Food Additives and Contaminants: Part B Vol. 4, No. 2, June 2011, 152–159
VIEW DATASET Polycyclic aromatic hydrocarbons in Brazilian commercial soybean oils and dietary exposure M.C. Rojo Camargo*, P.R. Antoniolli, E. Vicente and S.A.V. Tfouni Food Science and Quality Center, Institute of Food Technology – ITAL, Av. Brasil, 2880, 13070-178 – Campinas-SP, Brazil (Received 12 October 2010; final version received 28 April 2011) In this study the 13 polycyclic aromatic hydrocarbons (PAHs) identified as being genotoxic and carcinogenic by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) were determined in different brands of soybean oils available on the Brazilian market, totalling 42 samples. A solid-phase extraction (SPE) method for sample clean-up with a C18 cartridge, followed by reversed-phase HPLC with fluorescence detection, was used for determination. The method showed good recoveries for most PAHs studied with values between 74% and 111%. Good intra- and inter-day precisions (0.55RSD511.9) and high correlation coefficients (r240.999) were obtained. The presence of PAHs was detected in all 42 samples with mean summed PAH levels ranging from 10.4 to 112.0 mg kg1. The mean and maximum dietary exposures for total PAHs were estimated as 12.4 and 19.1 ng kg bw1 day1, respectively. Keywords: oils and fats; processed foods; PAH; process contaminants – PAHs
Introduction Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemical compounds that are formed during incomplete combustion of organic matter and have been found as contaminants, predominantly from environmental pollution and food processing, in a variety of foodstuffs, including vegetables, fruit, cereals, oils and fats, smoked fish and meat, coffee and tea (Weibhaar 2002; Camargo and Toledo 2003; Bishnoi et al. 2005; Lin et al. 2005; Moret et al. 2005; Purcaro et al. 2006; Tfouni et al. 2007; Djinovic et al. 2008; Rey-Salgueiro et al. 2008). Ever since PAHs were first recognised as being carcinogenic, the presence of these compounds in food has been a matter of concern worldwide. Studies carried out in different countries have identified oils and fats as important sources of PAHs in the diet (Lodovici et al. 1995; Pupin and Toledo 1996; Moret and Conte 2000; Camargo and Toledo 2002; Falco´ et al. 2003; Barranco et al. 2004; Lage-Yust and Davina˜ 2005; Purcaro et al. 2006). The presence of PAHs in vegetable oils is mainly attributed to direct drying of the oil seeds with combustion gases before oil extraction during processing, resulting in relatively high and variable levels of these contaminants in the final product. In view of the potential health effects, the necessity of establishing maximum limits for PAHs has been recognised by some countries in different opportunities. As consequence, the Codex Committee on Food *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2011.585244 http://www.informaworld.com
Additives and Contaminants (CCFAC) placed these contaminants in the priority list for the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) evaluation. During its 64th meeting the JECFA, based on the documents from European Union Scientific Committee on Food (SCF) and from International Programme on Chemical Safety (IPCS), concluded that 13 PAHs (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ j]fluoranthene, benzo[a]pyrene, dibenzo[a, h]anthracene, dibenzo[a, e]pyrene, dibenzo[a, h]pyrene, dibenzo[a, i]pyrene, dibenzo[a, l]pyrene, indeno[1,2,3-cd]pyrene and 5-methylchrysene) were clearly carcinogenic and genotoxic, even in low exposure doses (European Commission 2005; WHO 2005). Due to the genotoxicity of PAHs, it is not possible to assume a threshold mechanism and consequently a provisional tolerable weekly intake (PTWI) cannot be established (WHO 2008). Thus, in 2006, the CCFAC at its 38th session agreed to elaborate a proposed draft Code of Practice for the reduction of contamination of food with PAHs from smoking and direct drying processes (WHO 2008). Furthermore, the European Commission established maximum levels for benzo[a]pyrene in different food groups. For oils and fats the maximum limit has been restricted to 2.0 mg kg1 (European Commission 2006). In Brazil, the yearly consumption of vegetable oils account for roughly 3.72 million tons, 86% of which correspond to soybean oil (Nunes 2007). A preliminary
Food Additives and Contaminants: Part B study conducted by the present authors pointed relatively high and variable levels of PAHs in the most consumed brands of soybean oils. In this manner, considering the importance of this product in the Brazilian diet and the lack of information about possible sources of contamination, the results showed the necessity of extending the investigation to other brands of soybean oil available on the national market, once the Brazilian regulation for oils and fats does not establish a maximum level for any PAH. Therefore, focusing on all 13 PAHs identified as being carcinogenic and genotoxic by the JECFA, the present study had three purposes: (1) to validate a SPE-HPLC-FLD method for the simultaneous determination of these 13 compounds, (2) to determine the levels of PAHs in different brands of soybean oils commercially available in Brazil, and (3) to estimate the Brazilians dietary exposure to these contaminants through oil consumption.
