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.     

Extraction of polycyclic aromatic hydrocarbons from cookies: A comparative study of ultrasound and microwave-assisted procedures

The chromatographic determination of 15 polycyclic aromatic hydrocarbons (PAHs) in cookies has been improved in order to obtain a fast method with a low limit of detection through the combination of microwave-assisted extraction (MAE), oil saponification and solid-phase extraction clean-up before the injection of purified extracts in a C18 201TP52 (5 µm, 250 ×?2.1 mm) column. Using acetonitrile–water as mobile phase, with a 50% to 95% w/w acetonitrile gradient for a fixed flow of 0.250 ml min^?1, 15 PAHs were separated in 45 min. The column temperature was maintained at 15°C; and fluorimetric detection was made at a fixed excitation wavelength of 264 nm and emission measurements at the best wavelength for each analyte, from 352 nm for 11H-benzo[b]fluorene to 500 nm for indeno[1,2,3-c,d]pyrene. Recoveries for all 15 PAHs varied between 96 ±?4 and 105% ±?4%; and the limits of detection ranged from 0.015 ng g^?1 for chrysene to 0.7 ng g^?1 for phenantrene. Results were compared with those obtained by conventional Soxhlet extraction during 8-h refluxing with toluene, demonstrating that the methodology proposed is appropriate to quantify PAHs in cookies. Furthermore, the microwave-assisted method was faster and used less solvent than the conventional and ultrasound-assisted methods. The extraction time was reduced to 9 min compared with the 8 h required for Soxhlet extraction and 60 min required for ultrasound-assisted treatment, and the solvent consumption has been reduced to 25 ml compared with the 155 and 90 ml required using Soxhlet and ultrasound, respectively.     

Extraction of polycyclic aromatic hydrocarbons from cookies: A comparative study of ultrasound and microwave-assisted procedures

The chromatographic determination of 15 polycyclic aromatic hydrocarbons (PAHs) in cookies has been improved in order to obtain a fast method with a low limit of detection through the combination of microwave-assisted extraction (MAE), oil saponification and solid-phase extraction clean-up before the injection of purified extracts in a C18 201TP52 (5 µm, 250  × 2.1 mm) column. Using acetonitrile-water as mobile phase, with a 50% to 95% w/w acetonitrile gradient for a fixed flow of 0.250 ml min^-1, 15 PAHs were separated in 45 min. The column temperature was maintained at 15°C; and fluorimetric detection was made at a fixed excitation wavelength of 264 nm and emission measurements at the best wavelength for each analyte, from 352 nm for 11H-benzo[b]fluorene to 500 nm for indeno[1,2,3-c,d]pyrene. Recoveries for all 15 PAHs varied between 96  ± 4 and 105%  ± 4%; and the limits of detection ranged from 0.015 ng g^-1 for chrysene to 0.7 ng g^-1 for phenantrene. Results were compared with those obtained by conventional Soxhlet extraction during 8-h refluxing with toluene, demonstrating that the methodology proposed is appropriate to quantify PAHs in cookies. Furthermore, the microwave-assisted method was faster and used less solvent than the conventional and ultrasound-assisted methods. The extraction time was reduced to 9 min compared with the 8 h required for Soxhlet extraction and 60 min required for ultrasound-assisted treatment, and the solvent consumption has been reduced to 25 ml compared with the 155 and 90 ml required using Soxhlet and ultrasound, respectively.     

食品中多环芳烃及卤代多环芳烃的研究进展

综述了多环芳烃及卤代多环芳烃的性质、毒性及国内外食品中的污染情况和研究现状,并对目前的分析测定方法进行了介绍,希望为我国开展食品领域内多环芳烃和卤代多环芳烃的研究提供参考。     

Optimization of the procedure for the determination of polycyclic aromatic hydrocarbons and their derivatives in fish tissue: Estimation of measurements uncertainty

