Abstract
This study analyzed four polycyclic aromatic hydrocarbons (PAHs) from frequently consumed home meal replacement (HMR) products in Korea and evaluated their chemical analysis methods. The PAHs investigated were benzo[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene. Chrysene-d12 and benzo[a]pyrene-d12 served as internal standards. The sample was dissolved in dichloromethane before extraction. Liquid–liquid extraction, microwave extraction, alkali digestion and GC–MS were used for analysis. Method validation was conducted on four matrices: fatty solid, fatty liquid, non-fatty liquid, non-fatty solid. Linear correlation coefficients (R2) were all above 0.99 and accuracy ranged from 80.03 to 119.65%. The LOD and LOQ were in the range of 0.03–0.15 and 0.09–0.44 μg/kg, respectively. The recoveries varied from 81.09 to 116.42% and precision ranged from 0.07 to 10.73% in both intraday and interday analysis. All concentrations of total PAHs from HMR products were detected at relatively low concentration. This study could provide the PAHs content from HMR products in Korea.
Keywords: Polycyclic aromatic hydrocarbons, Home meal replacement (HMR), GC–MS, Analytical method
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are organic compounds composed of two or more fused aromatic rings of carbon and hydrogen atoms. These ubiquitous environmental contaminants (found in air, soil, water and various foods) are formed by incomplete combustion of organic matter (Moon et al., 2010). The predominant source of PAHs is emissions from anthropogenic activities, such as automobile exhaust gas, oil refinery, heating, power plants, garbage incineration, sewage sedimentation, oil/gasoline spill, cigarette smoke, barbecue smoke and packaging materials and manufacturing production (Moon et al., 2006). Due to their lipophilicity and hydrophobic characteristics, PAHs tend to accumulate in the food chain (Pensado et al., 2005; Veyrand et al., 2013). Diet contributes to more than 70% exposure to PAHs among non-smokers (Gilbert et al., 1994; Lodovici et al., 1995). PAH contamination of food can be attributed to diverse pathways that include natural (environmental) and synthetic sources (e.g., culinary practices and industrial food processing). After ingestion, absorption and transport, the initial biological receptor for PAH is the microsomal mixed-function oxidases (Conney et al., 1957).
There are more than 100 different types of PAHs. The Agency for Toxic Substances and Disease Registry (ATSDR) designated 16 species as priority pollutants based on carcinogenicity (ATSDR, 1995). Later, the U.S. Environmental Protection Agency defined chrysene (CHR), benzo[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), benzo[b]fluoranthene (BbF), indeno[1,2,3-c,d]pyrene (IcdP) and dibenzo[a,h]anthracene as Group B2 (probable human carcinogens) (EPA, 2002). In turn, the International Agency for Research on Cancer (IARC) confirmed that BaA, BaP, BbF, benzo[j]fluoranthene, BkF and IcdP have carcinogenic potential in humans (IARC, 2004). It has classified BaP into Group 1 (carcinogenic to humans) and CHR and BaA into Group 2B (possibly carcinogenic to humans) (Lyon, 1994). According to the European Union Food Science Committee (SCF), BaP may be used as a marker for the occurrence and effects of carcinogenic PAHs in food. European Commission (EC) Regulation No. 1881/2006 of 19 December 2006 listed the maximum levels for BaP in several foods (European Commission, 2007). However, in 2008, the european food safety authority (EFSA) concluded that a sum of either eight or four specific PAHs (PAH8 or PAH4, respectively) would be better indicators of both the occurrence and toxicity of the genotoxic and carcinogenic PAHs than BaP alone (EFSA, 2008). Home meal replacement (HMR) meant fully- or partially- prepared foods in the late 1990s and has been used interchangeably with the terms such as “convenience food” or “ready-to-eat meals” in the year 2000. HMR industry in United States has expanded continuously and domestic HMR market increased early 2000s (Baskin, 2016). However, there are far fewer studies and no standards for PAHs in HMR products in Korea.
The purpose of this study is to develop and validate an analytical method for determine PAHs in four different matrices (fatty solid, non-fatty solid, fatty liquid, non-fatty liquid) and evaluate the content of PAHs from home meal replacement (HMR) products.
Materials and methods
Materials
The 4 PAH standards, BaP, BaA, BbF, CHR and two internal standards (IS), chrysene-d12 (CHR-d12) and benzo[a]pyrene-d12 (BaP-d12), were purchased from Supelco, Inc. (Bellefonte, PA, USA). All solvents (water, ethyl alcohol, n-hexane, methanol, dimethylformamide and dichloromethane [DCM]) were HPLC-grade and purchased from Burdick & Jackson (Muskegon, MI, USA). Potassium hydroxide (KOH, > 95%) for saponification and sodium sulfate anhydrous (Na2SO4, > 99%) for dehydration were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Sep-Pak silica cartridges (Waters Corp., Milford, MA, USA) were used as solid-phase extraction (SPE) columns for purification. Hydrophilic syringe PTFE filters (13 mm, 0.45 μm) were purchased from Advantec (Toyo Roshi Kaisha, Ltd., Tokyo, Japan).
Sample preparation
Samples were collected and prepared from May to October 2020. Food samples were purchased randomly from supermarkets in Korea and categorized as fatty solid, non-fatty solid, fatty liquid and non-fatty liquid, according to their fat content and rheological characteristics. To validate the analysis of PAH4, sausage and salad dressing were used as a representative matrix of HMR samples applicable to the solid and liquid fatty group, respectively. Conversely, porridge and fruit juice was used to represent the HMR samples in the solid and liquid non-fatty group, respectively. Solid samples were homogenized uniformly after discarding the inedible portion using blender and liquid samples were used as it is. Before analysis, samples were stored at − 20 °C. The HMR groups were categorized into 40 food categories.