Materials and methods Standards and reagents PAHs standards were purchased from Supelco Inc. (St. Louis, MO, USA) (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a] pyrene, dibenzo[ah]anthracene and indeno[1,2,3cd]pyrene), Fluka (Munich, Germany) (benzo[ j]fluoranthene, dibenzo[al]pyrene, dibenzo[ae]pyrene and dibenzo[ah]pyrene), Cambridge Isotope Laboratories Inc. (Andover, MA, USA) (5-methylchrysene) and ChemService Inc. (West Chester, PA, USA) (dibenzo[ai]pyrene). Hexane, methanol and N,N-dimethylformamide (HPLC grade) were acquired from Tedia Brazil Ltda (Brazil). Acetonitrile (HPLC grade) was supplied by J.T. Baker (Mexico City, Mexico). Water was purified on a Milli-Q system, Millipore Corp. (Bedford, MA, USA). The SPE cartridges AccuBondII (500 mg, 3 ml) were from Agillent Technologies Inc. (Allentown, PA, USA). Stock standard solutions were prepared by diluting the PAHs standards in acetonitrile.
Samples Soybean oils commercially available in Brazil were purchased at supermarkets in the region of Campinas, SP, between 2008 (11 different brands, three batches each) and 2009 (three different brands, three batches each), totalising 42 samples. All were analysed in duplicate for benzo[a]anthracene (B[a]P), chrysene (Chy), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[ j]fluoranthene (B[ j]F), benzo[a]pyrene (B[a]P), dibenzo[a, h]anthracene (D[ah]A), dibenzo[a, e]pyrene (D[ae]P), dibenzo[a, h]pyrene (D[ah]P), dibenzo[a, i]pyrene (D[ai]P), dibenzo[a, l]pyrene
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(D[al]P), indeno[1,2,3-cd]pyrene (Indeno) and 5methylchrysene (5MeChy).
Extraction and clean-up Soybean oils (0.5 g) were weighed in an erlenmeyer flask and 5 ml of hexane were added. The mixture was transferred into a 60 ml separating funnel and PAHs were extracted twice with 5 ml of N,Ndimethylformamide–water (DMF-H2O) (9:1, v/v) (Grimmer and Bohnke 1975; Barranco et al. 2003). The combined extract was concentrated under a flow of nitrogen until it reached approximately 50% of its initial volume. Then, the resulting solution was diluted with 5 ml of water before solid-phase extraction (SPE) clean-up. The SPE cartridges were prepared by prewashing with 5 ml of methanol, followed by 5 ml of water using a Vacuum Manifold from SupelcoÕ (Bellefonte, PA, USA). Then the sample solution was applied and the column was washed with 10 ml of N,Ndimethylformamide–water (1:1, v/v), followed by 10 ml of water, all eluates being discarded. The cartridges were dried under vacuum for 20 min. PAHs were eluted with 10 ml hexane at a flow rate of 2 ml min1 and the eluate was taken to dryness under a nitrogen stream. The residue was diluted in 0.5 ml acetonitrile, filtered through a 0.45 mm filter (PVDF, Millex-HV, Millipore) into a HPLC vial and capped for the HPLC analysis.
