1692

Journal of Food Protection, Vol. 74, No. 10, 2011, Pages 1692–1699
doi:10.4315/0362-028X.JFP-11-140
Copyright G, International Association for Food Protection

Multiresidue Determination of Carcinogenic Polycyclic Aromatic

Hydrocarbons in Honey by Solid-Phase Extraction and
High-Performance Liquid Chromatography
LOURDES CORREDERA,1* SUSANA BAYARRI,1 CONSUELO PÉREZ-ARQUILLUÉ,1 REGINA LÁZARO,1
FRANCISCO MOLINO,2 AND ANTONIO HERRERA1

1Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Facultad de Veterinaria, C/ Miguel Servet 177,

50013 Zaragoza, Spain; and 2Unidad de Calidad y Seguridad Alimentaria, Centro de Investigación y Tecnologı́a Agroalimentaria de Aragón,
Avenida de Montañana 930, 50059 Zaragoza, Spain

MS 11-140: Received 21 March 2011/Accepted 6 May 2011

ABSTRACT
An analytical procedure based on solid-phase extraction, using ethyl acetate as the elution solvent, and high-performance
liquid chromatography with fluorescence and diode array detection was developed for the identification and quantification of
polycyclic aromatic hydrocarbons (PAHs) in honey. The method has been optimized and validated in accordance with
Commission Regulation 333/2007 and Commission Decision 2002/657/EC. This method allows the identification of the 15
PAHs that should be monitored in food matrices, as proposed in 2002 by the Scientific Committee on Food and later by the
European Union in the Commission Recommendation 2005/108/EC, because of their genotoxic and carcinogenic properties. The
results of the validation study were in agreement with quality criteria described in European legislation in terms of sensitivity,
accuracy, and ruggedness, and the method was applied to the analysis of 42 honey samples (21 from Spain and 21 from other
regions). The honey samples were not contaminated by PAHs at detectable levels and thus could be marketed without health risk.

