Article pubs.acs.org/JAFC
SPE-LC-FD Determination of Polycyclic Aromatic Hydrocarbon Monohydroxy Derivatives in Cephalopods Diana Lourenço,† Liliana J. G. Silva,† Celeste M. Lino,† Simone Morais,‡ and Angelina Pena*,† †
Group of Health Surveillance, Center of Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Sta. Comba, 3000-548 Coimbra, Portugal ‡ REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal ABSTRACT: A new analytical methodology, based on liquid chromatography with fluorescence detection (LC-FD), after extraction, enzymatic hydrolysis, and solid-phase extraction (SPE) through Oasis HLB cartridges, was developed and validated for the simultaneous determination of three monohydroxy derivatives of polycyclic aromatic hydrocarbons (PAHs). The optimized analytical method is sensitive, accurate, and precise, with recoveries between 62 and 110% and limits of detection of 227, 9, and 45 ng/g for 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Their levels were estimated in different cephalopod matrices (edible tissues and hemolymph). The methodology was applied to samples of the major cephalopod species consumed worldwide. Of the 18 samples analyzed, 39% were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected. Its concentration ranged from 786 to 1145 ng/g. This highly sensitive and specific method allows the identification and quantitation of PAH metabolites in forthcoming food safety and environmental monitoring programs. KEYWORDS: polycyclic aromatic hydrocarbons, monohydroxy derivatives, cephalopods, solid-phase extraction, liquid chromatography with fluorescence detection
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INTRODUCTION Studies have shown beneficial effects of marine species consumption in the prevention of chronic diseases.1 Among seafood species, cephalopods represent one of the most important groups captured in Europe. In Portugal, cephalopods represented, in 2008, only approximately 10% of landings; however, the corresponding values in terms of auction transaction were approximately 26% of the wholesale market registered for marine species.2 Diet is considered one route of human exposure to polycyclic aromatic hydrocarbons (PAHs), a large group of organic compounds that are included in the European Union and U.S. EPA priority pollutant list due to their mutagenic and carcinogenic properties.3,4 With the aim of minimizing harmful effects on human health, recently, maximum safe levels of some PAHs in seafood products were established through European Commission regulation.5 Although for most people fish and seafood represent only a small part of the total diet, the contribution of this food group to the daily intake of PAHs in some individuals may be comparatively important.6,7 In the marine environment, PAHs are bioavailable to marine species via the food chain, as waterborne compounds, and from contaminated sediments. As lipophilic compounds they can easily cross lipid membranes and have the potential to bioaccumulate in aquatic organisms.8−11 Invertebrates such as cephalopods, may accumulate and bioconcentrate PAHs,12 having the potential to act as bioindicators and sentinel species for ecological risk assessment and marine environment monitoring studies.2,13 Nonetheless, information on PAH levels in the major cephalopods species such as squids and octopus is scarce.2,14,15 © 2014 American Chemical Society
