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Determination of benzo(a)pyrene by GC/MS/MS in

retail olive oil samples available in Qatar
a a a a
A. H. W. Abdulkadar , A. A. M. Kunhi , Al-Jedah Jassim & Al-Ali Abdulla
a
Central Food Laboratory, Ministry of Public Health , PO Box 21266, Doha, Qatar
Published online: 21 May 2010.

To cite this article: A. H. W. Abdulkadar , A. A. M. Kunhi , Al-Jedah Jassim & Al-Ali Abdulla (2003) Determination of
benzo(a)pyrene by GC/MS/MS in retail olive oil samples available in Qatar , Food Additives & Contaminants, 20:12,
1164-1169

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 Food Additives and Contaminants, Vol. 20, No. 12 (December 2003), pp. 1164–1169
Determination of benzo(a)pyrene by GC/MS/MS in retail
olive oil samples available in Qatar
A. H. W. Abdulkadar, A. A. M. Kunhi, Al-Jedah
Jassim* and Al-Ali Abdulla
Central Food Laboratory, PO Box 21266, Ministry of Public
Health, Doha, Qatar
(Received 1 July 2002; revised 1 September 2003; accepted
12 September 2003)
A survey was carried out for the presence of benzo(a)-
pyrene (B(a)P) in olive oils following reports that
some batches of Spanish olive–pomace oil and its
products contained high levels of B(a)P. Three types
Downloaded by [FU Berlin] at 07:30 07 May 2015
of olive oils (1) virgin olive oil, (2) refined olive oil and
(3) olive–pomace oil, originating from France, Greece,
Italy, the Lebanon, Spain, Tunisia and Turkey, and
available on Qatar market, were analysed for B(a)P.
Determination was carried out by extraction from a
cyclohexane solution with N,N-dimethylformamide:
water (9:1), back extraction with cyclohexane,
followed by clean-up on a silica gel column and
quantification by GC/MS/MS. The recovery and limit
of quantitation of B(a)P in olive oils by this method
were estimated as 88% and 0.5 g kg–1, respectively.
B(a)P was detected in amounts below the permitted
level of 2 g kg–1 in all 31 virgin olive oil samples and in
13 refined olive oil samples. However, all seven samples
of Spanish olive–pomace oil produced and packed in
Spain and three samples of olive–pomace oil produced
in Spain, but packed in Saudi Arabia, were found to be
contaminated with B(a)P, the level ranging from 3.1 to
70.8 g kg–1. In two samples of olive–pomace oil
originating from Greece, B(a)P was not detected.
Keywords: benzo(a)pyrene, olive–pomace oil, GC/
MS/MS, polycyclic aromatic hydrocarbons
*
To whom correspondence should be addressed.
e-mail: rcfcm@ qatar.net.qa
Food Additives and Contaminants ISSN 0265–203X print/ISSN 1464–5122 online # 2003 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/02652030310001625626
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are
known to be carcinogenic, mutagenic and highly
stable environmental pollutants. They are commonly
found in many foods like smoked meat and fish,
grilled and roasted foods, roots and leaves of vege-
tables, fruits, grains and vegetable oils (Howard and
Fazio 1980, IARC 1983, 1987, Speer et al. 1990).
Benzo(a)pyrene (B(a)P), a potent carcinogenic PAH,
is often used as an indicator of the presence of PAHs
in water and foodstuffs, as it is well known that this
compound is usually generated together with many
other PAHs that contaminate food.
For foods treated with smoking-flavour agents, the
European Union (EU) has set a maximum permissi-
ble limit of 0.03 mg kg–1 of B(a)P (EEC Directive
1988). No such regulation exists for B(a)P in other
foods, though the EU intends to set a maximum
of 1 mg kg–1. This maximum level has already been
established in Australia, Israel, Switzerland and
Germany (Derache 1990). However, very recently in
the UK the Food Standards Agency proposed that
any edible oil should not have levels of B(a)P higher
than 2 mg kg–1 (Food Standards Agency 2001b, c).
The determination of B(a)P and other PAHs in
vegetable oils have been accomplished in Europe
and the USA (Howard et al. 1966, Ciusa et al. 1970,
Swallow 1976, Joe et al. 1979, Speer and Montag
1988, Speer et al. 1990, Menichini et al. 1991, Gertz
and Kogelheide 1994, Pupin and Toledo 1996).
In general, the detected levels of B(a)P in the olive
oils analysed have been in the range of a few mg kg–1
to a high of 164 mg kg–1. Recently, the Spanish autho-
rities detected high levels of PAHs, specifically B(a)P
in some batches of olive–pomace oil produced in
Spain. This resulted in market-withdrawal and tem-
porary import ban on Spanish olive–pomace oil in