Extraction and clean-up
Fatty solid and non-fatty solid samples
Aliquots of 10 g of homogenized fatty solid and non-fatty solid samples were weighed in 300-mL flat-bottomed flasks and then spiked with 1 mL of 100 μg/kg deuterated IS (CHR-d12, BaP-d12). Afterward, 100 mL of 1 M KOH in ethanol solution was added for saponification and heated by reflux at 80 °C for 3 h to either separate PAHs bound to sample or remove the interfering substances. After rapid cooling of the flask with cold water, the solution was filtered through filter paper (110 mm; Advantec) and transferred to separatory funnel-1. A 50 mL mixture of ethanol:n-hexane (1:1, v/v) and 50 mL n-hexane were transferred to separatory funnel-1 for liquid–liquid extraction. Briefly, this solution was extracted twice using n-hexane (50 mL), followed by three washes with 50 mL of distilled water, then dehydrated using anhydrous Na2SO4. The dried sample was concentrated to 2 mL on a vacuum rotary evaporator (N–N series SB-100, Eyela, Tokyo, Japan) at 37 °C. The concentrate was extracted by SPE using Sep-Pak silica cartridges. The PAHs were eluted by 5 mL n-hexane, followed by 15 mL of n-hexane:DCM (3:1). The resulting solution was vaporized at 37 °C using a stream of nitrogen gas and the resulting residue was dissolved in 1 mL DCM for 6 min using a vortex (Scientific Industries, Inc., Bohemia, NY, USA). The solution was filtered through a 0.45 μm PTFE membrane filter and transferred to 2 mL amber screw-cap vials (Agilent Technologies, Santa Clara, CA, USA). Finally, 1 μL was injected into the GC–MS system (7820A/5975 MSD, Agilent Technologies, Santa Clara, CA, USA).
Liquid − liquid extraction for fatty liquid samples
A 10-g portion of the homogenized fatty liquid sample (Italian salad dressing) was weighed and transferred to separatory funnel-1 containing 100 mL n-hexane. After that, 50 mL of N,N-DMF:water (9:1) mixture was added to separatory funnel-1 and only the N,N-DMF:water (9:1) layer was transferred to separatory funnel-2. The n-hexane solution left in separatory funnel-1 was extracted twice with 25 mL of N,N-DMF:water (9:1) and the N,N-DMF:water (9:1) layer transferred to separatory funnel-2. After an initial extraction of the solution in separatory funnel-2 with 100 mL of 1% Na2SO4 and 50 mL n-hexane, the hexane layer was transferred to separatory funnel-3. The solution remaining in separatory funnel-2 was extracted twice with 35 mL n-hexane. Each time, the hexane phase was transferred to separatory funnel-3. This process was followed by three washes with 50 mL of distilled water, then the sample dried using anhydrous Na2SO4 and concentrated by SPE using a Sep-Pak silica cartridge. Subsequent steps were the same as those described above for the fatty solid and non-fatty solid samples.
Ultrasonication for non-fatty liquid samples
For non-fatty liquid samples, ultrasonication was performed as an extraction method. Briefly, 1 mL of 100 μg/kg IS (BaP-d12, CHR-d12) was spiked into the homogenized non-fatty liquid sample (10 g). The sample was extracted with 50 mL n-hexane in an ultrasonic bath (60 Hz, 330 W; JAC-2010, Kodo Co., Ltd., Gyeonggi-Do, Korea) for 20 min. Afterward, 35 mL n-hexane was added and the sample ultrasonicated twice. After transferring the solution to a separatory funnel, it was washed three times with 50 mL of distilled water, dehydrated using anhydrous Na2SO4 and concentrated by SPE using Sep-Pak silica cartridges. Subsequent steps were the same as those described above for the fatty solid and non-fatty solid samples.
GC–MS analysis of PAHs
HMR products were analyzed for 4 PAHs using a GC–MS system (7820A/5975 MSD, Agilent Technologies) equipped with an HP-5MS UI column (0.25 mm × 30 m, i.d. 0.25 μm film thickness; J&W Scientific, Folsom, CA). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The injection was performed in split-less mode at 310 °C. The oven temperature program was held at 80 °C for 1 min, initially. Then, the temperature was elevated to 290 °C at 10 °C /min and maintained at 290 °C for 5 min. The ion source and ion quadrupole temperatures were 250 and 150 °C, respectively. The MSD was used in selective ion monitoring mode (SIM) to check for the absence of contamination. The SIM mode improves the sensitivity by limiting the mass of the ions detected to one or more specific fragment ions of known mass rather than screening over a wide range of masses. As a result, the SIM mode allows for the detection of a particular analyte of interest and it eliminates the large portion of noise that exists in the full scan mode. Typically, three ions (one target and two qualifier ions) are searched per compound and those ions are unique between the analytes of interest. The selected ions (m/z) were 226, 228 and 229 for BaA and CHR; 250, 252 and 253 for BbF and BaP; 263, 264 and 265 for BaP-d12; and 236, 240 and 241 for CHR-d12 (the target ions are underlined).
Identification and quantification of PAHs
The identification of PAHs was verified by comparing the retention time of each standard solution between each trial. Spiked samples also confirmed peak identity. The standard stock solution was dissolved in DCM and five working solutions, ranging from 1 to 20 μg/L, were used to construct the calibration curves.
Method validation and analytical quality assurance
Method validation was conducted on four matrices: fatty liquid, fatty solid, non-fatty solid and non-fatty liquid. Calibration curves were acquired by analyzing a series of standard solutions with 4 PAHs mixture in five concentrations of 1, 2, 5, 10 and 20 μg/kg. The standards were also mixed with 100 μg/kg IS (CHR-d12, BaP-d12) for determining the recovery values, one of the validation parameters. All standard mixtures (1 μL) were injected in triplicate to acquire calibration curves. By repeating the analysis three times a day (intraday) for three consecutive days (interday), accuracy and precision (%) were obtained. Precision (%) was expressed as the coefficient of variation (CV).
Statistical analysis
All experiments were performed in triplicate and the results presented as mean ± standard deviation. GC–MS data were acquired and analyzed by HP Chemstation software (Hewlett Packard, Sunnyvale, CA, USA) and Microsoft Excel spreadsheet (Microsoft Corporation, Redmond, WA, USA).