Monohydroxy derivatives of PAHs are generated in the human organism, as well as in fish and other aquatic organisms, possessing well-developed enzyme systems that efficiently convert PAHs to epoxides and hydroxylated derivatives during phase I metabolism. These derivatives are further converted, in phase II, into highly water-soluble conjugates, such as glucuronides or sulfates, to facilitate excretion.12 Although this mechanism produces a detoxification, some PAHs are metabolized to active mutagen or carcinogen substances, which are capable of attacking cellular DNA. Moreover, assays with animals have shown that some PAH metabolites are suspected to be endocrine disruptors acting like hormones.16,17 Consequently, 1-hydroxypyrene was already suggested as a biological index for the dose of pyrene and indirect indicator for all PAHs.18 In the past few years, improved methods of determining, simultaneously, several PAH metabolites have been proposed mainly for human biological samples.19 Nonetheless, until now, works that include the determination of PAH metabolites in foods are very limited.17 To the best of our knowledge, concerning the determination of PAH hydroxylated metabolites in cephalopod samples, no study was found. Furthermore, the evaluation of PAH metabolites in several cephalopods species will shed some light about the possibility of PAH detoxification by these species. The available information is far from being comprehensive.20 Received: Revised: Accepted: Published: 2685
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Materials and Equipment. Oasis HLB (Merck) solid-phase extraction (SPE) cartridges were used. The LC apparatus consisted of two Gilson pump models 305 and 321 coupled to a Gilson autoinjector, model 234 (Gilson Medical Electronics, Villiers-le-Bel, France). A model 0305 Ultrafluor scanning fluorescence detector (LabAlliance, USA) operated at variable-wavelength program was also used. The results were recorded on UniPoint software (Gilson Medical Electronics). A 250 × 4.6 mm i.d., 5 μm, PAH C18 chromatography column (Waters, Milford, MA, USA) maintained at 30 °C was used. All chromatographic solvents were filtered through a 0.20 μm nylon membrane filter (Whatman, Dassel, Germany) and degassed for 15 min in an ultrasonic bath. All standard solutions and sample extracts were filtered through Durapore PTFE syringe filters, 0.45 μm, from Millipore (Milford, MA, USA). Sample Collection and Characterization. Fresh squid samples were randomly acquired from November 24 to 30, 2011, at supermarkets in the city of Oporto, Portugal. All samples were Loligo gahi, from Argentina Southwest Atlantic Ocean (FAO: 41). After arrival at the laboratory, samples were minced and homogenized. Moreover, hemolymph samples were obtained from Octopus vulgaris specimen harvested live from the Northwest Atlantic Portuguese coast. Sample collection, biometric characterization (Table 1), and
Due to the complexity of food samples, analysis of PAH metabolites remains a challenging task. A preconcentration step, which empowers detection of low concentrations and interferent removal, is required and often performed through solid-phase extraction (SPE). Among the analytical methods described in the scientific literature, reverse-phase sorbents, such as C18 cartridges, are the most applied in the analysis of biological, food, and environmental matrices.12,17,21 Moreover, methods for the simultaneous analysis of different PAH monohydroxy derivatives are quite limited and concern mainly matrices such as foodstuffs,17,22−25 and none about cephalopod samples. Quantitation usually relies on liquid chromatography with fluorescence detection (LC-FD) or on liquid chromatography with tandem mass detection (LC-MS-MS).17 This study provides the first report on the optimal conditions for the simultaneous determination of three monohydroxy derivatives of PAHs, namely, 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, using SPE-LC-FD, for the analysis of different cephalopod matrices (edible tissues and hemolymph). The developed method was applied to samples of the major cephalopod species consumed worldwide.26 The developed methodology allows an improved assessment of consumers’ PAH exposure, as well as the possibility to explore more deeply the PAH metabolic capacity of marine species and, in particular, of cephalopods.
