Analysis of polycyclic aromatic hydrocarbons in fish: Optimisation and validation of microwave-assisted extraction

An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatography with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the samples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food.     

Simultaneous formation of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) in gas-grilled beef satay at different temperatures

This study investigated the simultaneous formation of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) in gas-grilled beef satay at different temperatures (150, 200, 250, 300, and 350°C). Solid-phase extraction (SPE) was used for sample clean-up. Fifteen PAHs were determined using high performance liquid chromatography with fluorescence detection (HPLC-FLD) and nine HCAs were quantified using liquid chromatography tandem-mass spectrometry (LC-MS/MS) with a gradient programme. The lowest significantly concentrations of PAHs and HCAs were generated at 150°C; the formation of PAHs and HCAs simultaneously increased with temperatures. Benzo[a]pyrene was detected in all samples and increased markedly at 300 and 350°C. The sums of 4 PAHs (PAH4) in marinated beef satay at 300 and 350°C exceeded the maximum level in Commission Regulation (EU) 2015/1125. Significant reductions of polar and non-polar HCAs (except PhIP) were found in marinated beef satay across all temperatures. Overall, PAHs and HCAs showed opposite trends of formation in beef satay with marination.     

Optimisation and validation of an HPLC method for determination of polycyclic aromatic hydrocarbons (PAHs) in mussels

A method for determination of 11 polycyclic aromatic hydrocarbons (PAHs) in mussels (Mytilus galloprovincialis), using HPLC coupled to a fluorescence detector, has been optimised and validated, according to European Community rules. Sample preparation involves alkaline digestion of mussel tissue, liquid–liquid extraction of organic compounds and solid phase clean-up. Accuracy and precision of the method were determined by a validation study, carried out to demonstrate that the method is useful for both screening purposes and confirmation. Commission Regulation (EC) No. 2007/333/EC stated the performance criteria for the analysis of the only benzo[a]pyrene (BaP) in food products of animal origin, since BaP is the most studied PAH. We extended the BaP analysis performance criteria to other 10 toxic PAHs (listed in Commission Recommendation (EC) No. 2005/108/EC) and the validation study was performed also in agreement with Commission Decision (EC) No. 2002/657/EC. The method was applied to investigate 27 mussel samples from actively producing shellfish plants located in Campania (Italy) and variable levels of PAHs were detected ranging from <0.2 to 16 μg/kg wet weight.     

Polycyclic aromatic hydrocarbons in milk powders marketed in Argentina and Brazil

The aim of this study was to quantify polycyclic aromatic hydrocarbon (PAH) levels in milk powder samples commercialised in Argentina and Brazil during 2012. Thirty-one samples were available from the retail market. An HPLC method for the determination of PAHs was applied involving a clean-up step with silica cartridges. Recoveries were greater than 79% for all PAHs analysed. Reproducible determination with adequate detection and quantification limits (LOD and LOQ) were attained by HPLC with fluorescence detection for 14 PAHs. Acenaphthylene was determined with a UV–VIS detector. There is no significant difference in any PAHs or in the sum of them between the Argentinean and Brazilian samples. Therefore, the samples were evaluated together. The highest concentration of benzo(a)pyrene (BaP) detected was 0.57 µg kg^?1 in milk powder. Contamination of samples expressed as the sum of 15 analysed PAHs varied between 11.8 and 78.4 µg kg^?1 and as PAH4 (BaP, chrysene, benzo(a)anthracene and benzo(b)fluoranthene) was between 0.02 and 10.16 µg kg^?1. The correlation coefficient for PAH2 (BaP and chrysene) and PAH4 groups was 0.95, for PAH2 and PAH8 it was 0.71, and for PAH4 and PAH8 it was 0.83. All the samples were below the regulatory limit for BaP, but 65% of commercial milk powders do not comply with the European Union limit for PAH4. This is the first report of PAH contamination in powder milk from Argentina and Brazil.     