Results and discussion
Analytical quality assurance for PAH analysis
The calibration curve was linear over the concentration range tested (1, 2, 5, 10 and 20 μg/kg). The method validation results, including calibration equations, linearity (R2), LOD, LOQ, accuracy, precision and recovery, are presented in Tables 1, 2, 3 and 4. The correlation coefficient of linearity (R2) shows the peak area with concentration of the calibration curve. As shigh linearity (R2 > 0.99) was observed for PAHs at all concentrations.
Table 1.
Calibration equations, linearity (R2), limit of detection (LOD) and limit of quantification (LOQ) of polycyclic aromatic hydrocarbons (PAHs)
| Matrix | PAHs | Calibration equation | Linearity (R2) | LOD (ng/mL)1 | LOQ (ng/mL)2 |
|---|---|---|---|---|---|
| Fatty solid matrix (n = 3) | B[a]A | y = 0.0271x − 0.0006 | 0.9996 | 0.12 | 0.36 |
| CHR | y = 0.0197x + 0.0008 | 0.9997 | 0.15 | 0.44 | |
| B[b]F | y = 0.0221x + 0.0008 | 0.9998 | 0.08 | 0.24 | |
| B[a]P | y = 0.0137x + 0.0036 | 0.9984 | 0.07 | 0.20 | |
| Fatty liquid matrix (n = 3) | B[a]A | y = 0.0254x + 0.0037 | 0.9995 | 0.06 | 0.18 |
| CHR | y = 0.0188x + 0.0027 | 0.9999 | 0.11 | 0.33 | |
| B[b]F | y = 0.0217x + 0.0039 | 0.9992 | 0.09 | 0.27 | |
| B[a]P | y = 0.0179x − 0.0006 | 0.9993 | 0.09 | 0.28 | |
| Non Fatty solid matrix (n = 3) | B[a]A | y = 0.0326x − 0.0007 | 0.9997 | 0.04 | 0.11 |
| CHR | y = 0.0155x + 0.0004 | 0.9996 | 0.04 | 0.11 | |
| B[b]F | y = 0.0204x − 0.0013 | 0.9997 | 0.09 | 0.29 | |
| B[a]P | y = 0.0157x − 0.001 | 0.9991 | 0.09 | 0.27 | |
| Non Fatty liquid matrix (n = 3) | B[a]A | y = 0.0247 x + 0.0044 | 0.9990 | 0.03 | 0.09 |
| CHR | y = 0.0191x − 0.0005 | 0.9995 | 0.04 | 0.13 | |
| B[b]F | y = 0.0121x − 0.0023 | 0.9996 | 0.07 | 0.21 | |
| B[a]P | y = 0.0124x + 0.0013 | 0.9999 | 0.05 | 0.15 |
Numbers indicate the mean values (n = 3)
1The signal to noise ratio (S/N) of the LOD is 3.3
2The signal to noise ratio (S/N) of the LOQ is 10
Table 2.
Comparison of accuracy for each of the 4 polycyclic aromatic hydrocarbons(PAHs) from four matrices (fatty solid, fatty liquid, non-fatty solid, non-fatty liquid)
| Matrix | Conc. (μg/L) | Accuracy (%)a | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Intraday (n = 3) | Interday (n = 3) | ||||||||
| BaA | CHR | BbF | BaP | BaA | CHR | BbF | BaP | ||
| Fatty solid matrix | 1 | 88.85 | 90.10 | 113.66 | 105.12 | 96.92 | 87.06 | 112.30 | 102.63 |
| 2 | 93.82 | 89.23 | 117.26 | 106.53 | 97.38 | 86.93 | 116.88 | 98.98 | |
| 5 | 91.77 | 102.76 | 112.87 | 92.54 | 92.10 | 100.71 | 112.85 | 90.68 | |
| 10 | 98.73 | 92.37 | 111.85 | 104.97 | 102.46 | 94.59 | 112.40 | 108.54 | |
| 20 | 90.55 | 94.63 | 100.60 | 88.53 | 97.95 | 91.31 | 102.17 | 89.02 | |
| Fatty liquid matrix | 1 | 86.32 | 108.58 | 118.50 | 119.65 | 84.50 | 110.97 | 118.18 | 118.38 |
| 2 | 97.34 | 110.90 | 118.09 | 118.10 | 95.16 | 110.74 | 115.42 | 114.68 | |
| 5 | 98.25 | 115.11 | 119.53 | 112.14 | 95.27 | 113.67 | 113.47 | 108.31 | |
| 10 | 91.77 | 107.32 | 112.21 | 118.40 | 92.11 | 109.66 | 114.34 | 118.79 | |
| 20 | 84.12 | 105.13 | 100.88 | 117.02 | 97.76 | 93.60 | 90.94 | 118.61 | |
| Non Fatty solid matrix | 1 | 105.91 | 89.60 | 103.42 | 108.65 | 107.89 | 91.52 | 104.23 | 108.83 |
| 2 | 104.71 | 82.75 | 105.38 | 106.68 | 108.64 | 86.88 | 108.11 | 105.05 | |
| 5 | 114.42 | 88.22 | 100.75 | 95.70 | 119.51 | 92.84 | 100.34 | 94.64 | |
| 10 | 115.42 | 88.24 | 103.49 | 107.83 | 116.23 | 93.25 | 100.93 | 107.67 | |
| 20 | 109.20 | 84.27 | 94.24 | 106.11 | 109.83 | 87.75 | 91.73 | 103.58 | |
| Non Fatty liquid matrix | 1 | 84.96 | 105.24 | 102.18 | 83.41 | 84.18 | 106.13 | 101.58 | 80.94 |
| 2 | 91.52 | 105.50 | 105.44 | 80.89 | 88.85 | 104.71 | 105.03 | 88.60 | |
| 5 | 97.69 | 108.14 | 106.59 | 80.89 | 96.50 | 108.86 | 106.87 | 81.00 | |
| 10 | 91.01 | 107.94 | 101.27 | 87.02 | 91.30 | 105.88 | 99.25 | 87.10 | |
| 20 | 80.84 | 105.58 | 97.57 | 80.03 | 82.52 | 104.06 | 99.80 | 81.18 | |
aAccuracy (%) = [1-(mean concentration of measured standard solution-concentration of spiked sample)/ concentration of spiked sample] × 100
Table 3.
