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Detecting Polycylic Aromatic Hydrocarbons in Food
Dedicated GC columns offer more accurate compound reporting
by Johan Kuipers, John Oostdijk, Claudia Schulz, and Ansgar Ruthenschroer
The Gulf of Mexico oil spill, the largest in U.S. history, has raised awareness of a food safety issue, namely contamination by polycyclic aromatic hydrocarbons (PAHs). In the future, analytical testing for PAHs in fish, crustaceans, and bivalves will undoubtedly become a routine procedure for many laboratories. PAH exposure, through either environmental pollution or contaminated foodstuffs, and its effects on human health have been the topic of many scientific studies. The recent oil spill again focuses attention on this toxic class of compounds.
PAHs comprise a large group of chemical compounds that are known cancer-causing agents. Some PAHs have been shown to be carcinogenic and mutagenic.
PAHs comprise a large group of chemical compounds that are known cancer-causing agents. Some PAHs have been shown to be carcinogenic and mutagenic. The scope of monitored and regulated PAHs is under constant change, influenced by international advisory bodies such as the World Health Organization (WHO) and the European Food Safety Authority (EFSA). Changes in regulations highlight the need for more accurate quantification and improved detail in separation to isolate key PAHs from possible interfering isomers. Gas chromatography (GC), in combination with mass spectrometry (MS), is one of the principal analytical techniques used for identifying and quantifying PAHs in environmental and food-related samples.
During GC/MS analysis, some co-eluting PAHs exhibit an identical MS fragmentation pattern. The possible chromatographic co-elution of some PAHs therefore requires special attention. To obtain unambiguous identification and highly accurate quantification of priority and regulated PAHs, an optimized capillary column is essential.
Here we discuss the possibilities offered by a new generation of dedicated GC columns for PAH analysis, which contribute to more accurate reporting of these compounds for both food and environmental monitoring.
Until recently, most analytical methods for PAH monitoring were established for analyzing the 16 priority pollutant PAH compounds recommended by the U.S. Environmental Protection Agency (EPA). They are naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-cd]pyrene (see Table 1, p. 38). This list is often targeted for measurement in environmental samples.
The concern about environmental PAH pollution arises from the bioaccumulation risk in the food chain. PAH exposure occurs mainly through air inhalation and food consumption. Sources of airborne PAH include traffic and industry as well as tobacco smoke and open fires. Dietary exposure to PAHs through food consumption has recently gained importance because of general concern about food safety in the European Union and the United States. The oil spill in the Gulf will lead to mounting concern for food safety risks associated with marine organism consumption.
PAH occurrence in food is influenced by the same physiochemical characteristics that determine their absorption and distribution in man. Relative solubility in water and solvents, as well as volatility, determines their capacity for transport and distribution and, consequently, influences their uptake by living organisms. PAHs have a lipophilic nature that contributes to their accumulation in the lipid tissue of plants and animals. The waxy surfaces of vegetables and fruit can concentrate low molecular mass PAHs, mainly through surface absorption. The deposition of small PAH-contaminated airborne particles is the principle route of contamination for vegetables. Atmospheric fallout is also responsible for contamination of less volatile PAHs, which end up in fresh water or marine sediments. PAHs are strongly bound to these sediments, which then become potential reservoirs for PAH release. Filter feeding bivalves have a low metabolic capacity for PAHs, which may lead to their bioaccumulation. Oil spills are the other main cause of PAH contamination of marine organisms.
Smoking and Food Processing
Raw foods rarely contain substantial levels of PAHs, reflected by the relatively low-level background contamination in unprocessed foods from remote rural areas. The produce contamination level is already elevated in more populated regions because of airborne PAH emission by industry and motor vehicles.
Drying, smoking, grilling, roasting, and frying are major PAH-generating food processes and can contribute to alarming PAH levels. Smoked fish and barbequed meat may contain up to 200 µg/kg of PAHs. Charcoal-grilled duck breast steaks were reported to contain up to 300 µg/kg PAH. Smoke-processed duck breast steak contained up to 53 µg/kg of carcinogenic PAHs.
Vegetable oils used for seasoning and cooking, as well as those incorporated into biscuits and cakes, are significant dietary sources of PAHs. Their occurrence and level varies widely depending on drying processes and refining.
In 2002, the former EU Scientific Committee on Food identified 15 PAHs as potentially carcinogenic and suggested benzo[a]pyrene as an indicator of the occurrence and effect of carcinogenic PAHs in food. In 2005, the Joint FAO/WHO Expert Committee on Food Additives confirmed these findings and proposed adding benzo[c]fluorine. This group of PAHs has become known as 15+1 EU priority PAHs (see Table 1, above).
Recent scientific opinion of the EFSA Scientific Panel on Contaminants in the Food Chain led to the adoption of an alternative and more limited list of PAHs for food risk characterization. Oral carcinogenicity data are only available for benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, dibenzo[a,h]anthracene, and indeno[1,2,3-cd]pyrene.
For some time, benzo[a]pyrene was thought to be a suitable marker for the occurrence and effects of carcinogenic PAHs in food and was, therefore, the only regulated one. The EFSA panel concluded, however, that these eight PAHs (PAH8), either individually or in combination, were the best indicators of PAH toxicity in food. More recently, benzo[a]pyrene, chrysene, benz[a]anthracene, and benzo[b]fluoranthene (PAH4) have been suggested by the EFSA panel as suitable PAH indicators. The PAH4 EU regulation will come into effect in 2010-2011.
Accurate quantification of individual PAHs and PAH sets, either as 16 EPA, PAH(15+1), or the subset PAH4, requires separation of individual PAHs from interferences either by mass ion and/or by chromatographic separation.