Polycyclic aromatic hydrocarbons (PAHs) are well-
known compounds consisting of two or more aromatic
rings. PAHs are considered persistent organic pollutants and
are formed during the incomplete combustion and pyrolysis
of organic matter from both anthropogenic (e.g., emissions
in the environment from vehicle exhausts, asphalt pave-
ments, and processing of coal, crude oil, or petroleum) and
natural (e.g., forest fires) processes (30, 48).
Humans can be exposed to PAHs through various
routes. For the general population, the major routes of
exposure are food and inhaled air. In smokers, the
contributions from smoking and food may be of a similar
magnitude (16).
Food can be contaminated by environmental PAHs
present in air, soil, or water, but PAHs also may form
directly in food during processing (drying and smoking) and
cooking at high temperatures (e.g., grilling, roasting, and
frying) (10).
Several research articles have been published on PAHs
in environmental samples (29, 33, 49) and some food
products such as oil (6, 32), fish (36, 44), meat (38, 42), and
vegetables, fruits, and cereals (21). However, very little
information is available on the PAH concentrations in honey
(3, 9), which is a complex food matrix that can be
contaminated as a result of environmental pollution (e.g.,
* Author for correspondence. Tel: (z34) 976761543; Fax: (z34)
976761590; E-mail: lourdesc@unizar.es.
forest fires, stubble burning, or location of beehives near
industrial sites) or through the practice of blowing smoke
into the beehives during handling to calm the bees and keep
them from attacking the beekeeper. Wood chips, pine
needles, corrugated cardboard pieces, and other vegetable
materials usually can be used safely for bee smoking, but oil
products or contaminated materials must be avoided. In
addition, the smoker must be cleaned after use to prevent the
accumulation of combustion residues.
The control of PAHs is very important. PAHs, coal tar,
combustion emissions containing these compounds have
been carcinogenic in experimental animals, and genotoxi-
city and mutagenicity has been noted both in vitro and in
vivo (10, 20, 25). In various epidemiological studies,
frequent consumption of meat and fish products that have
been smoked or grilled using traditional techniques has been
associated with the high incidence of stomach cancer
observed in certain populations (1, 4).
In the 1970s, the U.S. Environmental Protection
Agency (EPA) identified the most frequently encountered
PAHs in environmental samples, the EPA PAHs, and these
compounds were investigated by several authors (26, 28, 30,
33). New legislation was adopted in the European Union
(EU) in 2005 (12), providing a list of 15 PAHs (8 in
common with the EPA list) of major concern for human
health because of their carcinogenic properties. This
legislation required the analysis of these PAHs in food
matrices to provide further information on the levels of
J. Food Prot., Vol. 74, No. 10
contamination by these compounds in food. Several authors
contributed information on the contamination of various
food matrices by these organic pollutants (41, 43, 45, 51).
According to the European Scientific Committee on
Food (SCF) (10), benzo(a)pyrene (BaP) can be used as a
marker for the occurrence and effect of carcinogenic PAHs
in food, but further analyses of the relative proportions of
BaP in foods would be necessary to inform a future review
of the suitability of maintaining this compound as a marker.
Commission Regulation 1881/2006 (13) fixed maximum
levels for BaP in various food products, but honey was not
included in this regulation.
The European Food Safety Authority (EFSA) CON-
TAM Panel (16) concluded that BaP is not a suitable
indicator for the occurrence of carcinogenic and genotoxic
PAHs in foods. On the basis of new evidence, the panel
concluded that the sum of eight high-molecular-weight
PAHs, i.e., benzo(a)anthracene (BaA), chrysene (CHR),
benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF),
BaP, dibenz(a,h)anthracene (DbA), benzo(g,h,i)perylene
(BgP), and indeno(1,2,3-cd)pyrene (IP), would be more
suitable as an indicator than would BaP alone (16).
The use of optimized and validated analytical method-
ologies is essential to assure the reliability of the results
obtained and thus their usefulness for risk assessment and
the establishment of maximum levels of contaminants in
foods. Most of the methods used for the determination of
PAHs in food samples are based on liquid-liquid extraction
(9, 45), supercritical fluid extraction (24), and solid-phase
extraction (SPE) (5, 6, 37) followed by gas chromatography
with mass spectrometry (GC-MS) (28, 47) or by high-
performance liquid chromatography (HPLC) (17, 21, 27).
Both of these chromatographic methods are very sensitive
for determining PAH concentrations usually found in foods,
but GC-MS usually is less effective for separation and
analysis of some of the 15 EU PAHs (45, 47). However,
very few studies have included a complete evaluation or
validation of their methods according to international
requirements before conducting analyses of real samples.
Commission Decision 2002/657/EC (11), implement-
ing Council Directive 96/23/EC (15), established common
criteria that can be used in validation processes of analytical
methodologies used for determining the presence of various
contaminants or residues in foodstuffs. Commission Regu-
lation 333/2007 (14) specified the methods of sampling and
performance requirements for the official control of the
concentration of BaP in food.
The aim of this study was the optimization and
validation of a simple, fast, economic, and reliable
analytical methodology for the determination of PAH
concentrations in honey, following the guidelines from
Commission Regulation 333/2007 and Commission Deci-
sion 2002/657/EC. This procedure was used with various
honey samples to provide information on the concentrations
of the 15 EU PAHs in honey, including the group of 8
PAHs proposed by the EFSA as indicators of PAH presence
in food. Because limits for BaP concentrations in various
food products other than honey have been established in
European legislation, our study was conducted to provide
DETERMINATION OF PAHS IN HONEY 1693
data that could be used as part of a risk assessment for
establishment of such limits.
MATERIALS AND METHODS
Reagents and apparatus. The 15 PAH standards in acetonitrile
(10 mg liter21) were obtained from Dr. Ehrenstorfer GmbH
(Augsburg, Germany): cyclopenta(c,d)pyrene (CPP; CAS no. 27208-
37-3, 99.0%), BaA (CAS no. 56-55-3, 99.5%), CHR (CAS no. 218-
01-9, 99.0%), 5-methylchrysene (5MC; CAS no. 3697-24-3, 99.8%),
BbF (CAS no. 205-99-2, 99.0%), benzo( j)fluoranthene (BjF; CAS
no. 205-82-3, 99.0%), BkF (CAS no. 207-08-9, 99%), BaP (CAS
no. 50-32-8, 99.5%), DbA (CAS no. 53-70-3, 99.0%), dibenzo(a,l)-
pyrene (DlP; CAS no. 191-30-0, 99.5%), BgP (CAS no. 191-24-2,
99.5%), IP (CAS no. 193-39-5, 99.5%), dibenzo(a,e)pyrene (DeP;
CAS no. 192-65-4, 99.0%), dibenzo(a,i)pyrene (DiP; CAS no. 189-
55-9, 99.9%), and dibenzo(a,h)pyrene (DhP; CAS no. 189-64-0,
99.0%). Standard mix solutions containing the 15 PAHs at
concentrations ranging from 0.010 to 240 mg liter21 were prepared
in acetonitrile and stored at room temperature in the dark.
HPLC analysis grade ethyl acetate, methanol, and acetonitrile
were used. All the organic solvents were supplied by Labscan
(Dublin, Ireland). Ultrapure water was obtained from a Milli-Q
Plus apparatus (Millipore, Bedford, MA).
The C18 (EC) 6-ml cartridges contained 500 mg of
octadecylsilica (Isolute, International Sorbent Technology, Hen-
goed, UK). The Visiprep SPE vacuum manifold was supplied by
Supelco (Bellefonte, PA). We also used a Turbo Vap II (Caliper
Life Sciences, Hopkinton, MA).
The high-performance liquid chromatograph (HP1100) was
equipped with a fluorescence detector and a diode array detector
(Agilent, Karlsruhe, Germany).
The liquid chromatographic column was a Hichrom PAH2
(250 by 4.6 mm, 5 mm; Hichrom, Theale, England) maintained in a
column oven (90-2215 rev F, LabAlliance, State College, PA).
Development of the analytical method. The analytical
method developed in this work was focused on the 15 PAHs
included in the EU list of important compounds and regarded as
probably or possibly carcinogenic to humans: BaA, CHR, 5MC,
BbF, BjF, BkF, BaP, IP, DbA, BgP, DlP, DeP, DiP, DhP, and
CPP. The methodology consists of three steps: honey sample
preparation, extraction and purification of the analytes by SPE
(C18, 500 mg), and analyte identification using HPLC with
fluorescence detection and diode array detection.
All the material used during the preparation of the sample and
the extraction and purification step was made of glass. As
recommended by the EFSA, plastics such as polypropylene were
avoided because PAHs can adsorb onto these materials. All
glassware and sample containers were thoroughly washed with
detergent solution and distilled water and rinsed with acetonitrile
before and after use to minimize the risk of contamination.
Working solutions were stored in foil-wrapped volumetric flasks,
and spiked honey samples were prepared on the same day to
minimize the possible photodecomposition of the PAHs studied.
A commercial rosemary honey was analyzed to certify that it
did not contain detectable amounts of the contaminants under
investigation. This reference matrix was used (i) as a method blank
to determine whether other analytes or interfering substances were
present in the laboratory, reagents, or apparatus, (ii) to obtain
spiked honey samples to evaluate the analytical methodology, and
(iii) to prepare standard mix solutions to avoid the matrix effect.
To obtain the spiked honey samples, appropriate volumes of
standard mix solutions were added to the reference matrix at four
1694 CORREDERA ET AL. J. Food Prot., Vol. 74, No. 10