Comparison of precision(CV) for each of the 4 polycyclic aromatic hydrocarbons (PAHs) from four matrices (fatty solid, fatty liquid, non-fatty solid, non-fatty liquid)
| Matrix | Conc. (μg/L) | Precision (%)a | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Intraday (n = 3) | Interday (n = 3) | ||||||||
| BaA | CHR | BbF | BaP | BaA | CHR | BbF | BaP | ||
| Fatty solid matrix | 1 | 1.57 | 2.89 | 2.16 | 0.50 | 5.32 | 0.30 | 1.85 | 2.50 |
| 2 | 6.60 | 0.76 | 1.68 | 1.45 | 6.43 | 1.13 | 0.39 | 5.48 | |
| 5 | 2.01 | 1.17 | 0.46 | 1.93 | 2.73 | 3.56 | 0.86 | 0.88 | |
| 10 | 3.78 | 2.89 | 2.19 | 0.58 | 4.88 | 3.02 | 2.50 | 6.49 | |
| 20 | 0.81 | 5.70 | 0.50 | 0.88 | 6.67 | 6.04 | 1.72 | 0.89 | |
| Fatty liquid matrix | 1 | 0.64 | 0.84 | 0.73 | 1.42 | 4.41 | 2.69 | 0.61 | 1.42 |
| 2 | 0.66 | 0.52 | 0.51 | 2.49 | 2.91 | 0.39 | 4.51 | 2.49 | |
| 5 | 0.98 | 1.08 | 0.41 | 0.25 | 3.45 | 1.37 | 6.84 | 0.25 | |
| 10 | 0.88 | 0.08 | 1.38 | 0.76 | 2.29 | 2.30 | 3.49 | 0.76 | |
| 20 | 0.21 | 0.45 | 0.73 | 2.01 | 10.33 | 10.73 | 8.60 | 2.01 | |
| Non Fatty solid matrix | 1 | 1.86 | 2.02 | 1.36 | 0.79 | 2.02 | 0.86 | 0.30 | 0.97 |
| 2 | 2.22 | 1.14 | 0.22 | 1.86 | 5.31 | 3.92 | 3.33 | 1.46 | |
| 5 | 5.55 | 0.45 | 2.13 | 1.51 | 6.31 | 5.05 | 1.79 | 0.83 | |
| 10 | 0.95 | 0.16 | 1.08 | 0.97 | 1.70 | 4.30 | 1.97 | 0.86 | |
| 20 | 1.16 | 0.19 | 0.85 | 0.19 | 2.03 | 2.67 | 1.70 | 2.53 | |
| Non Fatty liquid matrix | 1 | 0.67 | 2.05 | 0.10 | 0.54 | 1.22 | 1.19 | 2.25 | 5.75 |
| 2 | 1.16 | 0.53 | 0.10 | 0.78 | 2.19 | 1.20 | 1.58 | 2.42 | |
| 5 | 1.36 | 1.45 | 0.26 | 0.99 | 0.53 | 2.35 | 0.54 | 0.80 | |
| 10 | 1.43 | 3.08 | 0.29 | 0.40 | 1.43 | 0.93 | 1.95 | 0.45 | |
| 20 | 1.00 | 0.57 | 0.50 | 0.07 | 3.72 | 1.85 | 2.43 | 1.99 | |
aPrecision (%) = (standard deviation/mean) × 100
Table 4.
Comparison of recovery for each of the 4 polycyclic aromatic hydrocarbons(PAHs) from four matrices (fatty solid, fatty liquid, non-fatty solid, non-fatty liquid)
| Matrix | Conc. (μg/L) | Recovery (%)a | |||
|---|---|---|---|---|---|
| BaA | CHR | BbF | BaP | ||
| Fatty solid matrix | 1 | 82.74 ± 2.75 | 81.9 ± 0.81 | 94.82 ± 2.70 | 92.33 ± 4.18 |
| 2 | 90.48 ± 5.81 | 86.38 ± 0.48 | 108.37 ± 1.94 | 99.09 ± 1.55 | |
| 5 | 90.88 ± 1.79 | 100.94 ± 1.74 | 110.47 ± 1.06 | 90.71 ± 1.31 | |
| 10 | 97.98 ± 3.64 | 91.97 ± 2.81 | 111.36 ± 2.87 | 103.87 ± 0.61 | |
| 20 | 90.99 ± 0.79 | 94.38 ± 5.49 | 99.75 ± 0.62 | 89.07 ± 0.69 | |
| Fatty liquid matrix | 1 | 81.93 ± 1.37 | 102.16 ± 0.86 | 84.36 ± 1.40 | 81.09 ± 1.40 |
| 2 | 94.77 ± 0.63 | 108.61 ± 0.56 | 101.62 ± 0.86 | 100.04 ± 3.05 | |
| 5 | 97.00 ± 1.27 | 116.29 ± 1.28 | 116.42 ± 0.49 | 106.01 ± 0.35 | |
| 10 | 91.76 ± 0.99 | 107.15 ± 0.07 | 110.12 ± 1.63 | 118.05 ± 0.89 | |
| 20 | 85.94 ± 0.19 | 105.04 ± 0.48 | 99.14 ± 0.74 | 118.40 ± 2.43 | |
| Non Fatty solid matrix | 1 | 84.42 ± 1.66 | 83.32 ± 1.75 | 95.84 ± 1.17 | 83.78 ± 5.08 |
| 2 | 93.59 ± 2.59 | 81.66 ± 1.02 | 102.27 ± 0.80 | 92.65 ± 1.99 | |
| 5 | 112.26 ± 6.41 | 87.95 ± 0.42 | 99.18 ± 2.21 | 91.25 ± 1.62 | |
| 10 | 115.93 ± 1.09 | 88.73 ± 0.14 | 102.85 ± 1.29 | 105.84 ± 0.65 | |
| 20 | 109.03 ± 1.29 | 86.04 ± 0.16 | 94.14 ± 0.77 | 105.26 ± 0.34 | |
| Non Fatty liquid matrix | 1 | 81.42 ± 0.86 | 80.90 ± 0.56 | 96.24 ± 0.58 | 80.13 ± 0.22 |
| 2 | 89.37 ± 0.93 | 93.75 ± 1.07 | 102.54 ± 0.24 | 88.035 ± 0.71 | |
| 5 | 96.58 ± 1.23 | 103.73 ± 1.79 | 105.83 ± 0.18 | 82.93 ± 0.83 | |
| 10 | 91.17 ± 1.31 | 106.10 ± 3.38 | 100.70 ± 0.27 | 87.92 ± 0.35 | |
| 20 | 83.63 ± 0.82 | 104.65 ± 0.63 | 97.35 ± 0.49 | 83.08 ± 0.08 | |
aRecovery rate was shown as mean ± RSD
In EC Regulation No. 333/2007, the stipulations for BaP are LOD < 0.3 μg/kg, LOQ < 0.9 μg/kg and recovery range of 50–120% (European Commission, 2007). Wretling et al. (2010) reported that recovery values of 15 carcinogenic PAHs were 84–113% and LOD and LOQ values were between 0.2 and 0.5 μg/kg. As shown in Table 4, the data obtained from the method validation in the current study confirm that GC–MS is a satisfactory method for the analysis and detection of 4 PAHs in samples of HMR products.