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Table 1. Squid and Octopus Sample Characterization sample
weight (g)
total length (cm)
L10A L10B L10C L18A L18B L18C L19A L19B L19C L24A L24B L24C
58.70 54.54 64.74 98.55 89.51 85.55 105.24 79.70 84.62 83.84 90.42 79.37
18.50 17.75 24.00 22.00 21.75 21.00 25.75 23.75 24.25 24.00 20.00 23.25
MATERIALS AND METHODS
Safety Considerations. The toxicity or carcinogenicity of each chemical and reagent used in this method was not precisely defined. However, each one must be treated as a potential health hazard, and exposure to these chemicals should be minimized. Some method analytes have been tentatively classified as known or suspected human or mammalian carcinogens. Pure standard materials and stock standard solutions of these compounds should be handled with suitable protection to skin, eyes, etc. Standards and Chemicals. The standards of 1-hydroxypyrene, 2hydroxyfluorene, and 1-hydroxynaphthalene were purchased from Sigma-Aldrich (St. Louis, MO, USA), all with ≥98% purity. Individual stock solutions were prepared at 2 mg/mL in acetonitrile/water (50:50). These solutions were stored at −20 °C, in the dark, for a maximum of 6 months. Individual intermediate standard solutions were prepared at 250, 50, and 5 μg/mL in acetonitrile/water (50:50). Working standard solutions were prepared weekly, kept in amber flasks at −20 °C, and used for calibration, accuracy, and repeatability assays of the validation study. Given the different fluorescence sensitivity of each compound, mixed standard working solutions were prepared at different concentrations for each compound. For linearity assays, the following concentration levels were used: 750, 850, 1000, 1500, and 2000 ng/ mL for 1-hydroxynaphthalene; 30, 40, 50, 60, and 75 ng/mL for 2hydroxyfluorene; and 150, 175, 200, 250, and 300 ng/mL for 1hydroxypyrene. HPLC grade acetonitrile and methanol were obtained from Carlo Erba (Milan, Italy), and ethyl acetate was from Panreac (Barcelona, Spain). Double-distilled water used throughout the experiments was prepared daily from a Milli-Q system (Millipore, Bedford, MA, USA). Sodium acetate and ascorbic acid were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich, respectively. Glacial acetic acid was obtained from Panreac, and β-glucuronidase/arylsulfatase from Helix pomatia (100,000 Fishman units/mL at pH 4.5 and 38 °C and 800,000 Roy units/mL at pH 6.2 and 38 °C, respectively) was purchased from Roche Diagnostics (Mannheim, Germany). A solution of sodium acetate buffer at 0.1 M was prepared by dissolving 1.36 g of sodium acetate in 250 mL of distilled water and adjusting the pH to 5.0 with acetic acid. An ascorbic acid solution at 1 mg/mL was prepared in methanol.
H48 H51 H52 H53 H54 H55 a
1025.0 578.8 927.9 654.0 868.8 965.3
48.5 48.5 46.5 42.0 46.5 49.5
mantle width (cm) Squid 14.5 6.00 10.0 12.3 11.0 11.0 12.3 11.0 11.0 10.5 6.00 9.75 Octopus 10.5 7.0 9.5 8.0 6.5 6.5
arm length (cm)
moisture ± SD (%; n = 3)
12.5 14.5 14.3 12.5 12.0 11.5 12.5 12.3 12.3 12.3 12.8 12.8
86.8 ± 85.3 ± NDa ND ND ND 84.9 ± ND ND 85.1 ± ND ND
1.0 1.5
39.0 39.0 37.0 33.0 38.0 39.5
80.7 82.8 81.0 80.8 83.9 81.9
± ± ± ± ± ±
0.5 0.4 1.0 1.3 0.5 0.7
0.5
0.2
ND, not determined.
preparation were performed in accordance with EC regulation no. 333/2007.27 Moisture of squids was evaluated using 10 g of homogenized sample according to Portuguese Standard NP 22821991 and the official AOAC method.28 Samples were kept frozen, in polycarbonate containers, at −20 °C, until analysis. Sample Extraction, Hydrolysis, and Solid-Phase Extraction. After defrosting, samples were minced and homogenized, and 1 g was added of 1 mL of ascorbic acid (1 mg/mL) and 6 mL of acetonitrile. Once vortex stirred, the sample was ultrasonicated for 5 min and extracted by centrifugation at 4 °C and 10000g for 10 min. The supernatant was collected and concentrated to dryness at 37 °C under a gentle N2 stream. For the enzymatic hydrolysis, the extract was redissolved in 4 mL of sodium acetate buffer. Then, 20 μL of β-glucuronidase/arylsulfatase was added and the oxygen atmosphere replaced by nitrogen. After incubation at 37 °C for 45 min, the enzymatic reaction was stopped by 2686