spiking levels (200, 20, 10, and 1 mg kg21), except for CPP and TABLE 1. Retention time and emission wavelength program used
BjF, for which only three spiking levels could be evaluated (200, for chromatographic identification of PAHs
20, and 10 mg kg21) because of the low chromatographic response
Retention time Absortion or
of these compounds. PAH (min) emission (nm)
A 12-g sample of honey was weighed into a 100-ml flask, and
30 ml water-methanol (90:10, vol/vol) was added. The flask was CPP 9.8 222a
then shaken, and the standard mix solution was added. The total BaA 9.7 420
volume of the honey mixture was diluted to 50 ml with water- CHR 10.4 370
methanol (90:10, vol/vol). 5MC 11.7 370
The SPE procedure was based on that described by Martı́nez BjF 12.9 500
et al. (29) and modified in our laboratory to adapt this technique for BbF 13.9 420
PAH determination in honey, with different elution solvents (15 ml BkF 15.3 420
of acetonitrile and 15 ml of ethyl acetate) and elution solvent BaP 18.2 420
volumes (15 ml of ethyl acetate and 25 ml of ethyl acetate). The DbA 20.9 420
final conditions were set up depending on the results obtained in DlP 24.4 420
recovery assays as follows. The SPE C18 cartridge (500 mg) was BgP 26.2 420
activated with 5 ml of ethyl acetate, then with 5 ml of methanol, IP 26.5 500
and finally with 5 ml of a water-methanol (98:2, vol/vol) mixture. DeP 32.0 420
A 5-ml aliquot of the spiked honey solution was transferred to the DiP 41.7 470
cartridge, washed with 5 ml of Milli-Q water, and dried for 5 min DhP 44.9 470
under vacuum. The PAHs were then eluted with 25 ml of ethyl a
acetate and concentrated to 100 ml at 40uC under nitrogen in a CPP was identified by diode array detection.
concentration workstation (Turbo Vap II). The extracts were not
evaporated to complete dryness because that could lead to a loss of Accuracy was determined based on recovery and precision
light PAHs (22, 42). The extracts were then reconstituted in 0.5 ml assays for honey samples at four spiking levels: 200, 20, 10, and
of acetonitrile. 1 mg kg21. For CPP and BjF, only the three highest levels were
HPLC for determination of analytes was carried out in reversed- evaluated. The individual PAHs were identified by comparing the
phase chromatograph with fluorescence and UV detectors as retention time and the spectrum of the substance in the sample with
described by Simon et al. (43) and modified as follows in our that of the respective compound in a standard mix solution
laboratory. A 20-ml extract aliquot was injected into the HPLC system, analyzed under the same conditions, and the recoveries of each
and the column was heated at 40uC in a column oven. The flow of the compound were determined by comparing the ratios of the peak
aqueous mobile phase (acetonitrile-water) was set to 1 ml/min, the areas obtained for the samples with those found for standard mix
gradient program for the mobile phase started with 80% acetonitrile solutions at the same concentration.
(0 min) and changed linearly to 85% (30 min) and 100% (32 min), Precision was determined by repeatability and reproducibility
and after 55 min (still 100%) the mobile phase was changed back to studies, expressed by the HORRAT value (Horwitz ratio: the
the initial composition (80% acetonitrile, 20% water). Table 1 shows observed relative standard deviation divided by the value estimated
the emission wavelengths used for detection and quantification (the from the Horwitz equation, according to Commission Regulation
excitation wavelength was set at 270 nm for all compounds). CPP 333/2007) of the recovery percentages from six spiked honey
cannot be analyzed by fluorescence detection because it is not samples analyzed the same day by the same operator (repeatability)
fluorescent; therefore, it was detected by diode array detection. and from six spiked honey samples extracted and analyzed on three
different days (within-laboratory reproducibility).
Evaluation of the analytical methodology. Based on According to the performance criteria for methods of analysis
international requirements (Commission Decision 2002/657/EC of BaP in foods, recovery must be between 50 and 120%, and for
and Commission Regulation EC No 333/2007, specific for precision assays the HORRAT values should be always less than 2.
sampling and analysis of the levels of BaP in foodstuffs), the A ruggedness study was carried out as defined by
evaluation of an analytical methodology includes the study of its Commission Decision 2002/657/EC to complete the evaluation
specificity, sensitivity, accuracy (recovery and precision), and of the analytical method. For this purpose, we selected seven
ruggedness. possible variables that could influence the measurement results:
To determine specificity, 20 blank samples were analyzed to brand of solvents used (A), SPE cartridge drying time (B), elution
make sure no interference was observed in the chromatogram for solvent volume (C), elapsed time between extraction and analysis
the retention times of the analytes under investigation. (D), room temperature (E), column temperature (F), and analyst
In the sensitivity study, the linear ranges of the detector for the (G). We varied each of them in an order of magnitude that matches
PAHs were studied, and the limits of detection (LODs) and limits the deviations that might occur among laboratories; the effect of
of quantification (LOQs) were established according to Commis- various solvent brands, a shorter drying time, quantitative errors in
sion Regulation EC No 333/2007. The LOD was calculated based pipette handling, the passage of time between extraction and
on the lowest concentration giving a response of three times the analysis (for example a weekend), different room temperatures
standard deviation of the mean for the blank samples, whereas the depending on the season, instrumental errors in the column oven,
LOQ was considered six times the standard deviation of the mean and different analysts were evaluated. The ruggedness test was
for the blank samples. then carried out with spiked honey samples (20 mg kg21) using the
Performance criteria for BaP require LODs of less than approach of Youden (experiment design shown in Table 2).
0.3 mg kg21 and LOQs of less than 0.9 mg kg21. For these tasks, Once the study was complete, the effect of each factor was
standard mix solutions at nine concentrations ranging from 0.010 calculated by subtracting the mean result obtained with the variable
to 240 mg liter21 were prepared and added to the reference matrix at high level (reference value) indicated by the uppercase letter and
to be injected into the HPLC apparatus in duplicate. the mean result achieved with the factor at low level (varied value),
J. Food Prot., Vol. 74, No. 10 DETERMINATION OF PAHS IN HONEY 1695

