Monitoring and assessment of polycyclic aromatic hydrocarbons in pizza samples (meat and chicken) using modified MSPE extraction and GC/MS method

doi:10.21203/rs.3.rs-4821025/v1

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Monitoring and assessment of polycyclic aromatic hydrocarbons in pizza samples (meat and chicken) using modified MSPE extraction and GC/MS method

https://doi.org/10.21203/rs.3.rs-4821025/v1

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The objective of present research was to assess the PAH (polycyclic aromatic hydrocarbon) concentrations in Pizza (chicken and beef) cooked, by technique of MSPE-GC/MS (magnetic solid phase extraction-gas chromatography mas spectrophotometry). The outcomes revealed the mean ± SD of PAH4, ∑PAHs and BaP in all pizza samples was 2.97 ± 1.82, 12.75 ± 2.1 and 0.18 ± 0.02µg/kg, respectively. Based on the present results, in meat pizza samples, the mean ± SD (min-max) of PAH4, total PAHs and BaP was 4.20 ± 0.9 14.90 ± 1.59 and 0.31 ± 0.02 µg/kg, respectively, and in chicken pizza samples was 1.5 ± 0.8 10.18 ± 1.96 and 0.03 ± 0.01, respectively. A Heat map was used to understand the individual similarities and distinctions between PAHs detected in each sample with color intensity. The Based on the Monte Carlo consequences achieved, the EDI (estimated daily intake) of PAH measured was ranked as B(k)F > Fl > B(a)P > B(b)F > B(a)A > Ph > CHR > I(1,2,3-cd)P > D(a,h)A. The ILCR (incremental lifetime cancer risk) for the children and adults was 2.28E-8 and 6.07E-9, respectively. The research’s outcomes indicate the contents of PAH in pizza (chicken and beef) do not pose a safety concern for consumers in Iran (ILCR < 10^− 6).

Meat

Chicken

Pizza

Polycyclic aromatic hydrocarbons (PAHs)

Risk assessment

Food safety

Various thermal processes that are carried out on food can bring the risk of producing side compounds that are harmful to health. A major method that can result in production of dangerous combinations like PAHs includes incomplete burning. One of the most important chemical contaminants from foods and the environment that are dangerous to health of human is the PAH group, including nearly 100 various chemicals. People may be exposed to complex mixtures of PAHs from ingestion, inhalation and dermal contact routes. Based on a report of the European Scientific Committee, four PAHs of benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), chrysene (CHR) and benz[a]anthracene (BaA) are indicators of carcinogenic PAHs in food products (Babaoglu et al. 2017, Kacmaz 2019, Park et al. 2017).

Technically, 15 PAHs can be addressed based on the guidelines by the European Union Scientific Committee for Food (EU SCF) to achieve clear evidence on carcinogenicity (3). The EU Standard No. 835/2011 reports that the permissible limits for the total PAH and BaP in food products such as smoked meat, meat products, fish and smoked muscle meat include 12 and 2 µg/kg, respectively (Commission 2011).

Several studies have shown that the most potential PAH that can cause cancers is BaP. Only 4PAHs has been assessed through the oral route, demonstrating positive indications of carcinogenicity. The major adverse effect of PAHs is cancer. Generally, all PAHs are genotoxic; however, their oral consumption can cause other diseases and toxicities such as hematopoietic and reproductive toxicities (Girelli et al. 2014, Naghashan et al. 2023, Shariatifar et al. 2021, Shariatifar et al. 2022).

Various studies have shown that smoking and the environment are not the only factors that transmit PAHs to people. It is estimated that, nearly 90% of exposure to PAH compound is through foodstuff intake in non-smokers (Khalili et al. 2023, Sharifiarab et al. 2022).