Influence of different smoking techniques on contamination by polycyclic aromatic hydrocarbons in traditional smoked Mozzarella di Bufala Campana

The levels of six polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene and three other PAH were determined in smoked Mozzarella di Bufala Campana. Different smoking techniques were compared to evaluate the risk associated with consumption of these cheeses. Although no maximum limits are set for PAHs in cheese, the levels found in some samples constitute a health risk. In fact, using corrugated cardboard, high levels were detected in the whole product or in the outer part. Also, the use of traditional materials involves the accumulation of PAHs in the outer part, but in the total product concentrations are significantly lower. No contamination was found using liquid smoke.     

Polycyclic aromatic hydrocarbons (PAHs) in yerba mate (Ilex paraguariensis) from the Argentinean market

Polycyclic aromatic hydrocarbons (PAHs) occurrence in 50 samples marketed in the main supermarkets from Argentina was surveyed. A high performance liquid chromatography (HPLC) method was applied with fluorescence detection (FLD) and UV—VIS diodes array detector (DAD) for the analysis of 16 PAHs in “yerba mate” (Ilex paraguariensis), with recoveries higher than 89% and limits of detection and quantification lower than that found by other methodologies in previous studies. Contamination expressed as the sum of 16 analysed PAHs ranged between 224.6 and 4449.5 μg kg^?1 on dry mass. The contamination expressed as PAH4 (sum of benzo(a)pyrene, chrysene, benzo(a)anthracene and benzo(b)fluoranthene) varied between 8.3 and 512.4 μg kg^?1. The correlation coefficient for PAH2 (sum of benzo(a)pyrene and chrysene) and PAH4 groups was 0.99, for PAH2 and PAH8 (sum of benzo(a)pyrene, chrysene, benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, dibenzo(a,h)anthracene and indeno(1,2,3cd) pyrene) 0.97 and for PAH4 and PAH8 0.98.     

Determination of polycyclic aromatic hydrocarbons in cachaça by HPLC with fluorescence detection

Cachaça is a distilled alcoholic beverage obtained from sugar cane fermentation. In this study, 25 brands of cachaça commercially available in Brazil were analysed for the presence of 5 PAHs (benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene and dibenz[a,h]anthracene). The methodology involved liquid–liquid extraction with cyclohexane and dimethyilformamide–water (9:1, v/v), clean up on silica gel column and determination by high performance liquid chromatography using fluorescence detection. PAHs peak identity was confirmed by gas chromatography–mass spectrometry (GC–MS). Variable levels of summed PAHs were detected in the analysed samples, ranging from not detected to 1.94 μg/L. The results confirm the presence of PAHs in cachaça and suggest that contaminated sugar cane may be the source of PAHs in sugar cane spirits.     

Rapid determination of polycyclic aromatic hydrocarbons in natural tocopherols by high-performance liquid chromatography with fluorescence detection

A simple and rapid method has been developed and validated for the determination of seventeen polycyclic aromatic hydrocarbons (PAHs) in natural tocopherol products. Samples were dissolved in n-hexane, cleaned by an alumina column, and separated and determined by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. The recoveries were greater than 77.9%, except for the lowest molecular weight PAHs (Na, 1-Me, 2-Me, AC, F) which were between 15.9% and 75.8%. The limits of quantification were less than 0.38 ng/g for the heavy PAHs and less than 1.50 ng/g for the light PAHs. Good repeatabilities were achieved with RSD less than 10.7% for all the objective compounds. This method has been applied to evaluating PAHs contents in various natural tocopherol products and controlling natural tocopherol product quality.     

PAHs content in sunflower,soybean and virgin olive oils: Evaluation in commercial samples and during refining process

Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogenic compounds that may contaminate vegetable oils and their levels can be reduced by refining. In order to understand the influence of the refining steps, the content of 15 PAHs was assessed throughout alkaline refining in soybean, sunflower and olive oil samples. Eight commercial brands of these oils were also analysed. The analytical method involved a liquid–liquid extraction, a solid-phase clean up (C₁₈ and Florisil) followed by RP-HPLC with fluorimetric detection. The total PAHs content in the studied samples can be considered generally low. The light PAHs (2–4 rings) were predominant. Virgin olive oils showed the highest values (max. 26 μg/kg). An evident decrease of PAHs contents during alkaline refining was observed (71%, 88% and 85% in sunflower, soybean and olive oils, respectively) being more pronounced in light PAHs. Neutralization and, particularly, deodorization were the more effective steps contributing to the PAHs decrease. Bleaching was responsible for a slight increase in the PAHs content in soybean and olive oils.     

