Polycyclic aromatic hydrocarbons (PAHs) are a large group of highly lipophilic organic molecules with two or more fused aromatic rings ^[1][2][1,2]. They contain only carbon and hydrogen atoms, unlike their parent category of polycyclic aromatic compounds (PACs). PACs include PAHs and their functional derivatives (alkyl, amino, chloro, cyano, hydroxyl, or thiol moieties) and may contain nitrogen, oxygen, or sulfur atoms in the aromatic structure (heteroatom PAHs) [3]. Given the limited state of knowledge regarding toxicological data on PACs, additional evaluation is required in order to provide an improved understanding of their toxicity [4].

PAHs are environmental and food-processing contaminants with suspected or confirmed carcinogenic properties ^[5][6][5,6]. Their conformational patterns, along with the respective ring number, determine, in a highly dependent manner, their physical and chemical properties [2].

Classification-wise, PAHs are generally divided based on their number of aromatic rings. As such, compounds containing two to four rings are classified as light PAHs (LPAHs) and are associated with increased (extremely high) volatility but low toxicity. Compounds with more than four rings are heavy PAHs (HPAHs), which are less volatile but more stable and exhibit higher toxicity ^[1][2][7][1,2,7].

In addition to unsubstituted PAH molecules, increasing attention has been given to PAH derivatives, which are perceived to exert greater toxicity than their precursor PAHs. These include substitutions on the aromatic ring and other groups of molecules, such as larger PAHs, alkylated PAHs, and/or compounds containing heteroatoms ^{[8][9][10][11][12]}[8,9,10,11,12].

When considering their toxicity, a great deal of information is accessible. As a summary of PAHs’ “history” in terms of analysis, research was initiated in the context of industrial sources of PAHs through environmental studies ^{[2][4][7][13]}[2,4,7,13]. Consequently, their migration to crops and food was found to be of great concern. Subsequently, regulatory bodies put in place guidelines with regard to the extent of their toxicity. In 1979, the United States Environmental Protection Agency (US-EPA) established a list of 16 PAHs that are considered priority pollutants. Later on (in 2008), given a series of analytical improvements, the 16 PAHs constituting the US-EPA list were deemed not representative enough for the entire PAH profile ^[2][8][13][2,8,13]. Accordingly, the European Food Safety Authority (EFSA) updated the list to align with the latest advances in the state of knowledge of toxicology [14]. The resulting EU 15+1 PAHs were established following a comprehensive exposure reassessment study (over 10,000 food samples from 18 European countries) and delineate the replacement of 8 LPAHs with another 8 HPAHs that manifest increased toxicity (Figure 1) ^[2][13][15][2,13,15].

PAHs are ubiquitous in the environment and are mainly generated from natural sources, including diagenetic processes (changes in sediments converting into rock) and anthropogenic sources (combustion of organic matter such as coal, wood, and vegetation) ^[2][3][2,3]. Consequently, PAH transport over long distances is accounted for by their airborne environmental contamination, enabled through their adsorption onto atmospheric particles, as well as their direct deposition onto soil and plants ^[2][16][17][2,16,17].

Moreover, PAHs undergo transformation processes in the environment over long periods, involving degradation reactions such as oxidation, nitration, and halogenation ^{[10][12][17][18]}[10,12,17,18]. As PAHs are exposed to light in the environment, they may undergo photochemical processes. Oxidation reactions generate oxygenated PAHs and quinones [19], as well as photodegradation products upon extended photooxidation, while nitro-PAHs result from reactions with NO₂ or NO₃ radicals [17]. Additionally, combustion processes result in the tandem emission of NPAHs and OPAHs during soot formation ^[18][20][18,20]. Further heterogeneous oxidation and incomplete combustion reactions promote their high abundance in polluted air and particulate matter ^[17][18][17,18]. Additionally, the interaction between environmental PAHs and halogen-containing compounds during food processing and photochemical processes might result in the production of halogenated PAHs (XPAHs) ^[10][12][21][10,12,21].