TABLE 2. Selected factors and experiment design for ruggedness study, with the combination of determinations used
Combination of determinations
Original Varied
Selected factor Abbreviation value value 1 2 3 4 5 6 7 8

Brand of the solvents used A, a 1 2 A A A A a a a a

SPE cartridge drying time B, b 5 min 1 min B B b b B B b b
Elution solvent vol C, c 25 ml 24.5 ml C c C c C c C c
Time between extraction and analysis D, d 1h 72 h D D d d d d D D
Room temp E, e 20uC 15uC E e E e e E e E
Column temp F, f 40uC 39.5uC F f f F F f f F
Analyst G, g 1 2 G g g G g G G g
Observed result R S T U V W X Y Z

marked with the corresponding lowercase letter. A statistical test
was used to evaluate the influence of each investigated factor. The
effect of the selected factors together was measured, comparing the
standard deviation of the differences (SDi) with the standard
deviation of the method. An SDi that is significantly larger than the
standard deviation of the method carried out under within-
laboratory reproducibility conditions indicates that all factors
together have an effect on the result, even when each single factor
does not have a significant influence.
To evaluate the results obtained, we applied a t test after
making sure that all the results followed a normal distribution
according the Kolmogorov-Smirnov test (a ~ 0.05).
Analysis of honey samples. Once the method was validated,
it was applied to the analysis of 42 commercial honey samples to
evaluate the presence of PAHs in honey from different geograph-
ical regions. The study included 21 samples of Spanish honey and
21 samples from other European and non-European regions that
were sold in several Spanish supermarkets. All samples presented a
homogeneous appearance, without phase separation, and all were
stored at room temperature in a dark place and analyzed within
1 month after collection.
RESULTS AND DISCUSSION
Optimization and evaluation of the analytical
methodology. As far as we know, no analytical method
combining these techniques for the determination of PAHs
in honey has been published. The methodology described
here provided resolution and identification of the 15 PAHs
studied. The HPLC elution profile of the spiked honey
samples containing the 15 PAHs is shown in Figure 1.
Table 3 provides the linearity and sensitivity results
obtained for each compound. The results of our study
satisfied the criteria of European regulations for BaP in
foodstuffs (Commission Regulation EC No 333/2007),
which require LODs below 0.3 mg kg21 and LOQs below
0.9 mg kg21. In our study, the LOD and the LOQ for BaP
were 0.08 mg kg21 in both cases. The calculated LOQs for
the rest of the PAHs met the performance criteria and were
below 0.9 mg kg21 in all cases, with LODs of 0.08 to
0.84 mg kg21, except for CPP (not fluorescent) and BjF (less
fluorescent). These average LODs are much lower than the
LODs published by Albero et al. (3) for honey. Dobrinas et
al. (9) reported LOQs of 0.15 mg kg21 in the same matrix,
lower than those obtained in our work. However, these two
research groups focused on the EPA PAHs and used GC-
MS for the analysis.
Our average recoveries ranged from 50 to 99% and
were above 80% at the two higher spiking concentrations
for most of the compounds (Table 3), with HORRAT values
always less than 2. These results meet the requirements of
Commission Regulation 333/2007 (recoveries between 50
and 120% and HORRAT values less than 2).
Like us, Albero et al. (3) obtained high recoveries (66
to 115%) for eight of the PAHs that we analyzed (CHR,
BaA, BbF, BkF, BaP, DbA, IP, and BgP) at high spiking
levels (20 to 800 mg kg21); however, these authors did not
report recovery assays for low spiking levels. Similarly,
Rivera et al. (38) studied various SPE sorbents for some
PAHs in meat, with recoveries always above 90%. Windal
et al. (51) studied PAHs in oils and dried plants, and their
recoveries ranged from 60 and 115%. Danyi et al. (8) also
reported high recoveries (63 to 118%) using cyclohexane-
dichloromethane as an extraction solvent for monitoring
these pollutants in food supplements. The recovery of heavy
dibenzopyrenes obtained in some is often low (43, 47).
Several authors who analyzed PAHs in food did not specify
the accuracy of their analytical methodologies (9, 21).
Regarding ruggedness, Table 4 shows the two-tailed P
values obtained for the t test for each factor in the PAHs
analysis and the SDi values and reproducibility standard
deviations for each analyte. The combined factors did not
significantly affect the method performance, and we can
assume that the analytical method is robust; of the 105
results obtained in the ruggedness study, only four had P
values below 0.05: the variables of analyst (factor G) and
SPE drying time (factor B) for DbA and BgP, and DlP and
IP, respectively. The influence of SPE drying time has been
specifically studied by several authors (18, 19, 23), who
concluded that the results obtained with each analytical
method were seriously influenced by this factor.
The determination of the ruggedness of an analytical
method is a laborious task, and very few studies have
included this kind of assay with regard to PAHs in food.
Serpe et al. (41) studied the ruggedness of their methodol-
ogy for analysis of PAHs in mussels and stated that in
general the method was not affected by slight variations in
some critical factors. However, they did not report the
specific results obtained for each compound. Norwood et al.
(35) reported ruggedness results validating an analytical
methodology for PAHs in metered dose inhaler drug
formulations; other authors have published results for
1696 CORREDERA ET AL. J. Food Prot., Vol. 74, No. 10

FIGURE 1. Chromatograms of a honey

sample spiked at 10 mg kg21. The 15 PAHs
were studied at different wavelengths: (a)
diode array detection Sig222nm; (b) lex
270/lem 370; (c) lex 270/lem 500; (d) lex
270/lem 470; (e) lex 270/lem 420.