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cooling to room temperature, and the extract was centrifuged at 10000g, for 10 min, at 4 °C. This supernatant was then loaded into 200 mg Oasis HLB cartridges, previously conditioned with 5 mL of water and 5 mL of methanol. The cartridges were then washed with 5 mL of water and eluted with 10 mL of methanol/ethyl acetate (50:50). Finally, the eluate was evaporated to dryness under a gentle stream of nitrogen, at 37 °C, and the dried extracts were analyzed. Chromatographic Conditions. Quantitation of the three PAH metabolites was carried out with LC-FD analysis. The dried eluate was dissolved in 1 mL of acetonitrile/methanol (50:50) and microfiltered. A 20 μL injection volume was used with a flow rate at 1 mL/min and a gradient elution of (A) (water) and B (acetonitrile) as follows: 0−5 min, 50% acetonitrile; 5−20 min, a linear gradient from 50 to 100% acetonitrile; 20−25 min, 100% acetonitrile; 25−30 min, a linear gradient from 100 to 50% methanol; and 30−32 min, 50% acetonitrile. A variable excitation/emission wavelength program was optimized to detect the different compounds as follows: 0−6.5 min, 266/348 nm; 6.51−9 min, 266/366 nm, acetonitrile; 9.01−30 min, 346/390 nm. Validation Assays. To verify that the analytical method complies with criteria for the performance characteristics as established by Commission Decision 2002/657/EC, the following described validation procedures were carried out. Linearity was evaluated through a calibration curve assembled by plotting each PAH metabolite peak area versus the concentration of standard solutions, at five concentration levels. The concentration levels ranged between 750 and 2000 ng/mL for 1-hydroxynaphthalene, between 30 and 75 ng/mL for 2-hydroxyfluorene, and between 150 and 300 ng/mL for 1-hydroxypyrene. Linearity was also evaluated in matrix-matched samples at 750, 850, 1000, 1500, and 2000 ng/g; 30, 40, 50, 60, and 75 ng/g; and 150, 175, 200, 250, and 300 ng/g 1hydroxynaphthalene,2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Detection and quantification limits (LODs and LOQs) were evaluated on the basis of the noise obtained with the analysis of unfortified blank samples (n = 6). LOD and LOQ were defined as the concentration of the analyte that produced a signal-to-noise ratio of 3 and 10, respectively, and were then tested experimentally by spiking blank samples at such levels. Recovery assays were performed on spiked samples to determine the accuracy and precision of the method, in triplicate, under the same analytical conditions. The accuracy was evaluated through spiking blank matrix at three levels of known concentrations: 750, 1000, and 2000 ng/g 1-hydroxynaphthalene; 30, 50, and 75 ng/g o2hydroxyfluorene; and 150, 200, and 300 ng/g 1-hydroxypyrene. Precision was evaluated though the RSD (%) of the fortified samples, in within- and between-day assays.
Given that the chromatographic separation achieved was inadequate, a gradient elution system of acetonitrile and water was established. For the simultaneous determination of the three compounds, a variable-wavelength program was optimized on the basis of previous studies.17,25 Because PAH metabolites are subject to thermal degradation and photodegradation, antioxidant addition should prevent or at least reduce these processes. Some authors recommended their use in the preparation of the mobile phase and standard solutions to avoid degradation and increase the stability of the analytes. The addition of ascorbic acid at 1 mg/mL to the mobile phase was tried; nonetheless, because no improvement in the standards’ stability was observed, this procedure was abandoned in further studies.9,33 The addition of tertbutylhydroquinone (TBHQ), a hydroquinone derivative substituted with a tert-butyl group, in the preparation of standard solutions has also been reported to avoid standards’ degradation.17 However, this compound shows fluorescence at the same wavelength as 2-hydroxyfluorene, causing an overload of the photomultiplier tube (PMT) of the detector. Because the aim of this study was to quantitate simultaneously 1hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, TBHQ usage was also excluded. To ensure standard stability, degradation studies on working standard solutions were performed during five consecutive days, and it has been concluded that the standard solutions were stable until the third day without antioxidant addition. Representative LC-FD chromatograms of a mixed standard solution and a spiked squid sample are shown in Figure 1. Extraction Procedure Optimization. Optimization of the extraction, enzymatic deconjugation, and purification/concentration steps was performed with a blank sample (without PAH metabolites).