Given their associated negative effects, the need for a systematic and comprehensive analysis of PAHs is increasing ^[4][22][4,22]. Accordingly, considerable efforts have been dedicated to screening the environmental transport and fate of PAHs in order to supply further insight with regard to their exposure and effects on human health ^[4][17][4,17]. For example, Andersson and Achten (2015) thoroughly explained how the priority PAHs (Figure 1) are standardized and widely accepted by scientists and routinely integrated into various environmental investigations. However, the team pointed out the difficulty of using a small number of representatives for a plethora of compounds (Figure 2) ^[2][8][2,8].

In terms of PAH compounds overseen by EU and US rules versus those not following such guidelines, future studies are encouraged to re-evaluate their genotoxicity and carcinogenicity and should include additional compounds pertaining to their occurrence in food.

Meat, edible oils, and cereal products are among the main food categories that are accompanied by relatively high daily intakes [23]. Given that they are usually consumed in large amounts, these foodstuffs represent concerning dietary exposure levels [24].

Current evidence addresses the amounts of PAHs stemming from food intake, which depends on both the initial food contamination (manufacturing/packaging) and the method of cooking ^[2][15][25][2,15,25]. However, under the combined action of mixed manufacturing and packaging processes, there are still uncertainties with regard to the source, fate, and health effects of PAHs. To this extent, a broad context is set for the continuous need for additional relevant research on the toxicity, occurrence, and analysis of PAHs and PAH derivatives (nitrated, oxygenated, halogenated, etc.) in meat, edible oils, and cereal products.