Europe (Food Standards Agency 2001a). Similarly, at
the same time it was reported that high levels of
B(a)P, up to 100 mg kg–1, were found in olive–pomace
 Determination of benzo(a)pyrene by in olive oil in Qatar
oil produced in Greece (Food Standards Agency
2001c). Following these reports from Europe, Qatar
and other GCC countries, also imposed a temporary
import ban on olive–pomace oil and a survey for the
occurrence of B(a)P in olive oils available in Qatar
market was conducted. The present communication
reflects the data obtained under such a surveillance
conducted in Qatar.
In general, the determination of PAHs in food prod-
ucts and smoke flavourings has been carried out
following schemes that include an alkaline treatment
of the sample, liquid/liquid extraction, clean-up of the
extract to eliminate interfering substances and finally
separation, identification and quantification of the
PAHs using chromatographic techniques such as
thin layer chromatography (TLC) (White et al. 1971),
high-performance liquid chromatography (HPLC)
(O’Hara et al. 1974, Gomaa et al. 1993, Ybiku et al.
1993, Pupin and Toledo 1996), gas chromatography
with a flame ionization detector (GC-FID) (Menichini
Downloaded by [FU Berlin] at 07:30 07 May 2015
et al. 1992) or gas chromatography-mass spectro-
metry (GC-MS) (Potthast 1979, Guillen et al. 2000).
Bearing in mind the advantage of MS detector for
unequivocal identification of chemical contaminants,
in the present investigation GC/MS was employed.
Materials and methods
Samples
Samples of olive oil were collected by food inspectors
from the local market. Samples were stored in a dark
and cool place until analysis.
Reagents
All reagents, except silica gel, were procured from
BDH Laboratories (Poole, UK). Cyclohexane, N,N-
dimethylformamide and sodium sulphate used for
extraction and clean-up were of AnalaR grade.
Silica gel 60 (70–230 mesh) for column chromatogra-
phy was from Merck (Darmstadt, Germany) (Art.
No. 7754). It was deactivated by drying at 160
C for
8 h followed by cooling and addition of 12% water.
It was allowed to equilibrate for 16 h (overnight)
before use, and was stored in an air tight container.
1165
Standards
Benzo(a)pyrene standard solution (100 ng ml–1) was
bought from Riedel-de Haen (Seelze, Germany).
From this solution, working standards of 25, 50,
100 and 200 pg ml–1 were prepared by serial dilution
with cyclohexane. Standards were stored in brown
vials under refrigeration.
Extraction and clean-up
Extraction and clean-up were done according to
Speer et al. (1990), with a slight modification as
described below.
Olive oil (10 g) was weighed into a beaker. After
adding 50 ml cyclohexane and dissolving it, the
material was transferred into a 250 ml separating
funnel. The beaker was rinsed three times with
50 ml cyclohexane and the rinsings added to the
separating funnel. This was extracted with portions
of 50, 25 and 25 ml N,N-dimethylformamide-water
(9:1, v/v). The extracts were pooled in a 500 ml
separating funnel and diluted with 100 ml 1% sodium
sulphate solution. This was re-extracted with portions
of 50, 35 and 35 ml cyclohexane. These extracts were
combined in another 250 ml separating funnel and
was washed twice with 40 ml distilled water, dried
with anhydrous sodium sulphate and concentrated
on a rotary evaporator to 2 ml at 40
C, under
vacuum.
Deactivated and equilibrated silica gel (5 g) were
suspended in cyclohexane and packed into a glass
column of 10  300 mm. Similarly, anhydrous sodium
sulphate (5 g) was packed above the silica gel. The
prepared extract (2 ml) was transferred on to the
top of sodium sulphate in the silica gel column and
was eluted with 85 ml cyclohexane. The first 10 ml of
the eluent were discarded and the pooled 10–85 ml
fractions were concentrated to about 1 ml in a rotary
evaporator, and dried under gentle flow of nitrogen.
The residue was dissolved in 1 ml cyclohexane
followed by analysis by GC/MS/MS.
Spiking and recovery
To judge the suitability of the method, an olive oil
sample known to contain no B(a)P was spiked with
 1166 A. H. W. Abdulkadar et al.
B(a)P at the level of 5 mg kg–1. This spiked sample was
analysed by three analysts in duplicate, and recoveries
were recorded. The same sample was used as check
sample, and analysed regularly once in 2 weeks.
GC/MS/MS analysis
For GC/MS/MS determination, Varian Chrompack
(International BV, Middleburg, The Netherlands)