methodologies applied to pesticides in crops (31), aflatoxin
M1 in milk (34), nitrofurans in poultry muscle tissue (46),
and tetracyclines in milk (39).
Determination of PAHs in honey samples. In the
honey samples analyzed, no detectable levels of the 15
PAHs were found with the validated methodology. The
absence of these pollutants in the analyzed samples could be
due to the low levels of environmental pollution in the
geographical region, correct smoking processes used by
beekeepers (in terms of smoking duration and combustible
material used), completely capped hive cells at the time of
honey extraction (beeswax can protect the honey in capped
cells from external pollutants), storage of honey in stainless
J. Food Prot., Vol. 74, No. 10 DETERMINATION OF PAHS IN HONEY 1697

steel containers (which must be clean and free from

200 mg kg21

7.5
7.8
7.6
5.6
6.5
8.2
7.1
7.5
5.7
8.3
4.2
8.2
5.3
8.4
7.9
contamination), and a prolonged period between hive
smoking and PAH analysis.

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Repeatability and reproducibility results were expressed as mean ¡ standard deviation of six replicates at each spiking concentration (1, 10, 20, and 200 mg kg21). NA, not available.
Our results are in agreement with those reported for

93
93
86
93
84
91
84
94
83
74
87
84
75
70
64
another Spanish study by Albero et al. (3), who did not find
any of the PAHs studied in the analyzed honey samples
Reproducibility recovery (%)

10.4
10.6

10.1

10.5

11.3
10.8
20 mg kg21

(four unifloral and one multifloral sample). However,

9.7
9.9
6.8
8.4

8.7

9.9
9.5

9.5
9.5
Dobrinas et al. (9) reported the presence of BaP (with
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concentrations of 0.5 to 141 mg kg21) and 13 other
90
94
80
90
87
94
88
85
80
65
78
84
59
62
70
hydrocarbons (concentrations up to 665 mg kg21 depending
on the compound) in several honey samples from Romania,

10.1
11.2

11.4
10 mg kg21

but these authors did not detect CHR above the LOQ in any
6.3
5.9
5.5
6.6
8.2
9.6
6.5
7.2
6.7

8.4
9.4

9.4
of the samples and noted significantly higher concentrations
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of PAHs in urban sites than in mountain and rural sites.
80
87
77
84
70
82
74
81
77
60
79
71
63
55
50
In studies of other food products exposed to environ-
mental contamination, PAHs were also present at low
69 ¡ 11.3

65 ¡ 11.0

62 ¡ 11.9

50 ¡ 12.5

72 ¡ 11.7
57 ¡ 10.9
53 ¡ 11.5
55 ¡ 12.3
concentrations. Rojo Camargo and Toledo (40) analyzed
21

56 ¡ 5.8

59 ¡ 8.5
58 ¡ 5.5

53 ¡ 8.4

59 ¡ 8.4
1 mg kg

NA

NA

the relation between air pollution and PAH concentrations in

fruits and vegetables and found concentrations of these
pollutants up to 17.93 mg kg21, whereas Kazerouni et al. (21)
reported BaP concentrations of 0.01 to 0.48 mg kg21 in fruits
21

and vegetables and up to 0.56 mg kg21 in cereals and grains.

5.5
6.4
4.6
5.3
5.2
6.7
4.8
6.7
5.1
6.7
7.1
5.2
6.3
6.9
5.4
200 mg kg

Other studies dealing with infant formulas, milk, and related

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products revealed that these kinds of compounds are less

99
93
88
92
90
93
87
95
82
73
84
85
80
72
63

frequently found in infant food; in most analyzed products,

none of the PAHs studied were detected (2, 7). However,
10.7
21

PAH concentrations were usually higher in animal products,

8.2

9.3
9.7
8.3
8.6
9.0
8.5
7.9
8.8
8.5
9.2
9.3
9.9
9.5
Repeatability recovery (%)

20 mg kg

especially those that were smoked, grilled, or barbecued.

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White et al. (50) investigated the effect of various cooking

87
88
81
86
85
89
82
83
80
63
75
80
72
68
69

processes on meat products and found high concentrations

(8.4, 9.8, and 20.4 mg kg21) of BaP in precooked beef burger
12.7

10.6
11.5
21

samples, whereas barbecued burger samples had the highest

9.2
8.4
6.5
8.4
6.8

6.5
4.0
7.4
8.1
6.2
4.9
8.1
10 mg kg

concentrations (29.1 and 30.6 mg kg21).

¡
¡
¡
¡
¡
¡
¡
¡
¡
¡
¡
¡
¡
¡
¡

The estimated maximum daily intake of BaP from food

85
82
76
85
76
96
74
87
74
62
77
68
65
56
53

is about 6 to 8 ng/kg of body weight per day, i.e., about five

to six orders of magnitude lower than the daily doses that
51 ¡ 11.3
52 ¡ 12.5
21

59 ¡ 5.2
58 ¡ 8.2
61 ¡ 6.6

51 ¡ 4.5
56 ¡ 9.4
58 ¡ 8.3
64 ¡ 7.8
52 ¡ 6.6
68 ¡ 9.8
66 ¡ 9.0
59 ¡ 7.9

have induced tumors in experimental animals. The SCF

1 mg kg

NA

NA

concluded that at these levels of intake noncarcinogenic

effects are not to be expected and the risk of heritable effects
from dietary exposure to PAHs is low (10).
TABLE 3. Validation parameters and linearity of the method a