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RESULTS AND DISCUSSION Chromatographic Method Optimization. A gradient reversed-phase LC-FD method was developed for the simultaneous determination of 1-hydroxynaphthalene, 2hydroxyfluorene, and 1-hydroxypyrene. Different chromatographic conditions such as stationary phase, mobile phase composition, with isocratic or gradient program, flow rate, injection volume, temperature, and wavelength program were optimized for the separation of the selected PAH derivatives. According to the scientific literature, C18 LC columns are the most employed.19,29,30 A C18 PAH column was chosen given their specificity for the LC analysis of these molecules and, as expected, separation and sensitivity were appropriate. During the optimization of the chromatographic method, the organic solvents most commonly used for PAH metabolite determination were evaluated. A combination of acetonitrile with water29,31 or methanol with water19,32 is reported by most authors, and both of these combinations were tried. In a preliminary study, an isocratic elution system, consisting of methanol or acetonitrile and water (95:5), was attempted.
Figure 1. (A) LC-FD chromatogram of a standard solution mixture of (1) 1-hydroxynaphthalene, (2) 2-hydroxyfluorene, and (3) 1hydroxypyrene at 1000, 50, and 200 ng/g, respectively. (B) LC-FD chromatogram of a spiked sample with (1) 1-hydroxynaphthalene, (2) 2-hydroxyfluorene, and (3) 1-hydroxypyrene at 1000, 50, and 200 ng/ g, respectively. 2687
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Table 2. Validation Data Obtained for 1-Hydroxynaphthalene, 2-Hydroxyfluorene, and 1-Hydroxypyrene recovery (%)
RSD within-day (%)
RSD between-day (%)
compound
LOD (ng/g)
LOQ (ng/g)
F1a
F2b
F3c
F1a
F2b
F3c
F1a
F2b
F3c
1-hydroxynaphthalene 2-hydroxyfluorene 1-hydroxypyrene
225.0 9.0 45.0
750.0 30.0 150.0
98.7 70.3 79.9
110.1 77.6 62.2
107.4 79.0 67.4
9.5 6.0 8.6
4.6 7.2 4.8
15.3 6.1 3.7
6.8 1.6 1.9
0.5 0.1 1.4
12.2 7.5 0.8
a
Fortification level 1 = 750/30/150 ng/g of 1-hydroxynaphthalene/2-hydroxyfluorene/1-hydroxypyrene. bFortification level 2 = 1000/50/200 ng/g of 1-hydroxynaphthalene/2-hydroxyfluorene/1-hydroxypyrene. cFortification level 3 = 2000/75/300 ng/g of 1-hydroxynaphthalene/2hydroxyfluorene/1-hydroxypyrene.