Oxidative stability, volatile components and polycyclic aromatic hydrocarbons of cold-smoked sardine (Sardina pilchardus) and dolphinfish (Coryphaena hippurus)

Although a wide variety of compounds are deposited during the smoking process, much more attention has been given to phenols and polycyclic aromatic hydrocarbons (PAHs). However, even though phenols have an important role to play as food antioxidants, some PAHs have cytotoxic and mutagenic effects. In the present work, two fish species differing in composition (dolphinfish (Coryphaena hippurus) and sardine (Sardina pilchardus)) were salted and cold-smoked. Two different smoking treatments were selected in order to compare phenol content and muscle antioxidant activity employing the ferric reducing power (FRAP) method. Both smoking treatments increased fish lipid oxidation stability compared with the salted muscle. The characterization of the volatile components of the headspace of the more intensely processed smoked products was carried out by SPME/GC/MS, which revealed the presence of typical phenol and carbonyl derivatives, as well as some oxidation products and PAHs of low molecular weight. PAH composition was further investigated by extracting and identifying the PAHs by GC/MS. Neither benzo(a)pyerene nor other high molecular weight PAHs were detected, naphthalene and its derivatives being the most abundant compounds in the smoked products.     

Minimal clean-up and rapid determination of polycyclic aromatic hydrocarbons in instant coffee

The essential aim of this work is the development of a simple, fast, quantitative and economic method for polycyclic aromatic hydrocarbons (PAHs) potentially generated by roasting coffee beans, which is the most important process in the coffee industry for the development of the characteristic flavour of the bean mix. The PAHs were chosen because they differed in the number of aromatic rings, had different polarity, have low residual limits, are commonly widespread in the environment and are generated by roasting. The key issue is whether or not the most polar PAHs, those with lower molecular weight or less rings, appear in the water extracts of ground roasted coffee beans, taking into account that those PAHs with lower molecular weight are those with higher volatality. The proposed analytical method is also broadly applicable to other roasted foods or their aqueous extracts. The method was evaluated by constructing calibration curves, measurement of recovery and precision, and the limits of detection. The method involves extraction with hexane, clean-up with a silica cartridge, concentration to dryness and injection of the acetonitrile solution of the residue for HPLC analysis with fluorescence detection. The method allowed to confirm or not the presence of the selected PAHs in instant coffees.     

Polycyclic aromatic hydrocarbons (PAHs) in meat products and estimated PAH intake by children and the general population in Estonia

The concentrations of benzo[a]pyrene and 11 other polycyclic aromatic hydrocarbons (PAHs) were analysed from 322 commercial, cured meat products and 14 home-grilled meat samples as part of the Estonian food safety monitoring programme during 2001-2005. The maximum acceptable concentration of 5 µg kg^-1 for benzo[a]pyrene was exceeded in 3.4% of samples. The highest PAH concentrations were detected in home-grilled pork samples. Using of disposable grilling unit resulted in 1.6 times higher PAH concentrations compared to the traditional wood-burning grill. The average intake of benzo[a]pyrene and sum of 12 PAHs from meat products was estimated for children (age 1-16 years) on the basis of an individual food consumption questionnaire and, for the general population, based on national food consumption data. The highest total PAH concentrations detected were 16 µg kg^-1 in smoked meat and ham, 19 µg kg^-1 in smoked sausage and 6.5 µg kg^-1 in smoked chicken samples. Since smoking and grilling are prevalent meat-cooking methods in Estonia, the impact of meat products is assessed to be significant in overall PAH intake.     

