Journal of Food Composition and Analysis 23 (2010) 469–474

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Article

Determination of polycyclic aromatic hydrocarbons in aquatic

products by HPLC-fluorescence
Hong Zhang *, Ming Xue, Zhiyuan Dai
College of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, No. 149, Jiaogong Road, Hangzhou 310035, China

A R T I C L E I N F O A B S T R A C T

Article history: This paper analyzed 12 polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene, benzo[b]fluor-
Received 8 April 2009 anthene, benzo[k]fluoranthene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene, fluoranthene, anthra-
Received in revised form 14 September 2009 cene, benzo[a]anthracene, chrysene, fluorene, phenanthrene and pyrene, in aquatic products by using a
Accepted 31 December 2009
fluorimetric-detection RP-HPLC method. The preliminary treatment procedure was based on
microwave-assisted extraction and neutral Al2O3 solid phase purification, and a selective isolation of
Keywords: PAHs from aquatic samples was carried out. The isolated fractions of PAHs were determined by HPLC
Polycyclic aromatic hydrocarbons (PAHs)
with fluorescence detection. The test results suggest that the detection limits for the PAHs were from
Aquatic products
Residue analysis
0.0048 ng/mL to 0.075 ng/mL based on a signal-to-noise ratio of 3:1. The recoveries were from
Food safety 85.26  5.12% to 104.63  0.95%. This proposed method was successfully applied to 35 aquatic samples, in
Contamination which variable levels of summed PAHs were detected, ranging from 0.0 to 57.76 mg/kg.
Food analysis ß 2010 Elsevier Inc. All rights reserved.
Food composition
Fluorescence detection
Method determination

1. Introduction The residue analysis of PAHs has been accomplished by many

different methods, such as HPLC, gas chromatography, capillary
Polycyclic aromatic hydrocarbons (PAHs) are chemical com- electrophoresis, and hyphenated techniques (Wei and Jen, 2007;
pounds which consist of two or more condensed aromatic rings. Garcia-Falcon et al., 2004; Tfouni et al., 2007; Crimmins and Baker,
Currently, more than 200 types of PAHs have been found, some of 2006; Vives and Grimalt, 2002; Itoh et al., 2006).
which are highly carcinogenic. PAH contamination of food results The purpose of this paper was to determine 12 PAHs in aquatic
not only from cooking processes such as smoking, grilling, baking products simultaneously by HPLC with fluorescence detection
and frying, but also from the air through deposits, soil through (HPLC-FLD). The preliminary treatment procedure mainly concen-
transformation and water through deposition and transfer (Fan trated on the optimization of microwave-assisted extraction
and Peng, 2000; Tfouni and Toledo, 2007). When ingested, PAHs (MAE) and solid phase purification. In addition, we systematically
can be absorbed by the gastrointestinal tract and distributed investigated the applicability of the developed preliminary
throughout the body, resulting in acute or chronic injury (Zhang treatment procedure coupled to HPLC-FLD to aquatic products.
et al., 2006; Aguinaga et al., 2007). Animal tests have shown that
long-term intake of PAHs can cause cancer and immature egg cell 2. Materials and methods
death (Benedetti et al., 2007).
Given the well-known toxicity of PAHs, there have been 2.1. Materials and reagents
numerous studies to determine residues of PAHs. However, the key
topics of analyzing and monitoring PAHs have generally focused on Standards: 12 PAHs were used for the study: benzo[a]pyrene
BaP, some selected PAHs, or all 16 PAHs highlighted by the US (BaP) (99.6%), benzo[b]fluoranthene (BbF) (99.5%), benzo[k]fluor-
Environmental Protection Agency (EPA). Most of these empirical anthene (BkF) (99.4%), dibenzo(a,h)anthracene (D[a,h]A) (99.6%),
investigations were based on smoked food rather than on aquatic benzo[g,h,i]perylene (B[g,h,i]P) (99.5%), fluoranthene (FLT) (99.6%),
products (Wenzl et al., 2006). anthracene (ANT) (99.5%), benzo[a]anthracene (BaA) (99.5%),
chrysene (CHR) (99.5%), fluorene (FLR) (99.5%), phenanthrene
(PHE) (99.5%) and pyrene (PYR) (99.5%) were purchased from the
* Corresponding author. Tel.: +86 571 8807 1024; fax: +86 571 8807 8598. Chemical Metrology & Division, National Institute of Metrology
E-mail address: [email protected] (H. Zhang). (Beijing, China). Standard mixtures (of 500 ng/mL, 250 ng/mL,

0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2009.12.016
470 H. Zhang et al. / Journal of Food Composition and Analysis 23 (2010) 469–474