HPLC analysis The analysis was carried out in a Shimadzu LC-20 A Prominence HPLC (Japan) equipped with quaternary pumps (model LC-10AT), on-line degasser, a model SIL-20 A autosampler and a model RF-10AXL fluorescence detector. A C18 Vydac 201 TP column (5 mm, 250 4.6 mm) at 30 C and a mobile phase composed of acetonitrile and water, at a flow rate of 1 ml min1, was used to separate the PAHs. The gradient elution programme started with a linear gradient from 70% to 75% acetonitrile in 20 min, followed by a 15 min linear gradient from 75% to 100% acetonitrile and maintained 100% acetonitrile isocratic until 55 min, when finally returned to the initial conditions and the column was re-equilibrated with the initial mobile phase composition for 15 min. The injection volume was set to 30 ml. The following excitation (ex) and emission (em) wavelength programme was used to determine the PAHs: 0.01 min (268/398 nm) for B[a]A, Chy, 5MeChy; 16.70 min (312/507 nm) for B[ j]; 18.20 min (290/430 nm) for B[b]F, B[k]F, B[a]P, D[al]P, D[ah]A; 32.40 min (300/500 nm) for Indeno; 34.90 min (297/403 nm) for D[ae]P and 45 min (304/ 457 nm) for D[ai]P, D[ah]P. Data were acquired and processed with LC solution software.
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Identification and quantification of PAHs The identification of PAHs was performed by comparison of their retention times with those obtained by injecting standards in the same conditions. Peak identity was also confirmed by spiking the extracts with pure standards (standard addition method). The compounds were quantified using the external standard plot method. A mixed standard stock solution with PAHs was prepared in acetonitrile and stored at 4 C for 3 months. From this solution, seven working ones, ranging from 0.5 to 250 ng ml1, were used to construct linear regression lines (peak area ratios versus PAH concentration).
Validation study The following parameters were used for method validation: accuracy, precision (inter- and intraday repeteability), linearity, specificity, limit of detection (LOD) and limit of quantification (LOQ), according to Institute of Metrology, Standardisation and Industrial Quality (Inmetro) guidelines, under ISO 17025 criteria (Inmetro 2007). The accuracy and repeatability of the method were evaluated by performing recovery tests. Recovery (accuracy) was determined by spiking a blank control sample of soybean oil with the PAHs studied at 0.5, 1.0, 1.5 and 5.0 mg kg1 (n ¼ 3 replicates for each level) and the values were reported as average per cent recovery. Inter- and intraday repeatabilities, expressed as the percentage of relative standard deviation (% RSD), were checked by analysing the same sample control spiked with PAHs standard, in triplicate, during the same day and in 3 different days. In addition, the precision of the chromatographic system was carried out by injecting the same oil sample extract, fortified with a working standard PAHs solution (1.2 mg kg1) five times (n ¼ 5), during three consecutive days (n ¼ 3) and then checking the RSD of retention times and peak areas. Linearity was tested by the square correlation coefficients (r2) of the calibration curves. Method LOD and LOQ were determined using spiked matrices. For this purpose, seven independent soybean oil sample (blank) spiked with PAHs at a level of 0.5 mg kg1 were analysed. LOD and LOQ were calculated as the analyte concentration corresponding to, respectively, mean sample blank þ 3 s and mean sample blank þ 5 s. Finally, specificity was confirmed by analysis of the blank oil sample control, which was produced with soybeans dried naturally in order to avoid any kind of contamination.
Statistical analysis The software Statistica for Windows 5.5 (StatSoft Inc., Tulsa, OK, USA) was used to perform the analysis of variance (ANOVA). PAHs contamination levels in
different periods of time were compared by Tukey test (95% confidence).