Three alternative procedures were employed for the isolation of polycyclic aromatic hydrocarbons (PAHs; 15 of 16 US EPA priority pollutants and benzo[e]pyrene), their methyl-derivatives and sulphur analogues from fish tissue: (1) Soxhlet extraction, (2) batch extraction enhanced by sonication, and (3) saponification of the sample followed by re-extraction of analytes into hexane. Soxhlet extraction using hexane-acetone (1:1, v/v) was the most efficient extraction technique, with analyte recoveries in the range 70-108%. Within optimization of the clean-up step, several types of gel permeation chromatography (GPC) systems were tested: two types of polystyrene divinylbenzene copolymer gels (PSDVB), both 'soft' gel type (Bio-Beads S-X3) and 'rigid' gels type (PL gel and Envirogel) in combination with various mobile phases were compared. Bio-Beads S-X3 and mobile phase chloroform were the most appropriate for purifying of crude extracts before the final determinative step. High-performance liquid chromatography with fluorimetric detection (HPLC/FLD) was used for identification and quantification of PAHs in purified fish extracts. The uncertainties of PAHs measurements were estimated by employing two alternative approaches. Both provided similar results: the expanded uncertainties obtained for individual PAHs by the 'top-down' approach were in the range 9-53%, their values resulting from application of the 'bottom-up' approach were in the range 16-52%.     

Occurrence of polycyclic aromatic hydrocarbons and their hydroxylated metabolites in infant foods

Eleven polycyclic aromatic hydrocarbons (PAHs) have been analysed in commercial milk formulae and infant cereals. Two hydroxylated PAHs metabolites (1-OH-Pyr and 3-OH-B[a]P) and their conjugates were also analysed in milk samples. To determine the selected PAH metabolites, a simple, fast quantitative and economic method was developed. This method comprising ultrasound-assisted solvent extraction, enzymatic hydrolysis, solid-phase clean-up and detection by liquid chromatography with fluorescence detection (LC–FD) and liquid chromatography tandem mass spectrometry (LC–MS/MS) as confirmatory technique. The method was evaluated by constructing calibration curves, measurement of recovery, precision and the limits of detection. The purpose of this survey was to determine the selected analytes, to assess the exposure of babies and infants and to produce data for comparison with proposed limits that were being considered at the time of the survey. The results showed that not only no samples would have exceeded the limit for benzo[a]pyrene which is used as an indicator for the presence of PAHs, but also no hydroxy PAH metabolites have been detected.     

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.     

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.     

Single-laboratory validation of a saponification method for the determination of four polycyclic aromatic hydrocarbons in edible oils by HPLC-fluorescence detection

An analytical method is reported for the determination of four polycyclic aromatic hydrocarbons (benzo[a]pyrene (BaP), benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and chrysene (CHR)) in edible oils (sesame, maize, sunflower and olive oil) by high-performance liquid chromatography. Sample preparation is based on three steps including saponification, liquid–liquid partitioning and, finally, clean-up by solid phase extraction on 2 g of silica. Guidance on single-laboratory validation of the proposed analysis method was taken from the second edition of the Eurachem guide on method validation. The lower level of the working range of the method was determined by the limits of quantification of the individual analytes, and the upper level was equal to 5.0 µg kg^?1. The limits of detection and quantification of the four PAHs ranged from 0.06 to 0.12 µg kg^?1 and from 0.13 to 0.24 µg kg^?1. Recoveries of more than 84.8% were achieved for all four PAHs at two concentration levels (2.5 and 5.0 µg kg^?1), and expanded relative measurement uncertainties were below 20%. The performance of the validated method was in all aspects compliant with provisions set in European Union legislation for the performance of analytical methods employed in the official control of food. The applicability of the method to routine samples was evaluated based on a limited number of commercial edible oil samples.     

HPLC determination of polycyclic aromatic hydrocarbons in olive oils

 In this paper, HPLC with spectrofluorimetric detection was applied to the determination of polycyclic aromatic hydrocarbons (PAHs) in olive oils. These compounds may sometimes contaminate vegetable oils because of their specific lipophilic characteristics, which are a significant problem for their extraction and purification from lipid matrices. Some improvements to previously published methods are introduced and satisfactory results for repeatability and recovery were obtained. Data on 51 authentic olive oil samples are reported and it was found that there is usually a limited presence of PAHs in extra virgin olive oils; furthermore, the analysis of some blends of refined and virgin oils shows that the distributions of light and heavy PAHs are different with the content of the former being lower in refined samples. As an example of this fact, two samples of lampante oil were followed throughout the refining step. Received: 23 September 1996 / Revised version: 17 December 1996     

Evaluation of polycyclic aromatic hydrocarbons content in different stages of soybean oils processing

A study was conducted in order to determine the levels of 13 polycyclic aromatic hydrocarbons (PAHs) in crude soybean oils produced in Brazil and to evaluate the influence of the refining process in their reduction. Analysis of intermediary products (neutralized, bleached and deodorized oils) showed that all compounds were reduced through refining (up to 88%). Neutralization and deodorization steps contributed effectively to the PAHs decrease. The mean total PAHs content in crude and deodorized oil samples ranged, respectively, from 10 to 316 and 3 to 69μg/kg. Since vegetable oils have been shown to be the major sources of PAHs in the diet, a monitoring program should be developed by the refining industries and the use of activated carbon during oil processing is highly recommended.     