100 ng/mL, 50 ng/mL and 5 ng/mL in acetonitrile) were prepared 2.4. Sample preparation
from standard stock solutions (each having a concentration of
1 mg/mL in methanol). All working standard solutions were freshly A sample of 2.0 g was weighed into a centrifugal tube and then
prepared prior to use. 10 mL of 0.4 mol/L KOH–ethanol:water (9:1, v/v) was added. The
HPLC-grade organic solvents methanol and acetonitrile (Merck, mixture was heated in water bath for 60 8C and stirred continu-
Darmstadt, Germany), and water from a simplified water ously. The solution was transferred into microwave extraction
purification system (Millipore, Vienna, Austria) were used. Toluol, tanks. The centrifugal tube was cleaned twice, using 5 mL of
acetonitrile, methylene dichloride, cyclohexane and absolute ethyl cyclohexane. The tank was heated for 10 min at 100 8C (with
alcohol (analytical-reagent grade) were purchased from QZJH microwave power at 230 kW), then cooled until 30 8C. The solution
(Zhejiang, China). The solid extraction columns filled with neutral was transferred into 30 mL glass tubes. After leaving to stand, the
Al2O3 (Waters, 500 mg, 3 mL) were purchased from SPJG (Dalian, supernatant fluid was transferred into 10 mL glass tubes. This
China). method was called hydrolysis-MAE.
The supernatant fluid was evaporated until dryness with N2 at
2.2. Apparatus 45 8C, and redissolved with 3 mL of water/methanol (90:10, v/v).
Then the mixture was loaded onto the neutral Al2O3 SPE column,
HPLC analysis of PAHs was performed using an Agilent 1100 which had been previously preconditioned with 4 ml of methanol
liquid chromatograph (America) equipped with a fluorescence and 3 ml of Milli-Q water, successively. The column was rinsed
detector (Agilent 1046A, America) and a 20 mL loop injector. The with 3 ml of water/methanol (80:20, v/v) in order to remove high
Microwave extraction system used was CEM-MARS (2450 MHz, polar impurities. The cartridges were dried for 15 min under
America). The analytical system included a Hanban Science & vacuum, and the retained compounds eluted using 4 mL of
Technology C18 column (5 mm particle size, 4.6 mm  260 mm cyclohexane. This volume was evaporated until dryness under a
I.D.) and a mixture of acetonitrile and water as the mobile phase. nitrogen stream (45 8C), and redissolved in 1 mL of methanol. The
The separations were performed at 30 8C under gradient condi- extract was filtered using a 0.45 mm PTFE filters before the HPLC
tions (flow rate: 0.8 mL/min, 0–12 min acetonitrile 85–95%; 12– analysis.
26 min acetonitrile 95%). The fluorescent detection was performed
by applying the following excitation (Ex) and emission (Em) 3. Results and discussion
wavelength program: 210/310 nm from 5.6 to 6.3 min (determi-
nation of FLR), from 7.5 to 7.8 min (PHE), from 8.1 to 8.5 min (ANT), 3.1. Optimization of microwave extraction efficiency
248/382 nm from 9.0 to 9.5 min (PYR), from 9.7 to 10.2 min (FLT),
268/382 nm from 10.5 to 11.2 min (BaA), from 12 to 12.8 min The extraction procedures liquid–liquid extraction, hydroly-
(CHR), 286/410 nm from 15.3 to 15.8 min (BbF), from 15.9 to sis-ultrasonic extraction and hydrolysis-MAE were compared.
16.2 min (BkF), from 17.5 to 18.1 min (BaP), from 18.2 to 18.7 min Fig. 2 shows the concentrations of 12 PAHs in the same sample,
(D[a,h]A), from 21.6 to 22.6 min (B[g,h,i]P) (Fig. 1). The qualitative obtained by three different extraction procedures. It is clear that
analysis of PAHs was compared with the retention times for the extraction efficiency of hydrolysis-MAE is higher than the
standard PAHs with appropriate components, which were identi- other two methods, which is primarily due to the particular
fied in the spiked and unspiked aquatic samples under the same characteristics of microwave extraction. On the one hand – as
conditions. Quantitative determination was performed by using an the microwave sample is heated simultaneously by ion
external calibration curve method and peak area measurement. migration and dipole rotation – heat is transferred selectively
to different components of the food matrix. On the other hand,
2.3. Food samples the microwaves would prompt the generation of molecular
collision energy and fast chemical action, but not destroy the
The experiments used fresh aquatic products. The analyzed molecular structure (Xie and Chen, 2006). Because of the above
samples, which included freshwater fish (8 species), saltwater fish reasons, analytes were extracted from samples efficiently by
(8 species), algae (4 species), shellfish (7 species), shrimp (4 hydrolysis-MAE (n = 3), giving the highest extraction efficiency
species) and crabs (4 species), were purchased from the local and the most satisfactory recoveries (96.17  1.73% to
[(Fig._1)TD$IG]
market. The samples were de-boned prior to preparation. 102.37  4.59%).