CP-3800 gas chromatograph equipped with Varian
Chrompack Saturn-2000 MS/MS detector, Varian
CP-8200 autosampler and IBM computer with
Saturn GC/MS workstation software version 5.51
was used. The column employed was capillary col-
umn CP-Sil CB low bleed/MS (30 m long  0.25 mm
i.d.  0.25 mm thickness, Cat No. CP 7860).
Instrument conditions were set according to Feigel
(2000) with slight modifications. The temperature
programme started at 60
C with a hold for 3 min
Downloaded by [FU Berlin] at 07:30 07 May 2015
followed by an increase at 15
C min–1 up to 230
C
without a hold. Finally, from 230
C the temperature
was raised at 10
C min–1 up to 300
C with hold of
1 min. The total run time for a single injection was
22.33 min. Helium with a purity of 99.999% was used
as the carrier gas. The flow rate through the capillary
column was 1 ml min–1. Both the injector and detector
temperatures were 250
C. The injection volume was
1 ml and the technique was splitless.
The automatic method development (AMD) feature
available on a Saturn GC/MS workstation software
was used to determine the MS/MS conditions. The
final conditions for MS/MS were: isolation parent ion
mass m/z 252, isolation window  3, dissociation
excitation storage level 111, excitation amplitude
2.3 V, waveform type – resonant.
Results and discussion
The MS was initially operated in full-scan mode and
the retention time of B(a)P under the above-
mentioned GC conditions was determined to be
21.1 min by injecting a B(a)P standard of a concen-
tration of 2 ng ml–1. The detection limit for B(a)P with
a detector operating in MS mode was found less
(500 pg ml–1), which was not enough to detect the level
of 2 mg kg–1 or less of B(a)P in olive oil by using the
above extraction and clean-up method. To increase
the detection limit, the MS/MS option was used.
The detection limit (LOD) of the instrument for a
B(a)P standard solution in MS/MS mode was up to
5 pg ml–1, which corresponded to level of quantifica-
tion (LOQ) of 0.5 mg kg–1 of B(a)P in olive oil. The
MS/MS spectrum obtained for B(a)P standard
solution and B(a)P in sample extract was as shown
in figure 1.
The identification of B(a)P was based on both the
retention time and MS/MS spectrum. However, for
calibration and quantification, the main ion of MS/
MS spectrum, which was m/z 250 for B(a)P was used.
The calibration curve was linear (r ¼ 0.9970) passing
through zero for the four different concentrations of
B(a)P standards mentioned above. Extracts from the
olive oil samples containing B(a)P above the level of
20 mg kg–1 gave a response on the detector outside the
calibration curve. Such extracts were diluted two to
five times based on the detector response and injected
once again to get the value of B(a)P within the
calibration curve for better quantification of results.
No background for B(a)P was observed in the chro-
matogram when a cyclohexane extract prepared from
a solvent blank (solvent without olive oil sample)
and a sample blank (sample free from B(a)P) were
analysed. The average obtained for recovery of B(a)P
from the spiked sample of olive oil at the level of
5 mg kg–1 was 88.3%. The repeatability (RSDr)
obtained after analysing the spiked samples of olive
oil (5 mg kg–1) nine times in duplicate was 8.9%. The
recovery obtained in the present investigation was in
close agreement with that reported (90.5%) by Pupin
and Toledo (1996) for the similar method of sample
preparation and clean-up.
The samples collected for analysis were manufactured
and packed in different countries. As per the informa-
tion mentioned on the label of each sample, these
samples were of three types: virgin olive oil, refined
olive oil and olive–pomace oil. The levels of B(a)P
found in these olive oil samples are presented in
table 1. The levels of B(a)P reported therein were
not corrected for recoveries. It is evident from the
data that all of the virgin olive oil samples (n ¼ 31)
and refined olive oil samples (13), regardless of the
country of origin and the two olive–pomace oil made
in Greece, were found to contain B(a)P at levels less
than the permitted level of 2 mg kg–1. However, all the
seven samples of Spanish olive–pomace oil packed in
Spain were found to contain B(a)P at levels ranging
from 3.1 to 70.8 mg kg–1 and all the three samples of
Spanish olive–pomace oil packed at Saudi Arabia
 Determination of benzo(a)pyrene by in olive oil in Qatar 1167