PAHs content in HMR products
The validated method was used to evaluate the contents of the 4 PAHs in various HMR food products classified into 40 categories, with four samples per category (Table 5). GC–MS chromatograms of the 4 PAHs standards and spiked samples are shown in Fig. 1. All experiments were performed in triplicate.
Table 5.
Polycyclic aromatic hydrocarbons contents in HMR products
| Matrix | HMR product | PAHs (μg/kg) | ||||
|---|---|---|---|---|---|---|
| BaA | CHR | BbF | BaP | PAH4 | ||
| Non-fatty solid | Triangular gimbap | NDa | ND | ND | ND | ND |
| Fatty solid | Lunch boxes | ND | ND | ND | ND | ND |
| Fatty solid | French fries | 0.04 ± 0.04 | 0.02 ± 0.03 | ND | ND | 0.06 ± 0.03 |
| Non-fatty solid | Sandwiches | ND | ND | ND | ND | ND |
| Fatty solid | Fried Chicken | ND | ND | ND | ND | ND |
| Fatty solid | Hamburgers | 0.08 ± 0.03 | 0.02 ± 0.02 | 0.06 ± 0.04 | ND | 0.16 ± 0.03 |
| Fatty solid | Donuts | ND | 0.04 ± 0.03 | ND | ND | 0.04 ± 0.03 |
| Non-fatty solid | Salad | 0.13 ± 0.05 | ND | ND | ND | 0.13 ± 0.05 |
| Fatty solid | Cheese Stick | 0.05 ± 0.03 | 0.01 ± 0.03 | ND | ND | 0.06 ± 0.03 |
| Fatty liquid | Samgyetang | ND | ND | ND | ND | ND |
| Fatty liquid | Kneebone Soup (Doganitang) | ND | ND | ND | 0.06 ± 0.02 | 0.06 ± 0.02 |
| Fatty liquid | Galbitang | ND | ND | ND | ND | ND |
| Non-fatty solid | Chicken stock | ND | ND | ND | 0.03 ± 0.00 | 0.03 ± 0.00 |
| Non-fatty liquid | Beef-bone broth (Sagolgomtang) | ND | 0.61 ± 0.33 | ND | 0.04 ± 0.01 | 0.65 ± 0.11 |
| Non-fatty liquid | Stock soup of bone and stew meat (Seolleongtang) | ND | 0.10 ± 0.12 | ND | 0.06 ± 0.03 | 0.16 ± 0.05 |
| Non-fatty liquid | Meat broth | ND | 0.07 ± 0.09 | ND | ND | 0.07 ± 0.09 |
| Fatty liquid | Hoy spicy meat stew (yukgaejang) | ND | 0.12 ± 0.13 | ND | ND | 0.12 ± 0.13 |
| Non-fatty liquid | Other Instant tangb | ND | 0.04 ± 0.09 | ND | 0.04 ± 0.02 | 0.08 ± 0.05 |
| Fatty solid | Small intestine of cattle | ND | ND | ND | 0.07 ± 0.11 | 0.07 ± 0.11 |
| Fatty solid | Spicy grilled chicken | ND | ND | ND | ND | ND |
| Fatty solid | Trotter(Jokbal) | 0.06 ± 0.01 | ND | ND | ND | 0.06 ± 0.01 |
| Fatty solid | Chopped roast chicken | 0.04 ± 0.02 | 0.03 ± 0.03 | ND | ND | 0.07 ± 0.03 |
| Non-fatty solid | Pork rinds | 0.12 ± 0.13 | 0.04 ± 0.04 | 0.06 ± 0.05 | 0.14 ± 0.20 | 0.36 ± 0.10 |
| Non-fatty solid | Instant rice | ND | ND | ND | 0.06 ± 0.12 | 0.06 ± 0.12 |
| Non-fatty solid | Kimchi stew | ND | ND | ND | 0.07 ± 0.02 | 0.07 ± 0.02 |
| Non-fatty solid | Soup | ND | 0.02 ± 0.01 | 0.08 ± 0.01 | ND | 0.1 ± 0.01 |
| Non-fatty solid | Bulgogi with Rice (Bulgogi-deopbap) | 0.03 ± 0.01 | 0.09 ± 0.05 | ND | ND | 0.12 ± 0.03 |
| Non-fatty solid | Korean Seaweed soup | ND | ND | ND | 0.04 ± 0.01 | 0.04 ± 0.01 |
| Non-fatty solid | Rice soup | ND | 0.02 ± 0.02 | 0.07 ± 0.01 | ND | 0.09 ± 0.02 |
| Non-fatty solid | Cup rice | 0.07 ± 0.01 | 0.04 ± 0.04 | ND | ND | 0.11 ± 0.02 |
| Non-fatty solid | Bean paste stew (doenjang stew) | ND | ND | ND | ND | ND |
| Non-fatty solid | Bibimbap | 0.05 ± 0.01 | ND | ND | ND | 0.05 ± 0.01 |
| Non-fatty solid | Instant gukc | 0.04 ± 0.10 | 0.02 ± 0.08 | 0.06 ± 0.12 | 0.05 ± 0.01 | 0.18 ± 0.08 |
All treatments were replicated 3 times and represented mean ± standard deviations
aND = not detected, below limit of detection
bInstant tang: Jangkuk, Perilia Seed Soup, Pork Back-bone Stew (Gamja-tang), Ox Blood Hangover Soup (seonji haejangguk), Braised Spicy chiken (Dak-bokkeum-tang), Spicy Hot pot (Maratang) etc.