Fig. 1. HPLC-FLD chromatogram of 12 PAHs (concentration of standards mixture: 100 mg/kg, injection: 5 mL).
[(Fig._2)TD$IG] H. Zhang et al. / Journal of Food Composition and Analysis 23 (2010) 469–474 471

Table 1
Mean recoveries of samples spiked with 12 PAHs (n = 3).

Analyte Added (mg/kg) Found (mg/kg) Recovery %  RSD

FlT 2.11 2.17 102.91  1.38

5.76 6.01 104.45  4.22
10.56 10.10 95.67  0.27

BkF 2.07 1.93 93.04  1.83

5.64 5.23 92.89  4.90
10.34 9.36 90.53  0.83

BbF 2.09 2.09 99.82  3.71

5.70 5.16 90.62  3.24
10.45 10.93 104.63  0.95

BaP 2.11 2.02 95.65  1.73

5.76 5.43 94.23  1.34
10.56 10.87 102.97  7.93

B[g,h,i]P 2.09 2.10 100.28  1.39

5.70 5.66 99.35  4.74
10.45 10.00 95.74  0.93

BaA 2.09 2.28 109.09  2.69

5.70 5.79 101.58  4.47
10.45 10.35 99.04  2.68
Fig. 2. Concentrations of 12 PAHs by 3 different extraction procedures.
CHR 2.09 1.99 95.22  1.79
5.70 4.94 86.67  5.59
To determine the microwave power (100, 150, 200, 230, 250 10.45 8.91 85.26  5.12

and 300 kW) necessary for optimum extraction efficiency by MAE, FLR 2.09 2.16 103.35  8.01
samples spiked with all 12 analytes at a 20 mg/kg level, were 5.70 4.87 85.44  2.95
subjected to the hydrolysis-MAE method (n = 3). The outcome 10.45 9.55 91.39  3.36

showed that samples with microwave power between 200 kW PHE 2.09 1.99 95.22  7.73
(RSD%: 3.73%) and 250 kW (RSD%: 1.98%) resulted in similar high 5.70 5.72 100.35  2.80
recoveries of more than 90.02%. Moreover, the maximum 10.45 10.38 99.33  5.19

recoveries were achieved at 230 kW (RSD%: 3.81%). Consequently, PYR 2.09 2.07 99.04  0.60
microwave irradiation at 230 kW delivered power is recom- 5.70 5.80 101.75  7.03
10.45 10.39 99.43  9.00
mended to assist the hydrolysis-MAE sampling.
ANT 2.09 2.11 100.96  4.21
3.2. Optimization of SPE purification 5.70 5.63 98.77  0.72
10.45 10.32 98.76  1.78