Cyclodextrin-Promoted Fluorescence Detection of Aromatic Toxicants and Toxicant Metabolites in Commercial Milk Products

The detection of polycyclic aromatic hydrocarbons (PAHs) and their metabolites in food and in agricultural sources is an important research objective due to the PAHs’ known persistence, carcinogenicity, and toxicity. PAHs have been found in the milk of lactating cows and in the leaves and stems of plants grown in PAH-contaminated areas, thereby making their way into both cow milk and plant milk alternatives. Reported herein is the rapid, sensitive, and selective detection of 10 PAHs and PAH metabolites in a variety of cow milks and plant milk alternatives using fluorescence energy transfer from the PAH to a high quantum yield fluorophore, combined with subsequent array-based statistical analyses of the fluorescence emission signals. This system operates with high sensitivity (low micromolar detection limits), selectivity (100% differentiation even between structurally similar analytes), and general applicability (for both unmodified lipophilic PAHs and highly polar oxidized PAH metabolites, as well as for different cow and plant milk samples). These promising results show significant potential to be translated into solid-state devices for the rapid, sensitive, and selective detection of PAHs and their metabolites in complex, commercial food products.     

Optimisation of microwave assisted extraction (MAE) for polycyclic aromatic hydrocarbon (PAH) determination in smoked meat

A rapid extraction method involving microwave assisted extraction (MAE), followed by sample clean-up on a silica cartridge, reversed-phase high performance liquid chromatography (RP-HPLC) and spectrofluorimetric detection, was optimised for polycyclic aromatic hydrocarbon (PAH) determination in smoked meat. Compared to solvent extraction assisted by sonication, MAE, carried out with n-hexane on 2g of lyophilised sample at 115°C for 15min, allowed to obtain better extraction efficiencies. Limits of quantification (LOQ, s/n=10) lower than 0.2μg/kg wet weight were found for all PAHs, except for Fl (0.3μg/kg), P (0.6μg/kg) and IP (0.4μg/kg). The optimised procedure, that presented good analytical performances (with recoveries ranging from 77% to 103%, and precision within 10% for most of the PAHs), was applied to determine PAH content in different smoked meat products from the Italian market.     

Lowering of concentration of polycyclic aromatic hydrocarbons in liquid media by sorption into polyethylene terephtalate—a model study

Rapeseed oil and water were spiked with polycyclic aromatic hydrocarbon (PAH) solutions at the total levels of 955.1 and 711.4 g kg^–1, respectively, filled into polyethylene terephtalate (PET) cylindrical receptacles and the PAH concentrations in both liquid media followed for more than 90 h by HPLC. During this time, the PAH concentrations decreased by 315.1 in oil and 212.7 g kg^–1 in water due to an interaction of PAHs with PET. Using a modified kinetic equation, the diffusion coefficients for PAHs in both liquid media were determined. Values of the diffusion coefficients obtained indicate that the polarity of medium did not affect the rate of PAH removal. Calculation of the area occupied by a PAH molecule on the PET surface suggests that either the multilayer adsorption or the diffusion of PAHs into PET bulk came into account as the decisive factor bringing about the decrease of PAH concentrations in both media.     

PAHs concentration in heat-treated milk samples

In this study the presence of residual levels of Polycyclic Aromatic Hydrocarbons (PAHs) in milk samples from Calabria was evaluated. A comparative analysis of PAHs concentrations was conducted on raw, pasteurized, UHT semi-skimmed and whole milk.Quantitative determination of PAHs was performed by HPLC using a fluorescence detector and analysis in HPLC-MS was conducted to confirm the presence of these compounds.Residual levels of PAHs were found in all milk samples analyzed, showing higher concentrations in pasteurized and UHT milk than in raw milk samples.The results obtained demonstrate that PAHs presence also in raw milk is dependent from environmental pollution but pasteurization and UHT treatments of milk can influence PAHs formation; the differences found between whole and semi-skimmed samples can be due to different fat content of milk.     

