2006 of 19 December 2006 set-
ting maximum levels for certain contaminants in foodstuff. Official
Journal of the European Union L 364, 20.12.2006.
Commission Regulation (EU) No 835/2011 of 19 August 2011 amending
Regulation (EC) No 1881/2006 as regards maximum levels for
polycyclic aromatic hydrocarbons in foodstuffs. Official Journal of
the European Union L 215, 20.8.2011
Commission Regulation (EU) No 836/2011 of 19 August 2011 amending
Regulation (EC) No 333/2007 laying down the methods of sampling
and analysis for the official control of the levels of lead, cadmium,
mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs.
Official Journal of the European Union L 215, 20.8.2011
Commission Regulation (EU) 2015/1125 of 10 July 2015 amending
Regulation (EC) No 1881/2006 as regards maximum levels for
polycyclic aromatic hydrocarbons in Katsuobushi (dried bonito)
and certain smoked Baltic herring. Official Journal of the
European Union L 184, 11.7.2015
Dmochowska H (ed) (2012) International statistics yearbook. Central
Statistical Office of Poland, Warsaw
Dost K, Ideli C (2012) Determination of polycyclic aromatic hydrocar-
bons in edible oils and barbecued food by HPLC/UV–Vis detection.
Food Chem 133:193–199. doi:10.1016/j.foodchem.2012.01.001
Dubois V, Breton S, Linder M, Fanni J, Parmentier M (2007) Fatty acid
profiles of80 vegetable oils with regard to their nutritional potential.
Eur J Lipid Sci Tech 109:710–732. doi:10.1002/ejlt.200700040
EFSA (2008) A report from the unit of data collection and exposure
on a request from the European Commission Findings of the
EFSA data collection on polycyclic aromatic hydrocarbons in
food. First issued on 29 June 2007 and revised on 31 July 2008.
Available on: http://www.efsa.europa.eu/en/scdocs/doc/33r.pdf
(November 2015)
Guillen M, Sopelana P, Palencia G (2004) Polycyclic aromatic hydrocar-
bons and olive pomace oil. J Agr Food Chem 52:2123–2132.
doi:10.1021/jf035259q
Food Anal. Methods (2017) 10:1078–1086 1085
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Jędra M, Starski A, Gawarska H, Sawilska-Rautenstrauch D (2008)
Benzo(a)pyrene contamination of vegetable oils. Rocz Panstw
Zakl Hig 59(2):131–138
Krygier K, Wroniak M, Ptasznik S (2009) The impact of the different
stages of production of rapeseed oil for its health benefits. In:
Krzymański J (ed) Rapeseed oil—the new raw material, new truth.
PSPO-Publishing, Warsaw, pp. 31–43
Krygier K, Wroniak M, Maszewska M (2011) The impact of technolog-
ical processes on the nutritional value of edible oils. In: Nowak D
(ed) Food quality and safety. Shaping the nutritional quality in in-
dustrial processes. SGGW-Publishing, Warsaw, pp. 65–75
Lage Yusty MA, Cortizo Daviña JL (2005) Supercritical fluid extraction
and high-performance liquid chromatography-fluorescence detec-
tion method for PAHs investigation in vegetable oils. Food
Control 16:59–64. doi:10.1016/j.foodcont.2003.11.008
Moret S, Conte LS (2000) Polycyclic aromatic hydrocarbons in edible
fats and oils: occurrence and analytical methods. Journal of
Chromatogr A 882:245–253. doi:10.1016/S0021-9673(00)00079-0
Moret S, Purcaro G, Conte LS (2005) Polycyclic aromatic hydrocarbons
in vegetable oils from canned foods. Eur J Lipid Sci Tech 107:488–
496. doi:10.1002/ejlt.200501060
PN-EN ISO 661 (2006) Animal and vegetable fats and oils. Preparation
of test sample
Rodríguez-Acuna R, Pérez-Caminomdel C, Cert A, Moreda W (2008)
Sources of contamination by polycyclic aromatic hydrocarbons in
Spanish virgin olive oils. Food Addit Contam A 25:115–122.
doi:10.1080/02652030701459855
Sadowska-Rociek A, Surma M, Cieślik E (2015) Determination of poly-
cyclic aromatic hydrocarbons in coffee and coffee substitutes using
dispersive SPE and gas chromatography-mass spectrometry. Food
Anal Methods 8:109–121. doi:10.1007/s12161-014-9876-9
Starski A, Jędra M (2011) Assessment of polycyclic aromatic hydrocar-
bons level in rapeseed oils manufactured in Poland. Bromatology
and Chemical Toxicology XLIV 4:1054–1060
Teixeira VH, Casal S, Oliveira MBPP (2007) PAHs content in sun-
flower, soybean and virgin olive oils: evaluation in commercial
samples and during refining process. Food Chem 104:106–112 .
doi:10.1016/j.foodchem.2006.11.007
Tys J, Rybacki R, Malczyk P (2003) Rapeseed contamination with
benzo(a)pyrene in the north part of Poland. Oilseed Crops 34:
627–636
Węgrzyn E, Grześkiewicz S, Popławska W, Głód BK (2006)
Modified analytical method for polycyclic aromatic hydrocar-
bons, using SEC for sample preparation and RP-HPLC with fluo-
rescence detection. Application to different food samples. Acta
Chromatogr 17:233–249
Wenzl T, Simon R, Klieiner J, Anklam E (2006) Analytical methods for
polycyclic aromatic hydrocarbons (PAHs) in food and the environ-
ment needed for new food legislation in the European Union. Trend
Anal Chem 25:716–725. doi:10.1016/j.trac.2006.05.010
World Health Organization, International Agency for Research on Cancer
(2010) IARC monographs on the evaluation of carcinogenic risks to
humans. Some non-heterocyclic polycyclic aromatic hydrocarbons
and some related exposures. IARC Lyon
Wu S, Yu W (2012) Liquid–liquid extraction of polycyclic aromatic hy-
drocarbons in four different edible oils from China. Food Chem 134:
597–601. doi:10.1016/j.foodchem.2012.02.155
Yu Y, Qingzhe J, Wang Y, Gu Y, Wang X (2014) Sources of polycyclic
aromatic hydrocarbons in soybean oil and its dynamic changes re-
fining processing. Adv J Food Sci Technol 6(1):42–47
Zachara A, Juszczak L (2016) Food contamination by polycyclic aromat-
ic hydrocarbons—legal requirements and monitoring. Food Science
Technology Quality 3(106):5–20
1086 Food Anal. Methods (2017) 10:1078–1086
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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