Dietary exposure Intakes were calculated using the mean and maximum PAHs levels determined in the samples, considering all brands available on the market, during a year of production. Food consumption data were obtained from a National Household Survey, conducted by the Instituto Brasileiro de Geografia e Estatı´ stica (IBGE; Brazilian Institute of Geography and Statistics 2005) among 48,470 selected households covering the urban and rural areas of all 27 Brazilian states, during 12 months (July 2002 to June 2003). Considering only the south-east of the country, where the region from which samples were collected is located, a daily soybean oil consumption of 24.9 g per person per day was assumed. Individual daily intakes of PAHs (ng kg bw1 day1) were estimated by multiplying the average daily intake (g day1) of the selected food item by the individual PAH concentration (mg kg1) determined in this product. Also, a worst-case scenario was drafted using the maximum PAH concentrations determined in the analysed oils. A consumer average body weight of 60 kg was considered in the calcultations.
Results and discussion Analytical procedure One objective of this study was to optimise and validate a methodology by SPE-HPLC-FLD to determine 13 PAHs in soybean oils with minimum of interferences. For this purpose, different variables involved in the extraction procedure and HPLC analysis were studied. The effectiveness of different types of silica-based C18 cartridges and different amounts of sample was assessed with regard to the quantity of co-extractives remaining after evaporation of the solvent that affected positive or negatively in the recovery. Satisfactory results were obtained using 500 mg of C18 sorbent and 0.5 g of oil sample. Among 500 mg cartridges tested, the best performance was achieved with the higher carbon loading, i.e. using the AccuBondII 500 mg, 3 ml. Once defined the SPE sorbent, the solvents (volume and flow rate) used during conditioning, rinsing and elution steps were evaluated. Best results were obtained using 5 ml of methanol, followed by 5 ml of water for pre-washing the cartridges and 10 ml of N,N-dimethylformamide–water (1:1, v/v), followed by 10 ml of water for washing after the sample was applied. To determine the ideal rinsing conditions, a series of washes varying from 10% to 50% methanol at 10% increments was applied. It was observed that an increase in the
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Figure 1. HPLC chromatogram of soybean oil spiked with a standard mixture of PAHs. Table 1. Analytical parameters of the method used for PAHs analysis in soybean oils.
PAHa B[a]A Chy 5MeCri B[ j]F B[b]F B[k]F B[a]P D[al]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P
LOD (mg kg1)b
LOQ (mg kg1)b
Linear range (mg kg1)
0.03 0.02 0.16 0.39 0.20 0.16 0.16 0.12 0.15 0.54 0.07 0.10 0.76
0.05 0.03 0.27 0.65 0.35 0.25 0.25 0.20 0.24 0.90 0.12 0.25 0.96
0.5–50 0.5–50 0.5–50 1.0–250 0.5–50 0.5–50 0.5–50 0.5–50 0.5–50 1.0–250 0.5–50 0.5–50 0.5–50
r2
Mean recovery RSD (%)c
Interday precision RSD (%)d
0.9994 0.9994 0.9998 0.9999 0.9993 0.9990 0.9998 0.9993 0.9984 0.9997 0.9999 0.9967 0.9993
93 3.7 85 7.4 111 5.6 85 4.4 84 3.5 82 3.0 101 4.5 82 11.9 93 10.7 74 6.5 94 4.9 64 5.0 94 3.8
2.7 2.9 2.9 6.1 4.1 4.9 0.5 1.1 2.2 4.5 3.4 5.8 2.5
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeCri, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b LOD, limit of detection; LOQ, limit of quantification. c n ¼ 9 determinations in the same day relative standard deviation (RSD, %), expressed as a percentage of the recovery for a sample blank spiked at three different concentrations according to PAH. d n ¼ 9 determinations in three different days. r2, regression coefficient.