2. PAH Sample Pretreatment in Food and Quantitative Analysis

Sample preparation requires comprehensive extraction followed by purification before detection [26], entailing consistent improvement, as well as alternative approaches [2]. To provide repeatable data and satisfy legal criteria, proper sample preparation is necessary. Taking into account the complexity of food matrices, as well as the trace quantities of PAH molecules in comparison to other constituents, sample pretreatment translates into laborious and time-consuming tasks ^[15][27][28][15,27,28]. The most common PAH extraction methods include Soxhlet extraction, ultrasonication, and stirring/agitation [2]. The outcomes of these techniques usually involve large amounts of solvent and significant measurement errors ^[15][29][15,29]. Given the advancement toward more sensitive and accurate analytical techniques, the development of sample preparation processes has gained increasing attention. Automated equipment, shorter analytical times, greater quality, environmentally friendly processes, and smaller sample sizes are all benefits of technique optimization ^[25][26][28][25,26,28]. Modern extraction techniques for PAHs in food have achieved popularity through their increased efficiency and include pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), high-temperature distillation (HTD), and fluidized-bed extraction (FBE) ^{[2][13][25][30]}[2,13,25,30]. The purification step is commonly achieved by column chromatography, gel permeation chromatography (GPC), or solid-phase extraction (SPE), along with dispersive liquid–liquid microextraction (DLLME), solid-phase microextraction (SPME), magnetic solid-phase extraction (MSPE), and QuEChERS ^{[2][13][25][28]}[2,13,25,28]. The advantages of these techniques include lower cost, less solvent, time savings, and increased yield through selective interaction with the molecules, thus ensuring great extraction performance ^[30][31][30,31]. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method was first developed to screen pesticides in diverse food and agricultural products [32]. The original QuEChERS approach is aimed at simplifying the extraction and clean-up steps, as compared to time-consuming and laborious conventional procedures [33]. In recent years, the QuEChERS strategy has been persistently adjusted and systematically employed in routine analytical determinations ^[13][34][13,34]. Its foundation is based on dispersive solid-phase extraction (dSPE) to remove potentially interfering compounds (fats, pigments, sugars, etc.). Apart from multi-residue pesticide analysis, the method has been successfully implemented in food and environmental matrices to separate various target analytes, including mycotoxins, antibiotics, persistent organic pollutants (POPs), hormones, and PAHs, consistently attaining high selectivity, sensitivity, and specificity ^{[2][13][28][34]}[2,13,28,34]. In addition to the original unbuffered method involving an optimal 1:4 ratio of the two salts (MgSO₄ and NaCl) for partitioning ^[32][35][32,35], two modified QuEChERS methods were established: (a) the EN official method—BS EN 15662:2008 standard (withdrawn) revised in 2018 [36], employing the addition of a citrate buffer (1 g of trisodium citrate dihydrate and 0.5 g of disodium hydrogen citrate sesquihydrate); (b) the AOAC official method [37], applying the addition of an acetate buffer (1.5 g of sodium acetate) and 6 g of MgSO₄ instead of 4 g. In the purification process, MgSO₄ is used as a drying agent, whereas a primary secondary amine (PSA) is typically applied as a weak anion exchanger targeting the removal of fatty acids, sugars, organic acids, lipids, sugars, and some pigments ^[33][34][33,34]. Besides PSA and MgSO₄, the major sorbents employed are octadecyl silica (C18), which is able to hold vitamins and minerals and is highly effective in removing fats, and graphitized carbon black (GCB), which is effective in eliminating co-extracted pigments (e.g., carotenoids and chlorophyll) [34]. Other approaches are being steadily explored for improved clean-up efficiency, such as alumina, Florisil^®, chitosan, diatomaceous earth, zirconia-based sorbents (Z-Sep and Z-Sep⁺), Enhanced Matrix Removal-Lipid (EMR-Lipid), LipiFiltr^®, ChloroFiltr^®, CarbonX, and Cleanert^® NANO ^[26][34][38][26,34,38]. Common analytical approaches for the identification and quantification of PAHs in a wide range of food matrices make use of high-performance liquid chromatography (HPLC) with an ultraviolet (UV) or photo-diode array (PDA) detector and gas chromatography (GC) coupled with a flame ionization detector (FID) ^[2][15][28][2,15,28]. However, these techniques are exceedingly costly, time-consuming, and labor-intensive and no longer match today’s needs regarding selectivity and sensitivity requirements ^[15][28][15,28]. In contrast, LC and GC techniques coupled with mass spectrometry (MS) surpass the performance of DAD and FLD methods, being widely used in PAH determination in food matrices ^[2][13][39][2,13,39]. Official methods employing LC determination include ISO 15302:2007 for benzo[a]pyrene (BaP) (withdrawn), revised in 2017 [40], in crude or refined edible oils and fats using reverse-phase HPLC-FLD; ISO 15753:2006 (withdrawn), revised in 2016 [41], for 15 PAHs in animal and vegetable fats and oils, involving a clean-up on C18 and Florisil cartridges followed by HPLC-FLD; ISO 22959:2009 [42] for 17 PAHs in animal and vegetable fats and oils using LC-LC coupling and on-line donor–acceptor complex chromatography (DACC) and FLD; and CEN/TS 16621:2014 [43] for BaP, benzo(a)anthracene (BaA), chrysene (Chr), and benzo(b)fluoranthene (BbF) in foodstuffs using HPLC-FLD, based on SEC clean-up. Methods for PAH determination employing GC techniques include CSN EN 16619:2015 [44] for BaP, BaA, Chr, and BbF in foodstuffs (extruded wheat flour, smoked fish, dry infant formula, sausage meat, freeze-dried mussels, edible oils, wheat flour) using pressurized liquid extraction (PLE); size exclusion chromatography (SEC) and SPE clean-up, followed by GC-MS; and PD ISO/TR 24054:2019 [45] for 27 PAHs (including 16 EPA) in animal and vegetable fats and oils using LL extraction and silica gel column clean-up, followed by GC-MS. In order to address the increasing need for more precise PAH confirmation, modern LC-MS/MS and GC-MS/MS techniques are being developed in addition to conventional analytical methods ^{[2][26][28][46]}[2,26,28,46].