When analyzing aquatic products, it is highly desirable to D[a,h]A 2.11 2.10 99.53  2.74
generate clean extracts in order to decrease interferences and 5.76 5.59 97.05  5.12
10.56 10.32 97.73  2.47
increase sensitivity. Neutral Al2O3 is a type of strongly polar
adsorbent, which binds some negatron compounds, such as
aromatic and aliphatic amine compounds (Chen et al., 2004; Hu
et al., 2004). Therefore, neutral Al2O3 SPE columns were used to quantization limits; intra-day and inter-day precision. The
determine 12 PAHs in samples. This cleanup protocol was developed solutions for calibration and fortification were prepared in
to be simple, rapid and efficient, and to fulfil several requirements, methanol and stored at 4 8C. Linearity, detection and quantiza-
such as the ability to study a large number of samples simulta- tion limits were established through the analytical curves obtained
neously, and provide good sensitivity and selectivity. by quintuplicate analysis of PAHs at five concentration levels (0.25,
Due to the wide-ranging analyte polarity, the optimum elution 2.5, 10, 50 and 100 mg/kg) (Table 2).
solvent system had to incorporate a series of solvents to elute all
the analytes effectively. Thus we loaded 1 mL of a mixed standard Table 2
solution containing all 12 analytes at 20 ng/mL level onto each Calibration parameters of 12 PAHs with the proposed method.
respective cartridge, and eluted using different elution solvent PAHs Linearitya (r) Detection limitb Quantitation
systems (methanol, cyclohexane, methylene chloride, n-hexane, (ng/mL) limits (ng/mL)
acetonitrile). Recoveries from cyclohexane (96.44  6.82% to FLR 0.9990 0.0331 0.110
105.18  0.14%) were superior to the other 4 solvents for all the PHE 0.9928 0.0750 0.250
analytes (n = 3). ANT 0.9957 0.0171 0.057
Samples spiked with different concentrations (2, 5, 10 mg/kg) PYR 0.9932 0.0141 0.047
FLT 0.9980 0.0048 0.016
were used to test the validity of the developed SPE protocol. The BaA 0.9968 0.0300 0.100
mean recoveries obtained by MAE-neutral Al2O3 extraction ranged CHR 0.9967 0.0084 0.028
from 85.26  5.12% to 104.63  0.95%, with satisfactory reproduc- BbF 0.9978 0.0421 0.14
ibility and no interference and correct background noise (n = 3) BkF 0.9946 0.0048 0.016
BaP 0.9972 0.0048 0.016
(Table 1).
D[a,h]A 0.9974 0.0138 0.046
B[g,h,i]P 0.9987 0.0222 0.074
3.3. Method validation a
Value of correlation coefficient r for plots recorded in the range of detection
limit to 10 ng for each standard.
The method was validated in house, using the following b
Detection limits (based on a S/N = 3) were determined using PAH standard
performance criteria: linearity and linear range; detection and mixtures loaded directly onto a column using a 20 mL loop injector.
 472
Table 3
PAH content (mg/kg) in various aquatic products (n = 3).

Name FLR PHE ANT PYR FLT BaA CHR BbF BkF BaP D[a,h]A B[g,h,i]P Species

Mandarin fish 14.28  3.62a 4.31  1.13 b

+c 0.34  1.57 0.84  3.71 2.47  0.18 Freshwater
fish
Bighead carp 1.64  0.54 0.75  2.89 0.52  2.67 0.66  2.61 2.19  0.67
Snakehead 57.76  0.21 1.87  0.68 6.65  3.72 2.46  5.81 3.42  1.96 0.89  1.43 0.34  4.41
Crucian carp 8.68  3.34 3.68  4.28 10.07  0.26 3.54  2.90 1.14  0.69 2.55  1.90
Karut wak 54.37  2.94 4.42  1.13 9.14  0.08 3.52  2.13 3.42  4.72 3.65  3.31
Grass carp 25.53  0.69 1.79  3.32 10.09  1.14 2.97  2.77 - 0.96  1.73 0.48  4.12 0.71  2.17
Yellow-headed catfish 10.14  2.73 2.81  1.13 + 8.56  0.83 3.96  0.22 0.33  0.28 0.55  5.80 0.48  0.38 0.46  0.69

H. Zhang et al. / Journal of Food Composition and Analysis 23 (2010) 469–474

Common sturgeon 1.75  0.26 11.38  0.69 3.76  1.53 0.84  1.67 1.38  1.26 1.68  5.02

Grey mullet 10.95  0.72 3.81  0.38 7.76  1.90 3.76  1.67 0.76  0.26 Saltwater
fish
Yellow croaker 7.48  0.22 5.76  2.66 8.03  2.17 + + 0.47  3.68 +
Southern Flounder 0.64  2.23 3.46  1.78 0.91  1.13 1.49  1.19 1.02  0.37
Japanese Spanish 1.84  0.07 3.44  0.72 0.81  0.46 0.94  2.30 0.49  4.12
mackerel
Turbot 3.71  5.12 1.74  0.72 17.68  0.28 1.43  0.95 8.19  0.48 2.17  2.35 2.88  1.19 0.36  1.03
belted beard grunt 4.52  0.43 0.88  3.33 9.47  4.02 1.23  3.31 0.06  2.86 -
Robust tonguefish 12.82  0.96 1.41  0.75 12.80  1.37 1.61  1.72 3.18  1.60 0.43  0.80 3.70  3.98 0.52  3.53 0.88  0.61 0.93  1.31 0.43  0.69
Small yellow croaker 3.49  3.20 14.86  1.90 1.82  6.02 0.48  0.08 2.51  3.26

Laver 1.01  0.19 1.76  2.75 0.76  2.50 0.46  0.31 Algae
Green moss 0.56  7.02 0.34  2.83 0.61  0.62
Sargassum fusiforme 2.89  0.26 1.86  1.13 1.82  5.29 3.95  1.46 1.36  0.42 1.61  0.77
Kelp 0.52  2.51