It should be addressed that process, transportation, storage and consumption at home can be the factors affecting quantities of PAHs in food products. Numerous traditional and modern heat treatment techniques have been reported that cause generation of PAHs in meats and meat products. Ultra-processed foods such as pizza, sausages and bacons are prone to higher quantities of PAHs than that traditional foods are because the former foods are subjected to frying, heating and grilling processes. Optimizing temperature and time in industrial processes can help decrease PAHs in foods. Technically, PAHs residues have been detected in various foodstuffs, comprising meat, poultry, fish and their associated food products, edible oils, fruit juices, cereal products, smoked meat sausages, milks and milk powders, processed meats and edible vegetable oils (Alomirah et al. 2011, Kiani et al. 2019, Moazzen et al. 2022, Shariatifar et al. 2020a).

The most commonly used procedures for the assessment of these compounds include GC/MS and high-performance liquid chromatography (HPLC)/fluorescence. Nowadays, there are several approaches to extract and pre-concentrate PAHs from various types of foods. The most common classical extraction procedures include microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), Soxhlet assisted extraction (SAE), liquid-liquid extraction (LLE), ultrasound assisted extraction (UAE) and solid-phase extraction (SPE) (Gorji et al. 2016, Khalili et al. 2023, Moazzen et al. 2013, Roudbari et al. 2021). The MSPE by using magnetic nanoparticles (NPs) have newly emerged as a potential sample preparation procedure. Magnetic adsorbents are routinely disseminated (directly) into the solution of sample in the MSPE procedure (15–17). Considering the toxicity and carcinogenicity of PAHs, significant efforts should be made to evaluate and control PAHs in foods. As previously stated, one of the foods that is prone to high quantities of PAHs is pizza (18).

Due to the short lifespan and high consumption of fast foods by the majority of people, monitoring quantities of PAHs in pizzas can help make correct choices of safe foods and develop food safety standards. Therefore, the aim of present investigation was to consider contents and quantities of PAHs in various types of pizza (meat and chicken) samples using MSPE extraction technology coupled to GC -MS as well as assessment of their human health risks through assessing estimated daily intake (EDI) as well as non-carcinogenic and carcinogenic risks.

2.1. Pizza sampling

For current research, a total of 72 samples (duplicated) were purchased from Tehran. The samples were in 2 different types of Pizza (beef and chicken) with 2 different methods of cooking (electric and gas oven). After buying, the samples (Pizza) were conveyed to the laboratory (in cool-box) and frozen at a temperature of -18 ºC until the time of analysis.

2.2. Chemical compounds

In this study all reagents and PAH reference standards (order number: CRM47930, QTMPAH-Mix, 2000 micrograms per milliliters) were attained from Company of Merck (Germany) and Company of Supelco (Bellefonte, PA, USA), respectively. The Multi Walled Carbon Nanotubes (MWCNTs was attained from Nanoshel Co. (Panchkula, India) and prepared according to the previous studies (Gorji et al. 2016, Kargarghomsheh et al. 2023, Khalili et al. 2023, Kiani et al. 2018, Kouhpayeh et al. 2017, Mehraie et al. 2022).

2.3. Blank sample preparation

In our study, pizza samples (1kg) was twice ground, divided into pieces of 100 g and each piece was microwaved at 2450 MHz frequency for five minutes with 900 W output power. The appropriateness of current technique has been mentioned by former investigations (Moazzen et al. 2022, Shariatifar et al. 2021, Shariatifar et al. 2022, Sharifiarab et al. 2022).

2.4. Standards preparation

Based on the former investigations, All standard solutions (including standards of internal, stock and mixed) were prepared and retained at 4°C (Kiani et al. 2021, Kiani et al. 2019, Roudbari et al. 2021, Shariatifar et al. 2020a).

2.5. Samples preparation

Pizza samples were first crushed and homogenized. Then, according to previous studies, they were prepared and injected into the GC (Agilent 6890)-MS (Agilent 5973) equipment (Palo Alto, CA, United States) (Gorji et al. 2016, Khalili et al. 2023, Moazzen et al. 2013).

2.6. Instrumental analysis

In current investigation, all GC-MS conditions and characteristics (like type of column, carrier gas, temperature of oven and injector, etc.) were based on the former studies (Moazzen et al. 2013, Roudbari et al. 2021, Shariatifar et al. 2021). Table S1 shows the time window, ions of confirmation and ion of quantification of PAH analytes in the GC-MS equipment.