percentage of methanol provided unsatisfactory results. Differently, the use of dimethylformamide– water (1:1, v/v), according to Barranco et al. (2003), followed by pure water improved the method recovery and reproducibility. Then chromatographic conditions using different mobile phase composition, flow rate, column temperature, gradient elution and wavelength programme were applied. Mobile phase composition and gradient elution were optimised in order to achieve a good separation of the organic compounds in the shortest time analysis. As can be observed in Figure 1, the chromatographic conditions established were satisfactory when separating the 13 PAHs studied. Fluorescence detection was optimised with wavelength
programming for excitation and emission. Based on each PAH fluorescence spectra the most appropriate wavelengths for each group of compounds during the chromatographic run were selected in order to detect all of them and keep the gradient programme applied. After establishing the extraction procedure and cromatographic conditions the validation of the analytical method was certified according to the figures of merit described above. Table 1 summarises the proposed method performance. The average recoveries obtained for the compounds (64–111%) were satisfactory for determinations at the mg kg1 level and were in agreement with the criteria for methods of analysis for benzo(a)pyrene provided by Regulation EC No. 333
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(Horwitz et al. 1980; European Commission 2007). Intra- and interday repeatability of the extraction procedure, expressed as the percentage of relative standard deviation (% RSD), ranged from 3.0% to 11.9% and from 0.5% to 6.1%, respectively. For the chromatographic system, the RSD of the detector response was less than 1.5% (including intra- and interday repeatability) and the RSD of the retention times was lower than 0.09% for each compound. The PAHs responses were linear over the concentration range studied, as showed in Table 1. The LOD and LOQ for the target compounds varied from 0.02 to 0.76 mg kg1 and from 0.03 to 0.96 mg kg1, respectively.
PAHs in Brazilian commercial soybean oils Table 2 presents the mean and maximum levels of PAHs determined in three different batches of eleven brands (A–K) of soybean oils available in the Brazilian market. Variable levels of contamination were found within different brands of oils and within different batches of the same brand. Only B[ j]F was not detected in three samples analysed. The mean levels of individual PAHs ranged from 0.2 to 26.1 mg kg1. Brand K showed the highest PAH contamination (112.0 mg kg1). Brand B also presented high levels of PAHs, with the maximum levels (61.4 mg kg1) almost three times higher than the mean content of summed PAHs (26.9 mg kg1). This should be a motive of concern since brand B is the same one identified as the most contaminated in preliminary studies and, additionally, is the second brand most consumed among Brazilians. Besides, five soybean oils (B, D, I, J and K) presented levels of B[a]P higher than those established by the European Union (2.0 mg kg1) (European Commission 2005). In oil K, the most contaminated one, the mean and maximum B[a]P concentration was almost eight times (15.8 mg kg1) the maximum permitted limit. A comparison with other studies showed that the present levels are higher than those reported by other authors for the same kind of oil, whose values ranged between 0.09 and 6.1 mg kg1 (Pupin and Toledo 1996; Pandey et al. 2004; Teixeira et al. 2007). A year later, the three most consumed brands (A–C) were re-evaluated. Figure 2 compares the results obtained for these selected oils in both years (2008 and 2009). As can be observed, there was a significant variation in summed PAHs levels among different brands and batches, indicating the seasonality of oils contamination by PAHs (Tukey, p50.05). In 2009, most samples evaluated showed higher levels of PAHs than the respective ones in 2008. These variability could be related to different soybean seed
drying processes used prior to oil extraction (Camargo and Toledo 1998; WHO 2008).
PAHs dietary intake through soybean oil consumption in the region of Campinas, SP, Brazil Table 3 shows the estimated PAHs exposure through soybean oil consumption. As can be observed, the dietary intake of a single PAH ranged between 0.2 and 2.8 ng kg bw1 day1, while the mean exposure calculated for the sum of all of them was 12.4 ng kg bw1 day1. When the worst case scenario was considered, the total daily intake raised to 19.1 ng kg bw1 day1. For comparison purposes, with regard to B[a]P intakes, the mean and maximum estimated values were 1.2 and 1.7 ng kg bw1 day1, respectively, which can be considered relatively higher than the daily intake from total diets described by two US (0.8– 2.3 ng kg bw1 day1) (Kazerouni et al. 2001) and six European Union (0.8–4.8 ng kg bw1 day1) (European Union 2002) surveys, once in this study only one region and only one product were taken into account. When the margin of exposure (MOE) was considered for risk assessment purposes, values of 83.333 and 58.823 were obtained for mean and high level intakes, respectively. These values were calculated using a BMDL10 of 0.1 mg B[a]P kg bw1 day1 established by the JECFA (WHO 2005). According to the Committee, MOEs of 25,000 and 10,000 for mean and high level intakes, respectively, have been suggested to be of low concern for human health. Although the soybean oil represents a small contribution of the average diet, vegetable oils generally show higher levels of PAHs than other kind of foods. Consequently, in the Brazilian diet, along with many countries, despite differences in dietary pattern, the oils are expected to be the major contributors to PAHs intake (Dennis et al. 1983; De Vos et al. 1990; Kazerouni et al. 2001; Camargo and Toledo 2002; European Union 2002). In this manner and considering that PAHs are both carcinogenic and genotoxic, their levels in edible oils, likewise their intake, should be reduced to as low as possible in order to reduce consumer exposure to these compounds.