The analytical methodology described here has advan-

(mg kg21)

tages for PAH determination in honey samples because it is

LOQ

4.20
0.21
0.21
0.21
4.20
0.21
0.08
0.08
0.42
0.84
0.84
0.42
0.42
0.84
0.21

fast, easy, and economic. Because it produces reliable

results in this type of food matrix, it could be used in routine
laboratory monitoring protocols. The analyzed honey
samples were not contaminated by PAHs at detectable
(mg kg21)

levels and thus could be marketed without health risk. These

LOD

4.20
0.08
0.21
0.21
4.20
0.08
0.08
0.08
0.21
0.84
0.84
0.42
0.42
0.84
0.21

results are consistent with those for other honey samples in

Spain and are far below the BaP maximum limits
established by the EU for foods. However, because honey
may be exposed to smoke during handling, further studies
Linear range

4.20–200
0.21–200
0.21–200
0.21–200
4.20–200
0.21–200
0.08–200
0.08–200
0.42–200
0.84–200
0.84–200
0.42–200
0.42–200
0.84–200
0.21–200
(mg kg21)

are needed to complete a risk assessment and obtain

contamination data, which could serve to establish specific
maximum PAH limits for honey.

ACKNOWLEDGMENTS
The authors thank the Government of Aragón for giving a scholarship
CHR
5MC

DbA
BaA
CPP
PAH

DhP

to L. Corredera and for its financial support (Grupo de Investigación

BbF
BkF

BgP

DeP
BaP

DlP

DiP
BjF

IP

Consolidado A01, Fondo Social Europeo and Ayudas Apı́colas).

a
1698 CORREDERA ET AL. J. Food Prot., Vol. 74, No. 10

TABLE 4. Statistical values obtained for each factor in the ruggedness study a
P values (two-tailed) for t tests

PAH A B C D E F G SDi SD reprod

CPP 0.53 0.15 0.93 0.53 0.93 0.06 0.66 6.82 9.70
BaA 0.90 0.18 0.39 0.50 0.97 0.32 0.13 4.44 9.98
CHR 0.78 0.05 0.43 0.27 0.90 0.52 0.52 6.64 6.80
5MC 0.84 0.69 0.42 0.84 0.54 0.22 0.54 5.13 8.40
BjF 0.58 0.05 0.15 0.41 0.89 0.89 0.18 8.61 10.40
BbF 0.24 0.68 0.68 0.06 0.54 0.93 0.35 5.98 10.63
BkF 0.87 0.08 0.45 0.53 0.65 0.96 0.14 6.81 8.75
BaP 0.12 0.28 0.56 0.71 0.56 0.53 0.25 4.92 10.18
DbA 0.29 0.85 0.98 0.77 0.50 0.74 0.02b 8.28 9.98
DlP 0.88 0.03b 0.93 0.98 0.98 0.17 0.39 11.63 9.50
BgP 0.34 0.94 0.72 0.83 0.72 0.73 0.01b 9.69 10.50
IP 0.89 0.04b 0.96 0.89 0.86 0.09 0.07 11.39 9.54
DeP 0.07 0.91 0.74 0.43 0.59 0.21 0.60 4.82 9.50
DiP 0.76 0.09 0.84 0.54 0.92 0.15 0.29 11.16 11.30
DhP 0.85 0.32 0.44 0.24 0.15 0.33 1.00 5.16 10.80
a
SDi, standard deviation of the differences; SD reprod, standard deviation of reproducibility.
b
P values less than 0.05 indicate that the factor could have a significant influence on the result.