cInstant guk: rice cake soup, beef radish soup, dried pollack hangover soup
Fig. 1.
GC–MS Chromatograms of 4 PAH standards in SIM mode (A), 10 μg/kg standard spiked sample (B) and two internal standards with blank in HMR products sample (C)
BaP was detected in the range of 0.03–0.14 μg/kg and was highest in the pork rinds category. Among the samples, PAH4 was in the range of 0.03–0.65 μg/kg and was highest in the beef-bone broth (Sagolgomtang) category. The mean concentration of BaP in the HMR samples was 0.06 μg/kg and the PAH4 subgroup was present at 0.12 μg/kg. In accordance with EC Regulation (EC) No. 835/2011 (European Commission, 2011), the maximum BaP level for meat, smoked fish, smoked aquatic products and smoked meat products is 5.0 μg/kg, which is also the maximum for the PAH4 subgroup (BaA, CHR, BbF, BaP).
PAH occurs in a variety of foods and the level can vary with the cooking process. Processing, such as drying, packaging and heat treatment, smoking, baking, roasting, grilling and frying, have been reported to contribute substantially to PAH formation (Ledesma et al., 2014; Orecchio et al., 2009). Free radicals generated during the burning of food at high temperatures undergo recombination to form "light" PAHs (2–4 rings) and then move to hydrophobic food chain compartments to form "heavy" (> 4 fused rings) PAHs that are ultimately retained in fat-rich foods (Luzardo et al., 2013a;2013b). In processed food products, especially in HMR, PAHs can be formed by direct processes and miscellaneous processes. PAH formation in food is caused by incomplete combustion during direct heating of food at high temperatures (Singh et al., 2016). Moreover, sucralose, an artificial food sweetener, loses stability at high temperatures above 119 °C and undergoes thermal decomposition to form toxic compounds, such as chloropropanols, polychlorinated dibenzo-p-dioxins and dibenzofurans. It is likely to be the successor to PAHs (Dong et al., 2013). Perelló et al. (2009) conducted a study on 16 PAHs in raw and cooked (fried, grilled, roasted, boiled) meat, fish, potato, chicken, steak, string bean, rice and olive oil. Of all boiled products, hake had the highest total PAH content (5.36 μg/kg) and potato had the lowest (3.15 μg/kg). Findings depended on the food item and the cooking process. When boiling rice, the amount of PAH doubled compared to the initial raw product. In addition, in another study, boiled ham had a total PAH of 1.22 μg/kg (Martorell et al., 2010). No significant difference was observed between grilled and boiled beef samples (1.87 and 1.09 μg/kg, respectively), but the amounts were notably higher than that detected in the raw sample (< 0.10 μg/kg). Therefore, meat processing practices, such as baking, smoking, roasting and boiling, must be carried out carefully to meet safety and quality aspects (Singh et al., 2016). Drying is a commonly used method to remove moisture from food grains, oilseeds, vegetables and tea. Corn drying is carried out in industrial and other small kilns. The future use of dried corn largely depends on the type of fuel used, the air method and the heating and composition of the combustion gases used during the process. Two processes accompany the burning of corn: i) pyrolysis leading to unstable PAH formation and ii) thermal synthesis, which favors transformation into highly complex PAHs. The surface of dried corn had higher values of adsorbed PAH compared to raw, uncontaminated corn seeds. Moreover, a diesel-based kiln drying resulted in higher BaP pollution than a natural gas-based kiln (Kiš et al., 2009). The smoke drying, dryness and refining or raffination processes of oilseeds lead to greatly varied PAH content between various batches and brands of vegetable oils and cold-pressed unconventional vegetable oils containing biologically active compounds (Ciecierska et al., 2013). Temperatures between 160 and 185 °C are achieved during discontinuous frying of French fries and fish (Perelló et al., 2009). Monounsaturated hydrocarbons present in oils or fats undergo aromatization and dehydrogenation processes to promote PAH formation and contamination of foods processed with these fats (Olatunji et al., 2014). The PAH16 concentrations were determined in fried products, such as sardines, tuna, veal, hake, chicken, pork, lamb and potato, which were higher in the same sample (13.30–35.42 μg/kg) (Perelló et al., 2009).