2.7. Optimization of Method

To optimize the technique, five milliliters of mixed working standard solution (0.5 µg / mL) was added to the 500 g of blank sample. Then, the mixture for thirty minutes was homogenized and stored for 24 h at 4°C and applied for optimization of procedure. Lastly, based on the protocol of "one factor at a time", the technique was optimized (Khalili et al. 2023, Shariatifar et al. 2020a, Sharifiarab et al. 2022).

2.8. Validation of the analytical method

According to the Table S2, coefficient of determination, the linear range, LOQs, LODs, and percent of recovery were varied from 0.985 to 0.998, from 0.050 to 20.000 µg/kg, from 0.36 to 1.68 µg/kg, from 0.12 to 0.56 µg/kg, and from 95.7 to 102.1%, respectively. An evaluation of the accuracy of procedure was conducted based on the interday precision through examination QC samples (quality control), which were assessed multiple times over three days (consecutive), with four levels. The precision percent of all PAH compounds was lower than 8.6% for values of inter-day and the percent of reliability was assessed varied from 5.1 to 11.3%.

2.9. Assessment of probabilistic health risk (MCS technique)

The MCS is frequently employed in assessing the potential risks to health of human resulting from exposure to food pollution (Karimi et al. 2021a). In this research, an approach of MCS was employed using Crystal Ball software to conduct an uncertainty quantitative investigation in public health risk assessments. The EDI of the identified PAH compounds through the consumption of pizza (meat and chicken) was calculated based on Eqs. (1 and 2) (Samiee et al. 2020a, Shariatifar et al. 2021):

$$\:BEC={\sum\:}_{i=1}^{n}{C}_{i}\times\:TEF$$

$$\:\text{E}\text{D}\text{I}\:=\:\frac{\text{C}\:\times\:\:\text{I}\text{R}\:i\times\:\:\text{E}\text{D}i\:\times\:\:\text{E}\text{F}i}{\text{B}\text{W}\:\times\:\:\text{A}\text{T}}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:$$

Here, EDI represents the estimated daily intake (mg/kg), BEC stands for the potency equivalent level of PAH compounds (mg/kg), and C denotes the average detected level of PAH compounds (mg/kg). The details and examples of these parameters can be found in Table 1. As probably or possibly carcinogenic (Group 2A) to humans, the benzo[a]pyrene (B[a]P) is classified. The ILCR resulting from exposure to BaP through the dietary intake of meat and chicken was computed using Eq. 3:

$$\:ILCR=\:\frac{\text{B}\text{E}\text{C}\times\:\text{E}\text{F}\times\:\text{E}\text{D}\times\:\text{S}\text{F}}{\text{B}\text{W}\:\times\:\:\text{A}\text{T}}$$

The variable SF represents the oral cancer slope factor of BaP, which has a geometric mean of 7.3 per mg/kg/d according to USEPA's Risk Assessment Guidance for Superfund: Volume III Part A and Risk Assessment in Washington. The details and examples of these parameters are provided in Table S3.

Table 1

Parameters used in the present study for health exposure assessment in meat and chicken Pizza sample.
Exposure parameters		Unit	Reference
$\:\mathbf{E}\mathbf{F}\varvec{i}$	Exposure frequency	days per year	(Karimi et al. 2021b)
C	level of PAH compounds	mg/Kg	--
AT	Average time	days	(Eghbaljoo-Gharehgheshlaghi et al. 2020)
EDI	Estimated daily intake	mg/kg
BEC	Benzo(a) pyrene equivalents concentrations by toxicity equivalency factors (TEFs).	---	(EPA 2011)
BW	Body weight (for children and adults is between 15 and 70)	kg	(Shariatifar et al. 2020b)
ED	Exposure duration	days	(Shariatifar et al. 2020b)
IR	Average daily intake (61 gr-day)	kg/day	(Kalantari N 2015)
SF	Carcinogenic slope factor of oral intake (7.3)	mg/(kg/d)	(USEPA. Risk assessment guidance for Superfund: volume III part A &risk assessment. Washington)
TEFs	Toxic equivalent factors	--	Table S3