appropriate. Because the analyzed compounds degrade in the presence of oxygen, it is important to highlight the need to displace the oxygen present in the test tube by a nitrogen atmosphere shortly after the addition of the enzyme. SPE Optimization. SPE is one of the most employed tools for the cleanup of PAHs21 and their metabolites.17,19,37 First, we attempted to choose the best sorbent to obtain higher recoveries. C18 cartridges, reversed phase silica-based sorbents, have several disadvantages such as low breakthrough volumes, instability at some pH values, and the presence of silanol groups, which can result in low recoveries for some polar compounds or lack of precision from column to column, resulting in irreproducible recoveries. PAH hydroxylated metabolites have quite polar characteristic, and it was difficult to extract them with traditional reversed-phase sorbents. Aware that the SPE method, and particularly the C18 cartridges, might impose some variability in PAH hydroxylated metabolite recoveries, hydrophilic polymeric sorbents have been chosen, and best results were obtained with the reversed phase polymeric-based cartridges Oasis HLB, which allow a hydrophilic−lipophilic balance. According to our knowledge, the use of Oasis HLB has never been reported for the analysis of PAH hydroxylated metabolites. The initial Oasis SPE procedure was based on the C18 SPE procedures previously attempted.17,19 However, because 1hydroxynaphthalene and 1-hydroxypyrene metabolites were inefficiently retained, with recoveries of 34 and 32%, respectively, parameters, such as washing and elution steps, were optimized using a mixed standard working solution at 1000, 50, and 150 ng/mL 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. The obtained results suggested that for the washing step water is the more suitable solvent, as it has enough solvent power to remove weakly absorbed interferents without removing the strongly absorbed analytes. Washing solutions containing methanol led to substantial losses and greatly decreased 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1hydroxypyrene recoveries, from 87, 68, and 75%, respectively, to 66, 40, and 22%, respectively. With regard to the elution step, to ensure high recoveries of all PAH metabolites, mixtures of solvents are usually recommended. 21 Different solvents and volumes were evaluated, and it was found that the presence of methanol was important; nonetheless, neither methanol nor dichloromethane, alone, eluted efficiently 1-hydroxynaphthalene (recoveries of 15 and 40%, respectively) and 1-hydroxypyrene (recoveries of 32 and 8%, respectively). In agreement with other authors,17 10 mL of methanol/ethyl acetate was the best approach to obtain greater recovery. Method Validation. The developed analytical method complied with criteria for the performance characteristics as established by Commission Decision 2002/657/EC.38
Most of the analytical methods for determining PAH metabolites were developed for human urine samples,19 blood, and other biological fluids such as fish bile.34 Although some analytical methods have been optimized for the determination of hydroxylated PAHs in milk and in infant foods,17 to our knowledge no extraction procedures have been previously reported for their determination in cephalopods. To optimize the extraction efficiency, different parameters were tested. First, as these compounds are light sensitive and easily degradable, the importance of the addition of an antioxidant to the sample was evaluated. In the present study, the action of two antioxidants, ascorbic acid and TBQH, was assessed. When ascorbic acid was used at a concentration of 1 mg/mL, better recovery rates were achieved when 1 mL of the solution was added to 1 g of sample. With regard to the extraction solvent, and given that PAH metabolites present high solubility in acetonitrile, this solvent was chosen. Because enzymatic hydrolysis in the following step is active in aqueous media, the extraction organic solvent must be evaporated to dryness. However, the temperature applied during the evaporation step may limit the recoveries of hydroxylated metabolites. Different temperatures were tested (35, 37, 40, and 42 °C) and, in agreement with other studies,17 the highest temperatures degrade these compounds and, therefore, 37 °C was selected. In addition, to decrease the time of the evaporation step, two volumes of extraction solvent were tested, 6 and 10 mL. Nonetheless, the best recovery results were attained with 6 mL because a reduction in the evaporation time was accomplished. It is worth noting the importance of avoiding light and maintaining a low temperature to minimize metabolite degradation. Good separation between the sample and supernatant was achieved when 10000g was applied for 10 min at 4 °C. Hydrolysis Optimization. Because hydroxy metabolites are biotransformed to phase II metabolites by conjugation