For smoked fish meat and smoked fish products, the maximum allowable content is 30.0 μg/kg. All food samples in this study met the criteria for BaP and the PAH4 subgroup of the EC Regulation (European Commission, 2011). Martorell et al. (2010) recorded BaP, PAH4 and PAH8 concentrations of 0.07, 0.57 and 0.88 μg/kg, respectively, in fish and shellfish. According to Alomirah et al. (2011), meat and meat products presented BaP at 0.14 μg/kg, PAH4 at 1.45 μg/kg and PAH8 at 1.71 μg/kg. In another study, the average BaP concentration in meat samples was 1.34 μg/kg, PAH8 was 13.11 μg/kg and chicken samples contained 0.80 μg/kg of BaP and 7.83 μg/kg of PAH8. Martí-Cid et al. (2008) reported BaP, PAH4 and PAH8 concentrations of 0.14, 1.16 and 1.85 μg/kg in fish and shellfish and 0.13, 1.47 and 1.88 μg/kg in meat and meat products, respectively. Chung et al. (2011) detected a BaP concentration of 0.04 μg/kg in smoked products. The average concentration of BaP in the beef samples was 0.15 μg/kg in the charcoal-grilled sample, 0.01 μg/kg in the charcoal-roasted sample and 0.003 μg/kg in the gas-grilled sample. Pork samples had an average BaP concentration of 2.90 μg/kg in the grilled sample, 0.02 μg/kg in the charcoal-roasting sample and 0.004 μg/kg in the gas-grilled sample. charcoal-grilled meat had the highest BaP levels.
Veyrand et al. (2013) noted BaP and PAH4 concentrations of 0.005 and 0.042 μg/kg in vegetables and 0.011 and 0.082 μg/kg in breakfast cereal, respectively. Voutsa et al. (1998) reported a BaP concentration of 0.05–0.096 μg/kg in cabbage, 0.096–0.22 μg/kg in carrots and 0.08–0.75 μg/kg in lettuce. Zhong et al. (2002) reported BaA levels in Chinese cabbage (5.46 μg/kg) and cucumber (2.33 μg/kg). Bishnoi et al. (2006) claimed a BaP concentration of 0.19 μg/kg in potato and BaP was not detected in radishes and apples.
As mentioned above, many previous studies have been conducted to analyze PAHs in foods. In this study, we developed an analytical method for measuring PAHs in four different matrices HMR products and investigated four PAH levels from frequently consumed HMR products in Korea. PAH levels in HMR products were found to comply with the European Union’s safety standards. However, there are no standards for PAHs in HMR products in Korea and far fewer published studies. It is necessary to prepare an accurate quantitative analysis method and standards in Korea and this study would be able to provides a scientific basis for safety management.
Declarations
Conflicts of interest
The authors of the manuscript declare that there is no conflict of interest regarding the publication of this study.
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References
- Alomirah H, Al-Zenki S, Al-Hooti S, Zaghloul S, Sawaya W, Ahmed N, Kannan K. Concentrations and dietary exposure to polycyclic aromatic hydrocarbons (PAHs) from grilled and smoked foods. Food Control. 2011;22:2028–2035. doi: 10.1016/j.foodcont.2011.05.024. [DOI] [Google Scholar]
- ATSDR. Toxicological profile for polycyclic aromatic hydrocarbons. US Agency for Toxic Substances and Disease Registry, Atlanta, GA (1995) [PubMed]
- Baskin E, Choi JW, Heo SY, Park SJ. Trends in the Home Meal Replacement Market. Journal of Distribution Science. 2016;14:5–15. doi: 10.15722/jds.14.6.201606.5. [DOI] [Google Scholar]
- Bishnoi NR, Mehta U, Pandit G. Quantification of polycyclic aromatic hydrocarbons in fruits and vegetables using high performance liquid chromatography. Indian Journal of Chemical Technology. 2006;13:30–35. [Google Scholar]
- Chung S, Yettella RR, Kim J, Kwon K, Kim M, Min DB. Effects of grilling and roasting on the levels of polycyclic aromatic hydrocarbons in beef and pork. Food Chemistry. 2011;129:1420–1426. doi: 10.1016/j.foodchem.2011.05.092. [DOI] [Google Scholar]
- Ciecierska M, Obiedziński M. Polycyclic aromatic hydrocarbons in vegetable oils from unconventional sources. Food Control. 2013;30:556–562. doi: 10.1016/j.foodcont.2012.07.046. [DOI] [Google Scholar]
- Conney A, Miller E, Miller J. Substrate-induced synthesis and other properties of benzpyrene hydroxylase in rat liver. Journal of Biological Chemistry. 1957;228:753–766. doi: 10.1016/S0021-9258(18)70657-1. [DOI] [PubMed] [Google Scholar]
- Dong S, Liu G, Hu J, Zheng M. Polychlorinated dibenzo-p-dioxins and dibenzofurans formed from sucralose at high temperatures. Scientific Reports. 2013;3:1–4. doi: 10.1038/srep02946. [DOI] [PMC free article] [PubMed] [Google Scholar]
- EFSA. Polycyclic aromatic hydrocarbons in food‐scientific opinion of the panel on contaminants in the food chain. European Food Safety Authority Journal 6:724 (2008) [DOI] [PMC free article] [PubMed]
- EPA. Polycyclic Organic Matter. Environmental Protection Agency, Washington, DC (2002)
- European Commission. Commission regulation (EC) No. 1881/2006 of 19 December 2006. Setting maximum levels for certain contaminants in foodstuffs (Text with EEA relevance). Official Journal of European Commission L 364/365: 323 (2006)
- Commission European. Commission Regulation (EC) No 333/2007 of 28 March 2007 laying down the methods of sampling and analysis for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPD and benzo (a) pyrene in foodstuffs. Official Journal of the European Union, L. 2007;88:29. [Google Scholar]
- European Commission. Commission Regulation (EC) No. 835/2011 of 19 August 2011 amending Regulation (EC) No. 1881/2006 as regards maximum levels for polycyclic aromatic hydrocarbons in foodstuffs. Official Journal of the European Union L215:7–8 (2011)