Determination of four priority polycyclic aromatic hydrocarbons (4PAHs) by GC-MS in traditional Turkish yoghurts

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are ingested with foods and cause toxicities for humans. Yoghurt is one of the main foods that mediate PAH ingestion. Yan?k yoghurt, a traditional strained yoghurt, can contain PAHs due to its production process. Therefore, the concentration and types of four polycyclic aromatic hydrocarbons were determined by gas chromatography-mass spectrometry (GC-MS). Yan?k yoghurt samples were collected from different shops in Denizli, Turkey. For the method validation, the Eurachem guide (second edition) was adopted. Accordingly, linearity, working range, limits of detection (LOD), quantification (LOQ), precision and trueness values were set. Due to the selected validation parameters, the limit for detection was between 0.03 ng g^?1and 0.05 ng g^?1while the limit for quantification was between 0.10 ng g^?1 and 0.16 ng g^?1. Recovery rates higher than 83.80 % were obtained for four PAHs at two spiked concentrations (2.0 and 5.0 ng g^?1). Five Yan?k yoghurt samples and one commercial concentrated yoghurt sample were successfully analysed using the validated method.     

Polycyclic aromatic hydrocarbons in smoked cheese

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) represent a group of organic compounds containing two or more aromatic rings. Their control in the human food chain is required due to the mutagenic and carcinogenic potential, exhibited in vertebrates. In the present study, the occurrence of PAHs in 36 cheeses smoked by various processes was investigated. RESULTS: PAH concentrations (sum of 15 US EPA PAHs) found in samples smoked under controlled industrial conditions were at level 0.11 µg kg^?1, whereas in ‘home‐made’ cheeses, the PAH content was up to 10 times higher. A similar trend was observed for B[a]P, a marker compound representing carcinogenic PAHs. While its levels in commercial products prepared by controlled smoking technologies were close to the limit of quantification (0.03 µg kg^?1); in household samples, the B[a]P content ranged from 0.6 to 0.9 µg kg^?1. Significantly higher amounts of PAHs (up to three to six times) were found in surface layers as compared to internal parts of cheese. CONCLUSION: Although smoked cheese is a popular food, only several papers have focused on PAH levels in these products. This paper evaluates the contribution of different smoking technologies to PAH contamination of several cheeses and thus can help in a risk assessment associated with their consumption. Moreover, the study shows the concentration ratios of selected PAHs, from which the type of smoking technology can be indicated. The results obtained in this study also supported the suggestion of the EU Scientific Committee on Food to use benzo[a]pyrene as an indicator of the occurrence of higher‐molecular mass PAHs. Copyright © 2008 Society of Chemical Industry     

Determination of polycyclic aromatic hydrocarbons in edible oils and barbecued food by HPLC/UV–Vis detection

Determination of nine polycyclic aromatic hydrocarbons in corn, sunflower, olive oils and barbecued meat and fish by HPLC/UV–Vis method is described. The extraction procedure included a saponification, liquid–liquid extraction and finally purification of PAHs through a house-made silica–alumina column. Chromatographic determination was based on separation of PAHs on ODS column and measurement at 254 nm. All polycyclic aromatic hydrocarbons were separated and analyzed in 12 min on reversed phase ODS column with acetonitrile/water mobile phase at 1.5 mL min⁻¹ flow rate. The detection limits of nine polycyclic aromatic hydrocarbons ranged from 0.26 to 1.15 μg L⁻¹ at a signal/noise ratio of 3. The linearity of the method was between 0.9951 and 0.9996. Oil samples contain different PAHs ranging from 0.44 to 98.92 μg L⁻¹. Barbecuing process increased the concentration (in the range of 2- to 8-fold) and caused the formation of PAHs in food samples.     

A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes

A GC/MS method for the determination of ten polycyclic aromatic hydrocarbons (PAH) with four to six condensed aromatic carbon rings (including the six carcinogenic PAH) in smoked meat products and liquid smokes has been developed. The method implies accelerated solvent extraction (ASE), gel permeation chromatography for efficient lipid removal without saponification and ¹³C-labelled PAH for quantification. The calibration curves showed a good linearity for all PAH in the concentration range 0.01–10,000 ppb, the repeatabilities (RSDs, n=6) of different PAH ranged from 3 to 12%. Using this method, the analysis of a standard reference material of the National Institute of Standards and Technology (mussel tissue, SRM 2977) resulted in a good accordance between measured and certified PAH concentrations. The determination of PAH contents in 26 samples of smoked meat products and liquid smokes further confirmed the analytical power of the new method and gave a first insight into the specific PAH patterns.