with glutathione, glucuronide, or sulfate, an enzymatic reaction with β-glucuronidase is crucial to liberate them and obtain free metabolites. As the enzymatic hydrolysis is a chemical reaction catalyzed by an enzyme that uses water to break molecules, the use of an aqueous buffer solution is essential. It will also keep the pH constant at the optimum value for enzyme activity. Therefore, several experimental conditions, such as pH (4.5, 5.0, and 5.5), buffer concentration (0.05, 0.1, and 0.5 M), enzyme volume, incubation time (40, 45, 50, and 60 min), and temperature (37, 40, and 42 °C) were optimized. In agreement with other authors,17,35 it was found that 4 mL of a 0.1 M sodium acetate buffer, at pH 5, at 37 °C for 45 min, gave the best results. On the basis of scientific evidence,17,19,36 the addition of 20 μL of β-glucuronidase/arylsulfatase from Helix pomatia to 4 mL of solution is enough for the reaction to occur extensively, which was tested in this study and revealed to be clearly 2688
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molecular weight compounds with two and three rings were the most predominant PAHs in all of the species. The detected concentrations in L. gahi (from the Atlantic Ocean) may mean additional carcinogenic risks for high cephalopod consumers. Moreover, in a survey that revealed the measurable presence of four low molecular weight PAHs (naphthalene, acenaphthene, fluorine, and phenanthrene) in three commonly consumed and commercially valuable fish species (sardine, chub, and horse mackerel) from the Atlantic Ocean, naphthalene was the most abundant PAH in sardine and horse mackerel, accounting for ca. 55% of the total PAHs.41 In contrast to our results, in a study22 that included the analysis of PAH metabolites in the bile of flounder (Platichthys flesus), the major PAH metabolite detected was 1-hydroxypyrene. 1-Hydroxyphenanthrene and 3-hydroxybenzo[a]pyrene were also detected in 70 and 24 samples, respectively, of the 87 samples analyzed. However, the concentrations of 1-hydroxyphenanthrene and 3-hydroxybenzo[a]pyrene were below or near the LOQ (0.002 mg/mL bile). The bile of flounder samples from the Gulf of Gdansk presented 1-hydroxypyrene concentrations ranging from 0.019 to 0.066 mg/mL.22 Rey-Salgueiro et al.37 also investigated the levels of 3hydroxybenzo[a]pyrene in the bile and plasma of Nile tilapia after waterborne exposure to benzo[a]pyrene (10 and 100 mg/ L). Metabolites were detected in bile and plasma, and conjugates of 3-hydroxybenzo[a]pyrene (glucuronide and/or sulfate conjugates) were the majority forms in both biological fluids, glucuronide 3-hydroxybenzo[a]pyrene being the main metabolite in bile. The same research group, in a previous study17 concerning food safety of commercial milk formulas and infant cereals, reported that no hydroxy PAH metabolites (1-hydroxypyrene and 3-hydroxybenzo[a]pyrene) or their conjugates were detected in the analyzed foodstuffs.
The linearity of the method was evaluated in solvent and in matrix-matched solutions. In the working standard solutions, at five concentration levels, correlation coefficients (r2) were 0.9987 for 1-hydroxynaphthalene and 0.9959 for 2-hydroxyfluorene and 1-hydroxypyrene. In matrix-matched solutions adequate r2 values of 0.9949, 0.9901, and 0.9903 for 1hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively, were obtained. Because no standard reference material is available for PAH metabolites in cephalopods, for accuracy and repeatability assays, recoveries were determined through a spiking matrix at three different levels, on three different days, and each extract was analyzed three times. Accuracy varied between 62.2 and 110.1%, with within-day and between-day repeatabilities ranging between 3.7 and 15.3% and between 0.1 and 12.2%, respectively (Table2). Application to Real Samples. The optimized and validated analytical methodology was applied to the analysis of 12 squid samples of Loligo gahi species and to 6 hemolymph samples obtained from Octopus vulgaris harvested live from the Northwest Atlantic Portuguese coast. L. gahi is widely distributed along the Pacific coast of South America. It is the second most important loliginid squid in commercial fisheries worldwide.39 A small part of the total catch is consumed in Europe, predominantly in Spain.2 With regard to the selected octopus, it corresponds to the major species consumed worldwide40 and was recently proposed as a sentinel species for the presence of contaminants in coastal waters.13 Of the 18 samples analyzed, 39% (7) were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected, ranging between 786 and 1145 ng/g (Table 3). Naphthalene, a low molecular weight PAH, which is
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Table 3. PAH Metabolite Concentrations from the Analyzed Samples sample
1-hydroxynaphthalene (ng/g)
L18A L18B L10B L10C L24A L24B L24C
830 786 788