2.10. Statistical analysis

The investigation results were presented as mean ± SD, and the data for levels of PAHs in pizza samples (meat and chicken) were assessed for normality (Kolmogorov-Smirnov test) and homoscedasticity (Levene's test) using SPSS software (version 24.0). When the PAH components weren’t detected in the samples, a value of 1/2 LOD was employed to calculate the average level. A heat map analysis was conducted to assess the relationship between meat and chicken contamination, utilizing a clustering procedure of average linkage and a distance procedure of Pearson, accessible online at https://biit.cs.ut.ee/clustvis/. In addition, an MCS procedure was utilized for uncertainty risk assessment through the use of Crystal Ball® software (Version 2000.2, Decisioneering, Inc., Denver, CO, USA).

3.1. PAHs contents in Pizza samples

Table 2 revealed that the quantity of PAHs in all samples of Pizza. Based on the information in Table 2, the mean ± SD (min-max) of PAH4, total PAHs and BaP was 2.97 ± 1.82 12.75 ± 2.1 and 0.18 ± 0.02µg/kg, respectively. Among the PAHs, the highest and lowest mean was related to Pyrene (2.33 ± 0.22) and Dibenzo(a,h)Anthracene (0.08 ± 0.05), respectively. According to the mentioned consequences, the average of BaP and 4PAH were less than the stated standard levels (the EU has suggested 2 and 12 µg/kg, respectively). The presence of PAH compounds contaminants in Pizza can be for reasons such as initial contamination of meat (beef and chicken), contamination during preparation (equipment, etc.), amount of fat in the sample and especially during cooking (Gorji et al. 2016, Karslioglu &Kolsarıcı 2023, Khalili et al. 2023, Moazzen et al. 2013, Sahin et al. 2020, Terzi et al. 2008)

Table 2

PAHs contents in pizza samples.
	All samples					Sig.
	Minimum	Maximum	Mean	Median	Std. Deviation	Sig.
Naphtalene	0.15	2.90	0.95	0.75	0.03	0.78
Acenaphtylene	0.00	1.96	0.32	0.14	0.02	0.50
Acenaphtene	0.15	4.13	1.01	0.66	0.07	1.07
Florene	N. D*	5.05	1.17	0.46	0.5	0.74
Phenanthrene	N. D*	2.96	0.35	0.00	0.2	0.72
Anthracene	N. D*	1.64	0.22	0.00	0.2	0.40
Fluorantene	N. D*	8.77	1.63	0.80	0.31	0.32
Pyrene	0.13	8.44	2.33	1.02	0.22	0.02
Benzo(a)ant	N. D*	1.65	0.51	0.35	0.2	0.51
Chrysene	N. D*	2.64	0.64	0.46	0.3	0.03
B(b)F	N. D*	15.33	1.64	0.57	0.48	0.25
B(k)F	N. D*	5.85	1.14	0.31	0.68	0.56
B(a)P	0.01	0.94	0.18	0.05	0.02	0.00
Dibenzo(a,h)Anthracene	N. D*	0.39	0.08	0.07	0.05	0.09
Benzo(g,h,i)Perylene	N. D*	0.93	0.29	0.28	0.1	0.28
Indeno(1,2,3-cd)Pyrene	N. D*	4.01	0.31	0.09	0.1	0.08
PAH4	0.45	17.04	2.97	1.84	1.82	0.07
Total PAHs	5.28	32.72	12.75	11.42	2.1	0.09
*(Non Detected)

Table 3 revealed that the quantity of PAH compounds in samples of meat Pizza. Based on the information in Table 3, the mean ± SD (min-max) of PAH4, total PAHs and BaP was 4.20 ± 0.9 14.90 ± 1.59 and 0.31 ± 0.02µg/kg, respectively.