- Gilbert J. The fate of environmental contaminants in the food chain. Science of the Total Environment. 1994;143:103–111. doi: 10.1016/0048-9697(94)90536-3. [DOI] [PubMed] [Google Scholar]
- IARC. Overall evaluations of carcinogenicity : an updating of IARC monographs, vol. 1 to 42. International Agency for Research on Cancer (2004)
- Kiš D, Šuper D, Rozman V, Jurić T, Voća N, Guberac V. A comparative analysis of Corn drying in Stabil 3000 dry kiln using different energy sources. Agriculturae Conspectus Scientificus. 2009;74:205–207. [Google Scholar]
- Ledesma E, Rendueles M, Díaz M. Benzo (a) pyrene penetration on a smoked meat product during smoking time. Food Additives & Contaminants: Part A. 2014;31:1688–1698. doi: 10.1080/19440049.2014.949875. [DOI] [PubMed] [Google Scholar]
- Lodovici M, Dolara P, Casalini C, Ciappellano S, Testolin G. Polycyclic aromatic hydrocarbon contamination in the Italian diet. Food Additives & Contaminants. 1995;12:703–713. doi: 10.1080/02652039509374360. [DOI] [PubMed] [Google Scholar]
- Luzardo OP, Rodríguez-Hernández Á, Quesada-Tacoronte Y, Ruiz-Suárez N, Almeida-González M, Henríquez-Hernández LA, Zumbado M, Boada LD. Influence of the method of production of eggs on the daily intake of polycyclic aromatic hydrocarbons and organochlorine contaminants: an independent study in the Canary Islands (Spain) Food and Chemical Toxicology. 2013;60:455–462. doi: 10.1016/j.fct.2013.08.003. [DOI] [PubMed] [Google Scholar]
- Luzardo OP, Zumbado M, Boada LD. Concentrations of polycyclic aromatic hydrocarbons and organohalogenated contaminants in selected foodstuffs from Spanish market basket: Estimated intake by the population from Spain. Journal of Food, Agriculture & Environment. 2013;11:4. [Google Scholar]
- Lyon F. IARC monographs on the evaluation of carcinogenic risks to humans. Some Industrial Chemicals. 1994;60:389–433. [PMC free article] [PubMed] [Google Scholar]
- Martí-Cid R, Llobet JM, Castell V, Domingo JL. Evolution of the dietary exposure to polycyclic aromatic hydrocarbons in Catalonia. Spain. Food and Chemical Toxicology. 2008;46:3163–3171. doi: 10.1016/j.fct.2008.07.002. [DOI] [PubMed] [Google Scholar]
- Martorell I, Perelló G, Martí-Cid R, Castell V, Llobet JM, Domingo JL. Polycyclic aromatic hydrocarbons (PAH) in foods and estimated PAH intake by the population of Catalonia, Spain: temporal trend. Environment International. 2010;36:424–432. doi: 10.1016/j.envint.2010.03.003. [DOI] [PubMed] [Google Scholar]
- Moon HB, Kim HS, Choi M, Choi HG. Intake and potential health risk of polycyclic aromatic hydrocarbons associated with seafood consumption in Korea from 2005 to 2007. Archives of Environmental Contamination and Toxicology. 2010;58:214–221. doi: 10.1007/s00244-009-9328-5. [DOI] [PubMed] [Google Scholar]
- Moon HB, Kannan K, Lee SJ, Ok G. Atmospheric deposition of polycyclic aromatic hydrocarbons in an urban and a suburban area of Korea from 2002 to 2004. Archives of Environmental Contamination and Toxicology. 2006;51:494–502. doi: 10.1007/s00244-006-0002-x. [DOI] [PubMed] [Google Scholar]
- Olatunji OS, Fatoki OS, Ximba BJ, Opeolu BO. Polycyclic aromatic hydrocarbons (PAHs) in edible oil: temperature effect on recovery from base hydrolysis product and health risk factor. Food and Public Health. 2014;4:23–30. [Google Scholar]
- Orecchio S, Ciotti VP, Culotta L. Polycyclic aromatic hydrocarbons (PAHs) in coffee brew samples: analytical method by GC–MS, profile, levels and sources. Food and Chemical Toxicology. 2009;47:819–826. doi: 10.1016/j.fct.2009.01.011. [DOI] [PubMed] [Google Scholar]
- Pensado L, Casais M, Mejuto M, Cela R. Application of matrix solid-phase dispersion in the analysis of priority polycyclic aromatic hydrocarbons in fish samples. Journal of Chromatography A. 2005;1077:103–109. doi: 10.1016/j.chroma.2005.04.087. [DOI] [PubMed] [Google Scholar]
- Perelló G, Martí-Cid R, Castell V, Llobet JM, Domingo JL. Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking. Food and Chemical Toxicology. 2009;47:709–715. doi: 10.1016/j.fct.2008.12.030. [DOI] [PubMed] [Google Scholar]
- Singh L, Varshney JG, Agarwal T. Polycyclic aromatic hydrocarbons’ formation and occurrence in processed food. Food Chemistry. 2016;199:768–781. doi: 10.1016/j.foodchem.2015.12.074. [DOI] [PubMed] [Google Scholar]
- Veyrand B, Sirot V, Durand S, Pollono C, Marchand P, Dervilly-Pinel G, Tard A, Leblanc JC, Le Bizec B. Human dietary exposure to polycyclic aromatic hydrocarbons: results of the second French Total Diet Study. Environment International. 2013;54:11–17. doi: 10.1016/j.envint.2012.12.011. [DOI] [PubMed] [Google Scholar]
- Voutsa D, Samara C. Dietary intake of trace elements and polycyclic aromatic hydrocarbons via vegetables grown in an industrial Greek area. Science of the Total Environment. 1998;218:203–216. doi: 10.1016/S0048-9697(98)00206-X. [DOI] [PubMed] [Google Scholar]
- Wretling S, Eriksson A, Eskhult G, Larsson B. Polycyclic aromatic hydrocarbons (PAHs) in Swedish smoked meat and fish. Journal of Food Composition and Analysis. 2010;23:264–272. doi: 10.1016/j.jfca.2009.10.003. [DOI] [Google Scholar]
- Zhong W, Wang M. Some polycyclic aromatic hydrocarbons in vegetables from northern China. Journal of Environmental Science and Health, Part A. 2002;37:287–296. doi: 10.1081/ESE-120002588. [DOI] [PubMed] [Google Scholar]