REFERENCES ing the performance of analytical methods and the interpretation of

1. Afolabi, O. A., E. A. Adesulu, and O. L. Oke. 1983. Polynuclear
aromatic hydrocarbons in some Nigerian preserved freshwater fish
species. J. Agric. Food Chem. 31:1083–1090.
2. Aguinaga, N., N. Campilloa, P. Vinas, and M. Hernández-Córdoba.
2007. Determination of 16 polycyclic aromatic hydrocarbons in milk
and related products using solid-phase micro extraction coupled to
gas chromatography–mass spectrometry. Anal. Chim. Acta 596:285–
290.
3. Albero, B., C. Sánchez-Brunete, and J. L. Tadeo. 2003. Determina-
tion of polycyclic aromatic hydrocarbons in honey by matrix solid-
phase dispersion and gas chromatography/mass spectrometry. J.
AOAC Int. 86:576–582.
4. Alonge, D. O. 1988. Carcinogenic polycyclic aromatic hydrocarbons
(PAH) determined in Nigerian kundi (smoke-dried meat). J. Sci. Food
Agric. 43:167–172.
5. Barranco, A., R. M. Alonso-Salces, A. Bakkali, L. A. Berrueta, B.
Gallo, F. Vicente, and M. Sarobe. 2003. Solid-phase clean-up in the
liquid chromatographic determination of polycyclic aromatic hydro-
carbons in edible oils. J. Chromatogr. A 988:33–40.
6. Bogusz, M. J., S. A. El Hajj, Z. Ehaideb, H. Hassan, and M. Al-
Tufail. 2004. Rapid determination of benzo(a)pyrene in olive oil
samples with solid-phase extraction and low-pressure, wide-bore gas
chromatography–mass spectrometry and fast liquid chromatography
with fluorescence detection. J. Chromatogr. A 1026:1–7.
7. Ciecierska, M., and M. W. Obiedzinski. 2010. Polycyclic aromatic
hydrocarbons in infant formulae, follow-on formulae and baby foods
available in the Polish market. Food Control 21:1166–1172.
8. Danyi, S., F. Brose, C. Brasseur, Y.-J. Schneider, Y. Larondelle, L.
Pussemier, J. Robbens, S. De Saeger, G. Maghuin-Rogister, and
M.-L. Scippo. 2009. Analysis of EU priority polycyclic aromatic
hydrocarbons in food supplements using high performance liquid
chromatography coupled to an ultraviolet, diode array or fluorescence
detector. Anal. Chim. Acta 633:293–299.
9. Dobrinas, S., S. Birghila, and V. Coatu. 2008. Assessment of
polycyclic aromatic hydrocarbons in honey and propolis produced
from various flowering trees and plants in Romania. J. Food Compos.
Anal. 21:71–77.
10. European Commission. 2002. Opinion of the Scientific Committee on
Food on the risks to human health of polycyclic aromatic
hydrocarbons in food. SCF/CS/CNTM/PAH/29 final. European
Commission, Brussels.
11. European Commission. 2002. Commission Decision 2002/657/EC of
12 August 2002 implementing Council Directive 96/23/EC concern-
results. Off. J. Eur. Communities L 221:8–36.
12. European Commission. 2005. Commission recommendation 2005/
108/EC of 4 February 2005 on the further investigation into the levels
of polycyclic aromatic hydrocarbons in certain foods. Off. J. Eur.
Union L 43:43–45.
13. European Commission. 2006. Commission Regulation (EC) No 1881/
2006 of 19 December 2006 setting maximum levels for certain
contaminants in foodstuffs. Off. J. Eur. Union L 364:5–24.
14. European Commission. 2007. Commission Regulation (EC) No 333/
2007 of 28 March 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.
Off. J. Eur. Union L 88:29–38.
15. European Council. 1996. Council Directive 96/23/EC of 29 April
1996 on measures to monitor certain substances and residues thereof
in live animals and animal products and repealing Directives 85/358/
EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC.
Off. J. Eur. Communities L 125:10–32.
16. European Food Safety Authority. 2008. Scientific opinion of the
Panel on Contaminants in the Food Chain on a request from the
European Commission on Polycyclic Aromatic Hydrocarbons in
Food. EFSA J. 724:1–114.
17. Falcó, G., A. Bocio, J. M. Llobet, and J. L. Domingo. 2005. Health
risks of dietary intake of environmental pollutants by elite sportsmen
and sportswomen. Food Chem. Toxicol. 43:1713–1721.
18. Ferreirós, N., G. Iriarte, R. M. Alonso, and R. M. Jiménez. 2006.
MultiSimplex and experimental design as chemometric tools to
optimize a SPE-HPLC-UV method for the determination of
eprosartan in human plasma samples. Talanta 69:747–756.
19. Gonzalez, O., G. Iriarte, N. Ferreirós, M. I. Maguregui, R. M. Alonso,
and R. M. Jiménez. 2009. Optimization and validation of a SPE-
HPLC-PDA-fluorescence method for the simultaneous determination
of drugs used in combined cardiovascular therapy in human plasma.
J. Pharm. Biomed. Anal. 50:630–639.
20. International Agency for Research on Cancer. 1987. Overall
evaluations of carcinogenicity: an updating of IARC monographs
volumes 1 to 42. IARC Monogr. Eval. Carcinog. Risks Hum. Suppl.
7. International Agency for Research on Cancer, Lyon, France.
21. Kazerouni, N., R. Sinha, C. Hsu, A. Greenberg, and N. Rothman. 2001.
Analysis of 200 food items for benzo[a]pyrene and estimation of its
intake in an epidemiologic study. Food Chem. Toxicol. 39:423–436.
22. Kiss, G., Z. Varga-Puchony, and J. Labia. 1996. Determination of
polycyclic aromatic hydrocarbons in precipitation using solid-phase
J. Food Prot., Vol. 74, No. 10
extraction and column liquid chromatography. J. Chromatogr. A 725:
261–272.
23. Kubota, R., Y. Endo, A. Takeuchi, Y. Inoue, H. Ogata, M. Ogawa, T.
Nakagawa, N. Onda, and G. Endo. 2007. SPE-GC/FTD determina-
tion of N-methyl-2-pyrrolidone and its metabolites in urine. J.
Chromatogr. B 854:204–210.
24. Lage Yusty, M. A., and J. L. Cortizo Daviña. 2005. Supercritical fluid
extraction and high-performance liquid chromatography–fluores-
cence detection method for polycyclic aromatic hydrocarbons
investigation in vegetable oil. Food Control 16:59–64.
25. La Rocca, C., L. Conti, R. Crebelli, B. Crochi, N. Iacovella, F.
Rodriguez, L. Turrio-Baldassarri, and A. di Domenico. 1996. PAH
content and mutagenicity of marine sediments from the Venice
lagoon. Ecotoxicol. Environ. Saf. 33:236–245.
26. Liu, Y., L. Chen, Z. Jianfu, H. Qinghui, Z. Zhiliang, and G.
Hongwen. 2008. Distribution and sources of polycyclic aromatic
hydrocarbons in surface sediments of rivers and an estuary in