Table 3

PAHs contents in meat pizza samples.
	Meat
	Minimum	Maximum	Mean	Median	Std. Deviation
Naphtalene	0.17	2.45	0.86	0.61	0. 4
Acenaphtylene	N. D*	1.96	0.40	0.15	0.1
Acenaphtene	0.16	3.22	0.84	0.26	0.2
Florene	N. D*	5.05	1.26	0.32	0.5
Phenanthrene	N. D*	0.39	0.10	0.10	0.04
Anthracene	N. D*	1.64	0.35	0.08	0.1
Fluorantene	N. D*	7.87	1.43	0.62	0.21
Pyrene	0.32	8.44	3.56	2.46	0.89
Benzo(a)ant	0.00	1.27	0.59	0.47	0.2
Chrysene	0.17	2.64	0.88	0.57	0.2
B(b)F	N. D*	15.33	2.42	0.57	0.61
B(k)F	N. D*	5.85	1.36	0.51	0.81
B(a)P	0.02	0.94	0.31	0.25	0.02
Dibenzo(a,h)Anthracene	N. D*	0.39	0.08	0.06	0.01
Benzo(g,h,i)Perylene	N. D*	0.72	0.32	0.32	0.08
Indeno(1,2,3-cd)Pyrene	N. D*	0.49	0.15	0.09	0.06
PAH4	0.59	17.04	4.20	2.01	0.9
Total PAHs	6.36	32.72	14.90	13.93	1.59
*(Non Detected)

Table 4 revealed that the quantity of PAH compounds in samples of chicken Pizza. Based on the information in Table 4, the mean ± SD (min-max) of PAH4, total PAHs and BaP was 1.5 ± 0.8 10.18 ± 1.96 and 0.03 ± 0.01µg/kg, respectively.

Table 4

PAHs contents in chicken pizza samples.
	Chicken
	Minimum	Maximum	Mean	Median	Std. Deviation
Naphtalene	0.15	2.90	1.05	0.75	0.85
Acenaphtylene	N. D*	0.85	0.22	0.14	0.1
Acenaphtene	0.15	4.13	1.21	0.93	0.5
Florene	0.08	2.74	1.05	0.97	0.35
Phenanthrene	N. D*	2.96	0.65	0.01	0.1
Anthracene	N. D*	0.27	0.06	0.00	0.03
Fluorantene	0.37	8.77	1.87	0.94	0.52
Pyrene	0.13	4.02	0.86	0.28	0.20
Benzo(a)ant	N. D*	1.65	0.43	0.33	0.21
Chrysene	N. D*	0.85	0.36	0.36	0.16
B(b)F	N. D*	2.13	0.70	0.57	0.09
B(k)F	N. D*	5.06	0.87	0.14	0.1
B(a)P	0.01	0.09	0.03	0.01	0.01
Dibenzo(a,h)Anthracene	N. D*	0.20	0.07	0.07	0.03
Benzo(g,h,i)Perylene	N. D*	0.93	0.26	0.20	0.08
Indeno(1,2,3-cd)Pyrene	N. D*	4.01	0.50	0.08	0.24
PAH4	0.45	2.90	1.50	1.43	0.80
Total PAHs	5.28	18.68	10.18	10.04	1.96
*(Non Detect)

A comparison of the outcomes of other investigates in other countries is shown in Table 5. Based on the information in this table, the level of PAH compounds in different studies was both similar and different.