油籽炒籽条件对油脂中多环芳烃含量影响的研究

以花生仁和芝麻籽为原料,研究了炒籽温度和炒籽时间对其油脂中16种多环芳烃含量的影响。结果表明,随着炒籽温度的提高及炒籽时间的延长,花生油和芝麻油中Bap、PAH4、PAH16的含量都呈明显上升趋势。对照GB2716及欧盟No 835/2011中对Bap、PAH4的限量规定,花生仁的合理炒籽温度为不超过160℃、炒籽时间不超过20 min,芝麻的合理炒籽温度为不超过180℃、炒籽时间不超过20 min。在优化的炒籽条件下,花生油中Bap、PAH4、PAH16含量(μg/kg)分别从原料中的0.31、4.60、16.69增加至1.07、11.98、48.86,芝麻油中Bap、PAH4、PAH16含量分别从原料中的0.63、5.23和21.84增加至0.93、8.28和47.95。     

食品中多环芳烃的提取、纯化、以及检测方法的研究进展

对食品中多环芳烃(PAHs)的提取、纯化机理和检测方法进行了概述,侧重介绍了样品提取、纯化和分离过程中影响PAHs回收率的各种因素。     

Determination of polycyclic aromatic hydrocarbons (PAHs) in commonly consumed Nigerian smoked/grilled fish and meat

Smoking and/or grilling, when carried out with traditional methods involving direct contact with wood combustion fumes, is responsible for high contamination levels with carcinogenic polycyclic aromatic hydrocarbons (PAHs). The aim of this work was to investigate the PAH content of different smoked or grilled meat and fish products commonly consumed in Nigeria. A rapid method involving microwave-assisted saponification and simultaneous extraction followed by solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) separation and spectrofluorometric detection was employed. Samples that were smoked or grilled using traditional systems, which use a wood fire, were heavily contaminated with benzo[a]pyrene (BaP) at levels ranging from 2.4 to 31.2 µg kg^?1 wet weight. Considerably lower contamination levels were found in samples smoked or grilled in the laboratory using a charcoal fire (BaP from 0.7 to 2.8 µg kg^?1 wet weight). The health risk associated with a daily consumption of 100 g of these products was also evaluated using the margin of exposure (MOE) approach. MOE values lower than 10,000 were obtained for all smoked/grilled commercial samples, indicating a potential concern for consumer health.     

四种多环芳烃低剂量联合暴露对大鼠毒性作用的初步研究

目的 初步研究4种多环芳烃(苯并[a]芘、苯并[a]蒽、艹屈和苯并[b]荧蒽)低剂量联合暴露的毒性作用。方法 购入50只8周龄SD雄性大鼠,随机分为5组,每组10只,分别按0、10、50、250、1000 μg/kg·BW剂量连续灌胃染毒,基于人体4种多环芳烃的实际暴露量,灌胃剂量的比例设定为苯并[a]芘∶苯并[a]蒽∶艹屈∶苯并[b]荧蒽=0.99∶2.92∶2.68∶1.68。大鼠于染毒30 d后处死,取血清、脏器等生物样本,计算脏器系数,进行大鼠肝脏病理切片观察病理学改变,并检测肝功能相关指标、氧化应激相关指标及脂代谢相关指标。结果 相较于对照组,染毒组大鼠肝脏结构有明显异常,肝窦扩大,肝细胞出现气球样变;1 000 μg/kg·BW组肝脏脏器比显著升高。染毒30 d后1 000 μg/kg·BW组大鼠血清谷草转氨酶均显著上升(P<0.05);染毒组大鼠血清谷胱甘肽过氧化物酶在30 d时显著升高(P<0.05);染毒30 d后,1 000 μg/kg·BW组大鼠血清高密度脂蛋白胆固醇显著下降并且肝脏胆固醇显著上升(P<0.05),250 μg/kg·BW组大鼠血清甘油三酯显著上升(P<0.05),50 μg/kg·BW及以上染毒剂量的染毒组肝脏中TG显著上升(P<0.05)。结论 PAH4低剂量联合暴露对SD雄性大鼠产生了一定程度的肝损伤、氧化应激及脂代谢紊乱等不良影响。     