GC/MS Analysis for PAH Monitoring
PAH analysis in food is typically performed using gas chromatography-mass spectrometry (GC/MS) operated in the selective ion-monitoring (SIM) mode. The SIM mode improves selectivity while increasing sensitivity. Given the rising number of PAH analytes targeted for routine monitoring, along with regulation changes and the fact that many PAH congeners exhibit identical MS ion fragmentation, the chromatographic separation and selectivity of the GC column used for PAH analysis has gained importance. Often, general-purpose GC columns do not deliver the degree of selectivity required by the new regulations for more detailed PAH analyses. The chromatographic separation of PAH congeners that have very similar chemical structures and molecular mass is challenging (see Table 2, left).
GC columns for PAH analysis should be able to distinguish the small structural differences that exist between PAHs with virtually similar physicochemical properties. The high-boiling nature of the 5/6-ring congeners requires high GC column elution temperatures, in excess of 325°C. Obviously, GC columns and liquid phases for the analysis of these 5/6-ring PAHs should therefore be highly temperature resistant.
Recently introduced columns based on ionic liquids lack the thermal robustness for elution of the 5/6 -ing PAHs and, hence, have a limited durability. The majority of liquid phases are based on polysiloxane backbone chemistry, which is generally more thermally resistant. In recent years, functional groups (i.e., phenyl) have been incorporated into the polysiloxane chain as arylene inclusions, increasing the thermal and oxidative resistance of the liquid phase. Columns coated with such phases can operate at higher temperatures. This increased thermal resistance is apparent at temperatures above about 300°C. These arylene low bleed columns support the elution temperatures necessary for high-boiling dibenzopyrenes.
Arylene- and phenyl-substituted liquid-phase columns provide a reasonable degree of selective PAH separation. The most popular PAH column choices are the non-polar 5% phenyl/arylene polysiloxane (DB-5ms, Rtx-5ms, VF-5ms, ZB-5ms) and mid-polar 50% phenyl/arylene polysiloxane phase columns (DB-17ms, Rtx-17ms, VF-17ms). However, both liquid phases will suffer from inaccurate quantification of key target EPA and EU PAHs due to co-eluting interferences such as benzo[j]fluoranthene and triphenylene.
Figure 1 (above) illustrates the separation and quantification difficulties for important priority EPA and EU PAHs. Chrysene cannot be measured accurately on non-polar or mid-polar columns because triphenylene co-elution may create biased results. In addition, benzo[b]fluoranthene cannot be quantified accurately on 5% phenyl/arylene columns due to co-elution with the benzo[j]fluoranthene isomer. Co-elution also occurs for the triplet indeno[1,2,3-cd]pyrene/benzo[b]triphenylene/dibenz[a,h]anthracene group on these 5% phenyl/arylene columns.
The combined separation of chrysene/triphenylene and the three benzofluoranthene isomers is a unique feature of the Varian Select PAH column and is not possible on other commercially available columns.
A new, selective GC column dedicated for PAH analysis, Varian Select PAH, was recently introduced that has the unique ability to isolate chrysene from the interfering triphenylene while simultaneously separating the three benzo[b,k,j]fluoranthene isomers. The liquid phase of this column incorporates highly selective selectors for PAH-isomer separation to overcome the limitations of other GC columns. The selective column can also separate other critical peak triplets such as indeno[1,2,3-cd]pyrene, benzo[b]triphenylene, and dibenz[a,h]anthracene (see Figure 2, below).
The improved separation characteristics of this column provide a more precise characterization of PAHs in various food matrices such as smoked haddock and salmon (see Figures 3-4, below and p. 42). Salmon was spiked with a mixture of EPA- and EU-regulated PAHs, as well as triphenylene as an important interference. The PAH concentration range varied from <0.5 parts per billion (ppb) up to 10 ppb for benzo[a]pyrene. For sample saponification, a potassium hydroxide in methanol solution was added to the homogenized and weighted salmon. After saponification, the PAHs were extracted with cyclohexane. The extract was concentrated and further cleaned using fully automated gel permeation chromatography directly coupled to an evaporation unit in the same analytical system. Dichloromethane was used as the eluent. The fraction containing the PAHs was concentrated and analyzed by GC/MS.
Fast, detailed analysis of PAH was obtained on a 15 m x 0.15 mm x 0.10 µm Select PAH column, with dibenzo[a,h]pyrene, the last PAH of interest, eluting at 28 min (see Figure 3, p. 40).
Chrysene and triphenylene were sufficiently separated to allow accurate chrysene quantification (see Figure 4, p. 42). Bias in chrysene quantification due to triphenylene interference has not been studied in detail in food matrices, mainly due their co-elution on most GC columns.
The higher molecular weight toxic dibenzopyrenes are usually less prevalent at low concentration levels. Dibenzopyrenes are prone to discrimination effects in the injector, and care must be taken to ensure complete evaporation to obtain higher responses. Their low volatility may also create adsorption effects in the MS interface and ion source.
Higher MS interface and ion-source temperatures can limit these phenomena, increasing responses and reducing peak tailing for these high molecular weight PAHs. Further significant enhancement of the analytical sensitivity for dibenzopyrenes, improvement of signal-to-noise (S/N) ratios, and faster elution can be achieved using thinner film columns of 0.1 – 0.15 µm as applied to the Select PAH column. The low-bleed performance and good S/N ratio of the Select PAH column at 350°C are apparent.
The presence of PAHs in vegetable oils is mostly related to contact with combustion gases from the seed drying processes. Refining methods such as deodorization and treatment with activated charcoal can reduce the PAH level significantly, but residues may remain.
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