Table 5

Comparison with other studies in previous.
Researchers		Analytes	Results	Ref.
Lee et al.	Some samples of ready to eat food products	B[a]P	They stated, the mean concentration of B[a]P in these foodstuffs was 0.64 µg/kg.	(Lee &Shin 2019)
Khalili et al.	Boiled chicken, chicken kebab and grilled chicken,	16 PAHs	They stated, the mean concentrations of ΣPAHs, PAH4 and BaP were 144 ± 8.2, 6.3 ± 1.2 µg/kg and nd, respectively in boiled chicken samples, 112.9 ± 7.2, 3.4 ± 0.8 and nd, respectively in chicken kebab and 183 ± 13.4 µg/kg, nd and nd, respectively in grilled chicken samples.	(Khalili et al. 2023)
Samiei et al.	Sausages and hamburger	16 PAHs	They indicated, the average concentration of 16 PAHs anaytes in hamburger and sausage was 8.08–29.55 and 10.18–29.85 µg/kg, respectively.	(Samiee et al. 2020b)
G. Falcó et al.	Meat and Meat Products	16 PAHs	They expressed, the mean concentrations of ΣPAHs, PAH4 and BaP was 13.43, 1.61 and 0.098 µg/kg, respectively.	(Falco et al. 2003)
Husseini et al.	Traditional grilled chicken	4 PAHs	They stated, the concentrations of 4 PAHs was 1.52 to 49.9 µg/kg.	(El Husseini et al. 2018)
Reinik et al.	Meat products	12 PAHs	They stated, the maximum levels of ΣPAH was 16 µg/kg in samples of smoked ham and meat, 19 µg/kg in smoked sausage and 6.5 µg/kg in samples of smoked chicken.	(Reinik et al. 2007)
Gorji et al.	Iranian Kebabs (beef and chicken)	16 PAHs	They stated, the concentrations of BaP, and ΣPAHs were varied 0.28–5.81 and 7.37–17.94 µg/kg, respectively.	(Gorji et al. 2016)
Cho et al.	Smoked foods	B[a]P	They stated, the mean concentration of B[a]P was 0.45 µg/kg and the highest concentrations of B[a]P was 2.87 µg/kg in samples of smoked salmon.	(Cho &Shin 2012)
Mastanjević et al.	Traditional dry fermented sausage	16 PAHs	They stated, the total level of 16 PAHs was 124–679 µg/kg (at the end of the production).	(Mastanjević et al. 2019)

Higher or lower PAH levels in pizza samples can be owed to various reasons like environmental contamination, pollution during production and preparation, and pollution of raw materials used (meat, vegetables, flour and other additives) (Gorji et al. 2016, Khalili et al. 2023, Moazzen et al. 2013).

3.2. Assessment of human health risk

Simulation models incorporating three health risk indexes (ILCR and EDI) were utilized to assess the carcinogenic and non-carcinogenic impacts of extended oral exposure to PAHs.

The MCS method was employed in the risk assessment to produce various risk descriptors for individual variables in accordance with EPA procedures.(Karimi et al. 2021b, Rezaei et al. 2021, Shariatifar et al. 2020b, Yaminifar et al. 2021).

Several studies have investigated contamination risk through the MCS method, examining aspects like carcinogenic risk and non-carcinogenic of PAHs in mushroom samples (1), health risks associated with sulfur residues in raisins (Saghafi et al., 2021), PAH contamination in cream and ice cream (6), health impacts of acrylamide in commercial nuggets (4), and PAH levels in commercial coffee and tea (5), carcinogenic risk and non-carcinogenic hazard quotient of PAHs in in cereal products (7). The results revealed that the rank order of EDI (percentile 95%) based on the both average and high consumption of polluted meat pizza samples was P > B(k)F > Fl > B(a)P > B(b)F > B(a)A > Ph > CHR > I(1,2,3-cd)P > D(a,h)A (Table 6). The types of PAHs in food vary by region and type of production.

Based on Fig. 3, the EDI (percentile 95%) of B(a)P in the children and adults was 5.12E-6 and 1.47E-6 mg/kg-d, respectively. According to the EPA guidelines, consumption of BaP exceeding 0.0002 mg/kg BW/day through human diet has been identified as a potential concern of human health. The study found that Estimated Daily Intake values were less than the threshold, demonstrating a safe health risk level for the general public.

Figure 1 indications the BaP, BbF and B(k)F are the three principal contributors to the total Benzo(a) pyrene equivalents concentrations (mg/kg), whiles the other PAH compounds comprised have a contribution of less than 21 percent.