Bladder moon + 2.52  1.83 2.77  0.90 0.83  2.52 0.77  1.94 0.64  1.09 Shellfish
Glant Pacific oyster 2.48  1.89 2.77  0.26 0.71  3.08 0.65  0.17
Constricted tagelus 0.27  6.72 2.44  3.22 2.39  3.73 0.55  0.44 1.11  5.32 2.98  0.75 1.38  2.43
Pocker-chip venus 0.17  1.41 1.53  2.64 1.42  0.09 0.54  5.13 1.04  1.68 0.98  0.64 0.78  3.69
Gulf scallop 0.96  0.26 0.42  6.99 1.06  2.38 0.70  0.53 2.81  0.52
Chinese cyclina 3.21  0.52 0.36  1.59 0.38  4.18 1.51  1.99 + +
Burnets Tellin 1.43  0.54 1.65  4.00 2.76  3.35 2.63  0.79 1.22  1.81 1.06  1.51 2.31  1.13 1.13  0.79 2.18  2.27 1.43  0.67

Greasyback shrimp 2.51  0.83 0.21  2.61 3.52  0.37 0.99  1.56 2.27  0.58 1.64  2.81 0.58  1.76 0.62  6.45 0.38  1.96 Shrimp
White-leg shrimp 6.34  2.32 5.75  1.75 0.73  1.42 2.44  0.75 1.16  2.05 0.57  1.48
Red Swamp Crawfish 5.28  0.08 4.42  0.93 0.73  3.39 2.06  4.20 1.07  1.83 0.62  0.45
Shrimp 2.33  2.09 0.79  0.26 0.53  1.78

Blue crab 9.89  1.31 0.12  0.67 3.83  1.18 2.18  1.13 1.65  0.86 Crabs
Portunid 14.02  1.00 0.17  1.18 4.12  0.52 1.55  0.25 1.06  0.86
Lake crab + 16.14  2.54 0.21  2.68 3.41  3.16 0.59  3.37 +
Chinese tiger 9.17  1.06 1.93  0.69 0.61  2.23 0.52  3.25
head crab
a
The mean content and RSD value of each sample were obtained by triplicate analysis.
b
‘‘ ’’represents ‘‘not detected’’.
c
Limit ‘‘+’’ indicates that the analytes were detected in samples, but the contents were less than the limits of detection.
 H. Zhang et al. / Journal of Food Composition and Analysis 23 (2010) 469–474 473

Precision is a measure of how close results are to one another, polynuclear aromatic compounds can be even higher, as BaP
and is evaluated by making repetitive measurements for the entire constitutes only 0.5–5% of the total amount of PAHs in food
method. The excellent intra-day precision of the method was samples.
expressed as the relative standard deviation of peak area
measurements, and evaluated through the results that were 4. Conclusions
obtained in one day, using three different fortification levels (RSD:
1.26–6.95%, n = 7) under the same conditions. The inter-day This paper has explored a method for the simultaneous
precision was determined at the same concentrations levels (RSD: determination of BaP, BbF, BkF, D[a,h]A, B[g,h,i]P, FLT, ANT, BaA,
0.40–4.82%, n = 7). CHR, FLR, PHE and PYR. The whole optimized process for the
extraction and separation of the various polycyclic aromatic
3.4. Analysis of PAHs in aquatic samples analytes was further verified by performing spiking experiments in
samples, showing low limits of detection, good recoveries and
The analytical determinations were carried out in triplicate for reproducibility. More importantly, the method was successfully
each sample. The fully optimized and validated analytical method applied to diverse aquatic products, which is vital for examining
was later applied to study the levels of BaP, BbF, BkF, D[a,h]A, undesirable food contaminants in complex food matrices. Quanti-
B[g,h,i]P, FLT, ANT, BaA, CHR, FLR, PHE and PYR in 35 aquatic tative results indicated that 12 PAHs were detected in the variety of
samples (Table 3). We observed that all the examined samples aquatic products, but their levels were below the specific
contained PAHs. The most represented PAHs were FLR, PHE and migration limits imposed by the European Commission.
ANT, detected in more than 50% of the analyzed samples. The 3
highest concentrations recorded for FLR, PHE and ANT were 57.76, Acknowledgements
16.14 and 17.68 mg/kg, respectively. Meanwhile, the concentra-
tions of almost all the other PAHs were lower than 5.00 mg/kg. The This work has been financially supported by the 2007AA091803
levels of all 12 PAHs examined in aquatic products were below the project of the National High-tech R&D Program (863 Program). The
specific migration limits imposed by the European Commission (EC authors would like to thank Xuemei Wu for her kind help and
208/2005), indicating that all the aquatic products tested were safe useful scientific discussions.
for consumption.
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