250
100%

75% A

50%

25% 226

135 155 168 179 197 206 241

125 150 175 200 225 250 m/z

250
100%

75% B

50%
Downloaded by [FU Berlin] at 07:30 07 May 2015

25%
206 224 241
135 155 168 179 197
125 150 175 200 225 250 m/z

Figure 1. (A) MS/MS spectrum of Benzo(a)pyrene standard solution (50pg/çl). (B) MS/MS spectrum of Benzo(a)-
pyrene, extracted from olive-pomace oil (8 çg/kg).

Table 1. Benzo(a)pyrene in different types of olive oil available on the Qatar market and originating from various
countries.

Country of Country of Type of Total Number of samples with B(a)P (mg kg 1) Highest level of
origin packing olive oil samples <0.5 0.5–2 >2 B(a)P found (mg kg 1)

France France virgin 4 2 2 – 1.23

Greece Greece virgin 4 3 1 – 0.83
pomace 2 – 2 – 1.58
Italy Italy refined 3 1 2 – 1.63
The Lebanon The Lebanon virgin 5 4 1 – 0.59
refined 3 2 1 – 0.68
Spain Spain virgin 6 4 2 – 0.54
refined 5 3 2 – 0.58
pomace 7 – – 7 70.83
Saudi Arabia virgin 4 2 2 – 0.78
pomace 3 – – 3 48.96
Tunisia Tunisia virgin 3 2 1 – 0.56
refined 2 1 1 – 0.63
Qatar virgin 3 1 2 – 0.67
Turkey Turkey virgin 2 1 1 – 0.59
Total virgin 31 19 12 – 1.23
refined 13 7 6 – 1.63
pomace 12 – 2 10 70.83
 1168 A. H. W. Abdulkadar et al.
were found to contain B(a)P ranging from 5.8 to
48.9 mg kg–1.
In the present study B(a)P was detected in virgin and
refined olive oil in the range 0.5–1.6 mg kg–1. Pupin
et al. (1996) reported the presence of B(a)P in the
range 0.6–1.2 mg kg–1 in olive oils manufactured and
packed in Europe. Menchini et al. (1991) found high
levels of PAHs in virgin and refined olive oils avail-
able in Italy. According to Speer et al. (1990), B(a)P
was found in olive oils at a median of 0.7 mg kg–1.
Levels up to 60 mg kg–1 B(a)P have been found in
virgin Italian olive oil from plants exposed to indus-
trial emission of pitch condensate (Corradetti et al.
1988). The presence of PAHs in vegetable oil can be
as a result of (1) contamination of plant material,
primarily through the air; (2) drying the plant
material with smoke before extraction; and (3) con-
tamination through the extraction solvent (Welling
and Kaandrop 1986, Larsson et al. 1987). Low levels
of B(a)P in virgin and refined olive oil samples avail-
Downloaded by [FU Berlin] at 07:30 07 May 2015
able on the Qatar market indicate that these oils were
extracted from good quality olives from plants not
exposed to industrial emission and vehicular exhaust.
In this investigation, high levels of B(a)P up to
70.8 mg kg–1 were detected in Spanish olive–pomace
oil (table 1). It is the oil made from the residue left
after producing virgin oil, and B(a)P may be intro-
duced through the solvent used for the extraction
(Welling and Kaandorp 1986, Larsson et al. 1987).
Recently in Spain, high levels of B(a)P, up to 60 times
higher than that commonly found as background
contamination in vegetable oils, were found in
olive–pomace oil (Food Standards Agency 2001a).
Also, at the same time, the levels of B(a)P up to
100 mg kg–1 were reported in olive–pomace oil from
Greece (Food Standards Agency 2001c). However,
in the present survey, B(a)P was not detected in
pomace olive oil from Greece. This may be because
the brand of product analysed in Qatar might be
different from the brand of product reported to be
contaminated.
In Qatar and other Gulf countries, olive oil is used
extensively in various food preparations. Since the
purchasing power of the population is high, the per
capita consumption of olive oil is high in Qatar.
Therefore, there is a need to monitor continuously
the levels of B(a)P in olive oils, especially in olive–
pomace oils imported in Qatar, to safeguard consumer
health. The present study and other reports on the
presence of B(a)P in solvent-extracted olive–pomace
oils also point to the necessity of studying other
solvent-extracted edible oils such as rice bran oil.
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