As is apparent from the data shown in Figs. 2, the ILCR indexes (95%) for children and adults in the meat pizza samples were 2.28E-8 and 6.07E-9, respectively. Figure 2 displays a histogram plot illustrating the continuous distribution of carcinogenic risk for the pizza samples. The qualitative hazard classification for carcinogens can be delineated into three categories: ILCR less than 10^− 6 falls within the safe zone, ILCR greater than 10^− 4 represents the threshold hazard limit, and ILCR exceeding 10^− 3 is classified as the significant hazard zone. The research conducted by Ding et al. in 2013 focused on studying the presence of PAH compounds in diverse vegetables sourced from China. Based on their findings, The Cancer Risk Index in females and males was 1.1 × 10^− 5 and 1.2 × 10^− 5, respectively, which aligns with the findings of this study.(Ding et al. 2013). In another investigation, Nie et al. (2014) found that the risk of PAHs exposure from vegetables, wheat flour, and fruits ranged from 9.07 × 10^− 4 to 1.12 × 10^− 4 in adults. Martorell et al. observed the cancer risk associated with different food items among male adults in Catalonia was 4.50 × 10^− 6(Martorell et al. 2010), a finding that aligns with the current research outcomes.

However, the risk of PAHs exposure from pizza can result from various foodstuff processing procedures, like curing, heating, smoking, drying, grilling, and barbecuing, as well as from environmental pollutants that contaminate the air, soil, and water (Samiee et al. 2020a).

Table 6. Uncertainly analysis for the EDI of PAHs residues in pizza samples (mg/kg day).

	Adults				Children
Percentiles	5%	50%	75%	95%	5%	50%	75%	95%
Ph	2.00E-7	3.01E-7	3.55E-7	4.48E-7	7.10E-7	1.03E-6	1.25E-6	1.59E-6
A	1.25E-7	1.91E-7	2.21E-7	2.77E-7	4.50E-7	6.53E-7	7.77E-7	9.71E-7
Fl	9.50E-7	1.42E-6	1.68E-6	2.19E-6	3.25E-6	4.91E-6	5.82E-6	7.52E-6
P	1.32E-6	2.02E-6	2.36E-6	3.02E-6	4.65E-6	7.04E-6	8.24E-6	1.05E-5
B(a)A	2.97E-7	4.51E-7	5.34E-7	7.13E-7	1.02E-6	1.55E-6	1.83E-6	2.26E-6
B(b)F	3.59E-7	5.47E-7	6.49E-7	8.37E-7	1.28E-6	1.94E-6	2.26E-6	2.91E-6
B(k)F	9.63E-7	1.41E-6	1.64E-6	2.10E-6	3.33E-6	4.92E-6	5.77E-6	7.33E-6
B(a)P	6.59E-7	9.82E-7	1.16E-6	1.47E-6	2.29E-6	3.44E-6	4.07E-6	5.12E-6
D(a,h)A	1.03E-7	1.58E-7	1.88E-7	2.39E-7	3.67E-7	5.60E-7	6.55E-7	8.40E-7
D(a,h)A	4.51E-8	6.64E-8	7.83E-8	1.01E-7	1.52E-7	2.33E-7	2.78E-7	3.63E-7
I(1,2,3-cd)P	1.64E-7	2.55E-7	2.98E-7	3.80E-7	5.83E-7	8.71E-7	1.02E-6	1.29E-6
(CHR)	1.78E-7	2.62E-7	3.11E-7	3.95E-7	6.09E-7	9.27E-7	1.09E-6	1.41E-6

3.3. Heat map analysis

The heat map provides a visual representation of the relationships among PAH profiles in pizza and chicken samples. Furthermore, by displaying a comprehensive pattern of changes in variables across similar columns and rows for related measurable factor, heat maps offer valuable insights. Additionally, utilizing heat maps as a classification method enabled the differentiation of samples (meat and chicken) based on PAH compounds.

As depicted in Fig. 3, the first cluster in meat pizza samples (Fig. 3A) comprises Indeno(1,2,3-cd)Pyrene, B(b)F, Benzo(g,h, i)Perylene, and Anthracene, while the second cluster consists of two subgroups with PAH congeners including Fluoranthene, Naphthalene, Pyrene, Fluorene, Acenaphthylene, Acenaphthene, Phenanthrene, Benzo(a)anthracene, B(k)F, B(a)P, Dibenzo(a,h)anthracene, Chrysene, and Anthracene.