Conclusion Various sources are responsible for the presence of PAHs in vegetable oils resulting in a great variability of the contamination levels. Although the use of active carbon in the refining process is highly efficient to reduce the PAHs in oils, especially the ‘heavy fraction’ (four to five rings), oil refineries in Brazil do not use this adsorbent. Thus, it is recommended that a rigid
4.0 (9.3) 4.4 (11.0) 1.3 (2.5) 5LQ (4.5) 2.1 (8.5) 0.8 (2.7) 1.4 (5.5) 5LQ (0.3) 1.8 (4.1) 5LQ (5.2) 0.6 (1.3) 0.8 (2.6) 5LQ 17.2 (57.5)
A
4.2 (10.6) 5.9 (10.6) 1.7 (1.0) 1.7 (3.5) 2.3 (6.0) 1.1 (2.6) 2.1 (7.0) 0.2 (0.4) 3.4 (8.5) 3.1 (6.4) 0.5 (1.0) 0.7 (3.8) 5LQ 26.9 (61.4)
B 1.7 (4.9) 2.5 (4.4) 1.3 (5.5) 5LQ 0.6 (1.5) 0.4 (0.6) 0.5 (1.0) 5LQ (0.9) 0.9 (1.2) 2.0 (2.4) 0.5 (1.2) 5LQ (0.7) 5LQ 10.4 (24.3)
C 7.1 (13.0) 7.9 (11.3) 2.0 (2.4) 5LQ 2.6 (3.5) 0.8 (1.2) 2.2 (2.4) 0.6 (0.2) 1.2 (1.4) 2.2 (2.3) 0.7 (0.8) 1.0 (0.7) 1.7 (4.8) 30.0 (44.0)
D 1.2 (2.4) 2.0 (3.0) 0.7 (1.2) n.d. 1.3 (1.6) 0.5 (0.5) 1.0 (1.3) 0.7 (1.9) 1.6 (2.1) 2.0 (2.4) 0.5 (0.6) 0.3 (0.4) 5LQ 11.8 (17.4)
E 3.7 (4.6) 4.0 (4.7) 1.2 (2.1) n.d. 2.8 (3.7) 0.6 (0.8) 0.7 (0.8) 0.3 (0.7) 1.4 (2.3) 2.5 (3.9) 0.7 (0.9) 0.5 (0.9) 5LQ 18.4 (25.4)
F 3.0 (5.9) 3.6 (5.2) 1.2 (2.3) n.d. 1.6 (2.3) 0.6 (0.7) 0.9 (1.4) 0.2 (0.2) 1.1 (1.2) 5LQ (2.2) 0.7 (0.8) 0.3 (0.4) 1.4 (2.5) 14.6 (25.1)
G 5.2 (7.9) 5.0 (7.0) 1.0 (1.5) 5LQ 2.9 (5.2) 0.7 (1.1) 1.2 (1.8) 5LQ (0.2) 0.9 (1.1) 2.0 (2.8) 0.4 (0.5) 0.3 (0.3) 5LQ 19.6 (29.4)
H
5.7 (12.6) 8.2 (10.4) 0.9 (1.1) 2.6 (3.8) 4.5 (6.0) 1.9 (2.6) 4.8 (5.0) 5LQ (0.3) 4.4 (5.6) 5.5 (9.4) 1.0 (1.6) 5LQ (0.3) 5LQ 39.5 (58.7)
I 4.2 3.8 0.7 1.7 2.6 1.0 2.5 0.4 4.0 3.9 0.6 0.4 5LQ 25.8
(4.5) (4.3) (0.7) (1.9) (2.7) (1.3) (3.0) (1.0) (5.8) (6.1) (0.7) (0.9) (1.1) (34.0)
J 13.6 26.1 1.1 10.3 11.1 6.5 15.8 0.6 22.9 5LQ 1.2 1.2 1.6 112.0
(14.3) (27.3) (1.1) (11.8) (11.2) (6.8) (16.2) (0.8) (26.2) (2.1) (1.2) (1.2) (2.1) (122.3)
K
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeCri, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b Mean of three samples (in duplicate). n.d., not detected; 5LOQ, lower than the limit of quantification given in Table 1.