3. PAH Formation—Mechanistic Features

Given their chemical diversity, PAH formation and development occur through a complex set of reactions, mainly through condensation and cyclization from smaller organic molecules; under an array of specific conditions (carbon source, environment), complex approaches entailing PAH structural expansion have been investigated ^[3][6][47][3,6,47]. As such, the endeavor to provide an appropriate growth model has yielded several representations. The most important types of PAH formation mechanisms identified over the last few decades are based on acetylene addition reactions, vinylacetylene addition reactions, and radical reactions (Figure 3) ^{[11][47][48][49]}[11,47,48,49]. Hydrogen abstraction and acetylene or carbon addition (HACA). This mechanism was originally presented by Frenklach et al. (1984) [50] and later introduced as HACA [51] in the framework of PAH growth in ethane-, ethylene-, and acetylene-fueled flames. The mechanism is depicted as a two-step process involving hydrogen abstraction (activation of the aromatic molecule), followed by the addition of acetylene (C₂H₂) in the radical position. The advantages of HACA stem from its continuous PAH formation/growth. This is, in turn, attributed to the relatively low reversibility of the reaction and the increased affinity of hydrogen atoms due to low energy barriers throughout the process [52]. Nonetheless, given its reaction rate, the HACA growth mechanism is argued to underperform in comparison to the relatively quick PAH formation process ^[47][53][47,53]. Other pathways similar to HACA have been proposed in order to understand PAH chemistry. One of the alternatives is the Bittner–Howard process, involving the sequential addition of two C₂H₂ molecules to afford a C₄H₄ chain, which subsequently leads to additional ring formation [54]. However, these alternative pathways have been deemed unrealistic or limited under low-pressure or high-temperature flame conditions in terms of the residence time of unstable radicals ^[47][55][47,55]. The Diels–Alder mechanism, traditionally involving the reaction of acetylene with an olefin to afford a cyclic compound [56], was proposed by Siegmann and Sattler (2000) [57]. It involves the formation of a Diels–Alder adduct by the cycloaddition of acetylene, followed by the loss of hydrogen and the resulting bay region ring closure [57]. However viable, it should be considered that the DA mechanism is accompanied by high energy barriers throughout the process, along with low reaction rates ^[47][58][47,58]. Hydrogen abstraction and vinylacetylene addition (HAVA). Similar to acetylene, vinylacetylene is widely available in combustion flames and involved in PAH formation through the hydrogen abstraction vinyl acetylene addition mechanism, hence the HAVA terminology. It can be achieved with vinylacetylene (VA), without the need for additional carbon species, through the addition of reactive VA (double and triple bonds) to a PAH radical, followed by cyclization ^[59][60][59,60]. Explorations of the HAVA mechanism deemed it considerably viable under different conditions (low energy barriers of reaction, wide temperature and pressure ranges) [59]. Methyl addition cyclization (MAC). The methyl radical is the most abundant alkyl radical in combustion flames among potential radical species. Due to the prevalence of the methyl radical, MAC plays an important role in PAH formation ^[61][62][61,62]. It is initiated with the formation of ethyl or propyl chains on the PAH structure (addition of 2–3 methyl radicals), followed by hydrogen elimination and subsequent ring closure [63]. As observed in a study simulating the formation of pyrene from phenanthrene, MAC appears not to be competitive with HACA, given that C₂H₂ surpasses the methyl radical in occupying the armchair position radical sites [64]. Ethynyl radical addition (HAERA). Although less likely to occur, another alternative to HACA is the mechanism involving the ethynyl radical, which could represent a potential reaction route at low temperatures [65]. The formation of phenylacetylene from benzene and the ethynyl radical was predicted by considering the ethynyl addition mechanism (EAM) [66]. More recently, the formation of benzo(a)pyrene from chrysene was predicted by using the hydrogen abstraction ethynyl radical addition (HAERA) [67]. Vinyl radical addition (HAVA*). Similar to the HAVA reaction, hydrogen abstraction vinyl radical addition (HAVA*) can also lead to the formation of PAHs. It was assessed through pyrolysis studies, and its suitability for the formation of cyclopentafused PAHs was pointed out, as well as its significance in the formation of pyrogenic PAHs at moderate temperatures ^[68][69][68,69]. Phenyl addition cyclization (PAC). During toluene pyrolysis, the proposed route involves the addition of the phenyl radical, hydrogen abstraction, dehydrocyclization, and subsequent conversion into thermally stable condensed PAHs [70]. It is considered not competitive with HACA due to the low prevalence of benzene in flames, as opposed to acetylene [71]. Its most important features are attributed to the formation of asymmetric PAHs, with high efficiency if the phenyl radical is present, and the potential for continuous growth by enabling multiple fusion sites during each reaction step ^[52][72][73][52,72,73]. Resonantly stabilized radicals (RSR). The combustion environment is a suitable setting for recombination reactions (radical–radical, radical–neutral) to occur involving resonantly stabilized radicals (RSR), being relatively stable and in high concentrations in flames. Computational studies involving RSR pathways pointed out three important mechanisms: the propargyl radical ^[74][75][76][74,75,76], cyclopentadienyl radical ^{[77][78][79][80]}[77,78,79,80], and indenyl radical ^[81][82][83][81,82,83].