In chicken pizza samples (Fig. 3B), the first cluster includes B(b)F, Fluorene, and Benzo(g,h, i)Perylene, whereas the second cluster has two subgroups with PAH congeners such as Fluorene, Naphthalene, Acenaphthylene, Pyrene, Acenaphthene, Phenanthrene, Benzo(a)anthracene, Chrysene, B(a)P, Anthracene, and Indeno(1,2,3-cd)Pyrene, B(b)F. When considering the various PAH congeners in the samples, these combinations can be categorized into separate subgroups based on their quantities, leading to a clear differentiation between meat and chicken samples. In meat samples, Anthracene, Indeno(1,2,3-cd)Pyrene, and B(b)F, Benzo(g,h, i)Perylene were positioned closer together, indicating higher similarity and correlation among these variables. While, in chicken samples, B(b)F, Fluorene, and Benzo(g,h, i)Perylene were in closer proximity to each other. Statistical analysis revealed distinct classifications for each sample group in terms of PAH compounds.

Considering the danger of contaminants in food for human health, present research investigated the concentration of PAH compounds in cooked pizza (chicken and beef). Based on the present results, in beef pizza samples, the mean (min-max) of PAH4, total PAHs and BaP was 4.20, 14.90 and 0.31µg/kg, respectively, and in chicken pizza samples was 1.5, 10.18 and 0.03, respectively, which shows that beef pizza is more contaminating than chicken pizza. Heat maps used visually demonstrate the similarities and distinctions between PAHs, aiding in the interpretation of meat and chicken pizza contamination data. The health risk assessment findings, utilizing the Monte Carlo approach, indicated that the ILCR index was less than 10^− 6. Consequently, the analyzed meat and chicken samples don’t pose a significant carcinogenic risk to consumers.

Acknowledgments and funding: This article was supported by Food Safety Research Center and National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, IR, Iran (No. Grant 03-43009075).

Ethical Approval: Not applicable.

Consent to Participate: Not applicable

Consent to Publish: Not applicable

Competing interests: The authors have no relevant financial or non-financial interests to disclose

Availability of data and materials: All data generated or analyzed during current study are included in this article. The datasets analyzed during this study are available from the corresponding author on reasonable request.

Author’s contributions: Nabi Shariatifar and Zahra Hadian: Conceptualization, Supervision, Design of study, Writing- Reviewing and Editing. Majid Arabameri: Methodology, Software, Validation, Data curation, Writing- Original draft preparation. Mojtaba Moazzen: Writing- Original draft, Design of study, Methodology, Writing- Reviewing and Editing. Parisa Shavli: Visualization, Investigation, Methodology, Software, Validation.

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Monitoring and assessment of polycyclic aromatic hydrocarbons in pizza samples (meat and chicken) using modified MSPE extraction and GC/MS method

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Pizza sampling

2.2. Chemical compounds

2.3. Blank sample preparation

2.4. Standards preparation

2.5. Samples preparation

2.6. Instrumental analysis

2.7. Optimization of Method

2.8. Validation of the analytical method

2.9. Assessment of probabilistic health risk (MCS technique)

2.10. Statistical analysis

3. Results and discussion

3.1. PAHs contents in Pizza samples

3.2. Assessment of human health risk

3.3. Heat map analysis

4. Conclusions

Declarations

References

Supplementary Files

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Exposure parameters		Unit	Reference
\(\:\mathbf{E}\mathbf{F}\varvec{i}\)	Exposure frequency	days per year	(Karimi et al. 2021b)
C	level of PAH compounds	mg/Kg	--
AT	Average time	days	(Eghbaljoo-Gharehgheshlaghi et al. 2020)
EDI	Estimated daily intake	mg/kg
BEC	Benzo(a) pyrene equivalents concentrations by toxicity equivalency factors (TEFs).	---	(EPA 2011)
BW	Body weight (for children and adults is between 15 and 70)	kg	(Shariatifar et al. 2020b)
ED	Exposure duration	days	(Shariatifar et al. 2020b)
IR	Average daily intake (61 gr-day)	kg/day	(Kalantari N 2015)
SF	Carcinogenic slope factor of oral intake (7.3)	mg/(kg/d)	(USEPA. Risk assessment guidance for Superfund: volume III part A &risk assessment. Washington)
TEFs	Toxic equivalent factors	--	Table S3