8种多环芳烃联合暴露致大鼠肝脏毒性及BMDL推导

目的 探究8种多环芳烃（PAH8）联合暴露的大鼠肝脏毒性,利用基准剂量法（BMD）获得PAH8致肝脏毒性的基准剂量95%置信区间下限值（BMDL）。方法 雄性SD大鼠随机分为5组,每组10只,分别按照0、10、50、250和1 000μg/kg·BW剂量的PAH8连续染毒30 d后处死大鼠,计算脏器系数,进行肝脏病理学检测和油红O染色,检测血清谷丙转氨酶、谷草转氨酶、丙二醛、谷胱甘肽过氧化物酶（GSH-Px）、甘油三酯（TG）、总胆固醇（TC）的水平及肝脏TG、TC含量。选择具有统计学意义、毒理学意义和剂量效应趋势的肝脏毒性数据,利用BMDS 3.2软件进行BMD分析,选择最佳拟合模型得到BMDL值。结果 1 000μg/kg·BW剂量组大鼠肝脏系数较对照组显著升高（P<0.05）。PAH8染毒后部分大鼠肝脏出现细胞水肿、炎性浸润、脂肪变性等病理改变。10～250μg/kg·BW剂量组血清GSH-Px较对照组显著升高（P<0.01）,但1 000μg/kg·BW剂量组GSH-Px显著降低（P<0.001）。肝脏中TC含量呈现剂量效应趋势,1 000μg/kg·BW剂量...     

食品中多环芳烃的研究进展

简述了近年来关于食品中多环芳烃的形成机理、分析方法及控制措施的研究进展,以期为解决食品中多环芳烃的污染问题提供依据。     

Formation of polycyclic aromatic hydrocarbons in beef and lamb kokorec: Effects of different animal fats

Traditional kokorec, which is one of the products of offal (edible by-products) and consumed enthusiastically in Turkey, is produced from fresh and washed lamb and calf small intestines. Some health risks can occur unless hygiene and sanitation rules are followed and proper cooking procedures are applied during the production process of kokorec. One of the most important among these risks is the microbial origin hazard and another is the formation of polycyclic aromatic hydrocarbons. In this study, the aim was to determine the concentrations of eight polycyclic aromatic hydrocarbons formed during the cooking of kokorec produced from beef and lamb small intestines by adding various animal fats. Polycyclic aromatic hydrocarbonformation was specified in ready-to-consume kokorec samples in which eight polycyclic aromatic hydrocarbon concentrations varied between 3.07 and 40.11 µg/kg. The consumption of lamb kokorec could be recommended to consumers because its average total eight polycyclic aromatic hydrocarbon concentration was lower than that of beef kokorec.     

Application of accelerated solvent extraction followed by gel performance chromatography and high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons in mussel tissue

Accelerated solvent extraction (ASE) has been evaluated as a fast alternative to methanolic saponification for the extraction of 12 polycyclic aromatic hydrocarbons (PAHs) from mussel tissue. Several solvent systems and different operating conditions were investigated. The mixture dichloromethane-acetone (1:1, v/v) gave the best recoveries at 125°C and 1500 psi, in a total time of 10 min. No yield difference was found between freeze-drying (Fd) or drying the wet mussel with diatomaceous earth (Ded) prior to extraction. The ASE method was validated using the standard reference material SRM 2977, a freeze-dried mussel tissue with naturally present organic contaminants. The performance characteristics of the ASE method (trueness: 70-110%; precision: 4-14% and limit of quantification (LOQ): 0.1-0.25 µg/kg) meet the criteria established by the European Union for quantitative methods of analysis for official control of organic residues and contaminants. ASE provides a 24 times faster extraction than MSE and reduces 12 times the volume of solvent required.     

Sources of contamination by polycyclic aromatic hydrocarbons in Spanish virgin olive oils

The presence of polycyclic aromatic hydrocarbons (PAHs) in virgin olive oils results from contamination on olive skins and the oil itself during processing. Determination of nine PAHs was carried out by isolation of the hydrocarbon fraction and subsequent clean-up by solid phase extraction, followed by RP-HPLC analysis using a programmable fluorescence detector. Contamination of olive skins depends directly on environmental pollution levels and inversely on fruit size. In the oil mill, PAHs levels were increased by contamination from combustion fumes during the extraction process. Other procedures, such as washing or talc addition during extraction, did not affect PAHs levels. High concentrations of PAHs were only found as a consequence of accidental exposure to contamination, such as direct contact of olives with diesel exhaust and oil extraction in a polluted environment.