Shanghai, China. Environ. Pollut. 154:298–305.
27. Mäkelä, M., and L. Pyy. 1995. Effect of temperature on retention
time reproducibility and on the use of programmable fluorescence
detection of fifteen polycyclic aromatic hydrocarbons. J. Chroma-
togr. A 699:49–57.
28. Martı́-Cid, R., J. M. Llobet, V. Castell, and J. L. Domingo. 2008.
Evolution of the dietary exposure to polycyclic aromatic hydrocar-
bons in Catalonia, Spain. Food Chem. Toxicol. 46:3163–3171.
29. Martı́nez, E., M. Gros, S. Lacorte, and D. Barceló. 2004. Simplified
procedures for the analysis of polycyclic aromatic hydrocarbons in
water, sediments and mussels. J. Chromatogr. A 1047:181–188.
30. Masclet, P., H. Cachier, C. Liousse, and H. Wortham. 1995.
Emissions of polycyclic aromatic hydrocarbons by savanna fires. J.
Atmos. Chem. 22:41–54.
31. Maštovská, K., J. Hajšlová, and S. J. Lehotay. 2004. Ruggedness and
other performance characteristics of low-pressure gas chromatogra-
phy–mass spectrometry for the fast analysis of multiple pesticide
residues in food crops. J. Chromatogr. A 1054:335–349.
32. Moret, S., and L. S. Conte. 2000. Polycyclic aromatic hydrocarbons
in edible fats and oils: occurrence and analytical methods. J.
Chromatogr. A 882:245–253.
33. Moret, S., G. Purcaro, and L. S. Conte. 2007. Polycyclic aromatic
hydrocarbon (PAH) content of soil and olives collected in areas
contaminated with creosote released from old railway ties. Sci. Total
Environ. 386:1–8.
34. Muscarella, M., S. L. Magro, C. Palermo, and D. Centonze.
2007.Validation according to European Commission Decision
2002/657/EC of a confirmatory method for aflatoxin M1 in milk
based on immunoaffinity columns and high performance liquid
chromatography with fluorescence detection. Anal. Chim. Acta 594:
257–264.
35. Norwood, D. L., D. Prime, B. P. Downey, J. Creasey, S. K. Sethi, and
P. Haywood. 1995. Analysis of polycyclic aromatic hydrocarbons in
metered dose inhaler drug formulations by isotope dilution gas
chromatography/mass spectrometry. J. Pharm. Biomed. 13:293–304.
36. Pensado, L., M. C. Casais, M. C. Mejuto, and R. Cela. 2005.
Application of matrix solid-phase dispersion in the analysis of
priority polycyclic aromatic hydrocarbons in fish samples. J.
Chromatogr. A 1077:103–109.
37. Rey-Salgueiro, L., M. S. Garcı́a-Falcón, E. Martı́nez-Carballo, and J.
Simal-Gándara. 2008. Effects of toasting procedures on the levels of
polycyclic aromatic hydrocarbons in toasted bread. Food Chem. 108:
607–615.
DETERMINATION OF PAHS IN HONEY 1699
38. Rivera, L., M. J. C. Curto, P. Pais, M. T. Galceran, and L. Puignou.
1996. Solid-phase extraction for the selective isolation of polycyclic
aromatic hydrocarbons, azaarenes and heterocyclic aromatic amines
in charcoal-grilled meat. J. Chromatogr. A 731:85–94.
39. Rodrı́guez, N., M. C. Ortiz, and L. A. Sarabia. 2009. Study of
robustness based on n-way models in the spectrofluorimetric
determination of tetracyclines in milk when quenching exists. Anal.
Chim. Acta 651:149–158.
40. Rojo Camargo, M. C., and M. C. F. Toledo. 2003. Polycyclic
aromatic hydrocarbons in Brazilian vegetables and fruits. Food
Control 14:49–53.
41. Serpe, F. P., M. Esposito, P. Gallo, and L. Serpe. 2010. Optimisation
and validation of an HPLC method for determination of polycyclic
aromatic hydrocarbons (PAHs) in mussels. Food Chem. 122:920–
925.
42. Simko, P. 2002. Determination of polycyclic aromatic hydrocarbons
in smoked meat products and smoke flavouring food additives. J.
Chromatogr. B 770:3–18.
43. Simon, R., S. Palme, and E. Anklam. 2007. Validation (in-house and
collaborative) of a method based on liquid chromatography for the
quantitation of 15 European-priority polycyclic aromatic hydrocar-
bons in smoke flavourings: HPLC-method validation for 15 EU
priority PAH in smoke condensates. Food Chem. 104:876–887.
44. Stołyhwo, A., and Z. E. Sikorski. 2005. Polycyclic aromatic
hydrocarbons in smoked fish—a critical review. Food Chem. 91:
303–311.
45. Stumpe-Vı̄ksna, I., V. Bartkevičs, A. Kukāre, and A. Morozovs.
2008. Polycyclic aromatic hydrocarbons in meat smoked with
different types of wood. Food Chem. 110:794–797.
46. Verdon, E., P. Couedor, and P. Sanders. 2007. Multi-residue
monitoring for the simultaneous determination of five nitrofurans
(furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in
poultry muscle tissue through the detection of their five major
metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by liquid
chromatography coupled to electrospray tandem mass spectrome-
try—in-house validation in line with Commission Decision 657/2002/
EC. Anal. Chim. Acta 586:336–347.
47. Veyrand, B., A. Brosseaud, L. Sarcher, V. Varlet, F. Monteau, P.
Marchand, F. Andre, and B. Le Bizec. 2007. Innovative method for
determination of 19 polycyclic aromatic hydrocarbons in food and oil
samples using gas chromatography coupled to tandem mass
spectrometry based on an isotope dilution approach. J. Chromatogr.
A 1149:333–344.
48. Vila-Escalé, M., T. Vegas-Vilarrubia, and N. Prat. 2007. Release of
polycyclic aromatic compounds into a Mediterranean creek (Catalo-
nia, NE Spain) after a forest fire. Water Res. 41:2171–2179.
49. Wang, J., Y. Bi, G. Pfister, B. Henkelmann, K. Zhu, and K.
Schramm. 2009. Determination of PAH, PCB, and OCP in water
from the Three Gorges Reservoir accumulated by semipermeable
membrane devices (SPMD). Chemosphere 75:1119–1127.
50. White, S., A. Fernandes, and M. Rose. 2008. Investigation of the
formation of PAHs in foods prepared in the home and from catering
outlets to determine the effects of frying, grilling, barbecuing,
toasting and roasting. FD 06/13.1. Food Standards Agency, Central
Science Laboratory, Sand Hutton, UK.
51. Windal, I., L. Boxus, and V. Hanot. 2008. Validation of the analysis
of the 15 z 1 European-priority polycyclic aromatic hydrocarbons by
donnor-acceptor complex chromatography and high-performance
liquid chromatography–ultraviolet/fluorescence detection. J. Chro-
matogr. A 1212:16–22.
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