B[a]A Cri 5MeCri B[ j]F B[b]F B[k]F B[a]P D[al ]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P HPAs
PAHa
Soybean oils, mean (maximum) levels (mg kg1)b
Table 2. Mean and maximum PAHs levels in commercial soybean oils.
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Figure 2. Mean summed PAHs levels in commercial soybean oils (brands A–C) during 2008 and 2009.
Acknowledgements Table 3. PAHs dietary intake from soybean oils in Campinas, SP, Brazil.
Financial support from FAPESP (Proc. 05/59974-8) and scholarship from CNPq are gratefully acknowledged.
Intake (ng kg bw1 day1) PAHa B[a]A Chy 5MeChy B[ j]F B[b]F B[k]F B[a]P D[al ]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P HPAs
Means
Maximumsb
2.0 2.8 0.5 0.6 1.3 0.6 1.2 0.1 1.7 0.9 0.3 0.2 0.2 12.4
3.4 3.7 0.8 1.1 2.0 0.8 1.7 0.2 2.2 1.7 0.4 0.5 0.6 19.1
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeChy, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b Worst-case scenario: ingestion of foods with the maximum PAH concentration and taking values5LOD to be equal to the LOD.
monitoring programme for PAHs in soybean oil be initiated by the refining industries and a maximum or guideline level for B[a]P in vegetable oils established in the national standards. Additionally, a more comprehensive study should be adressed in order to evaluate the possible influence of each step of the refining process on the decrease of PAH contamination.
References Barranco A, Alonso-Salces RM, Bakkali A, Berrueta LA, Gallo B, Vicente F, Sarobe M. 2003. Solid-phase clean-up in the liquid chromatographic determination of polycyclic aromatic hydrocarbons in edible oils. J Chromatogr A. 988:33–40. Barranco A, Alonso-Salces RM, Crespo I, Berrueta LA, Gallo B, Vicente F, Sarobe M. 2004. Polycyclic aromatic hydrocarbon content in commercial Spanish fatty foods. Food Protect. 67(12):2786–2791. Bishnoi NR, Mehta U, Sain U, Pandit GG. 2005. Quantification of polycyclic aromatic hydrocarbons in tea and coffee samples of Mumbai city (India) by high performance liquid chromatography. Environ Monit Assess. 107:399–406. Camargo MCR, Toledo MCF. 2002. Avaliac¸a˜o da contaminac¸a˜o de diferentes grupos de alimentos por hidrocarbonetos policı´ clicos aroma´ticos. Braz J Food Technol. 5(76):19–26. Camargo MCR, Toledo MCF. 2003. Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits. Food Contr. 14(1):49–53. Camargo MSFO, Toledo MCF. 1998. Efeito do processamento na contaminac¸a˜o de o´leo refinado de milho por benzo(a)pireno. Braz J Food Technol. 1(1–2):97–106. De Vos RH, Van Dokkum W, Schouten A, JongBerkhout P. 1990. Polycyclic aromatic hydrocarbons in Dutch total diet samples (1984–1986). Food Chem Toxicol. 28(4):263–268. Dennis MJ, Massey RC, McWeeny DJ, Knowles ME. 1983. Analysis of polycyclic aromatic hydrocarbons in UK total diets. Food Chem Toxicol. 21(5):569–574.
Food Additives and Contaminants: Part B Djinovic J, Popovic A, Jira W. 2008. Polycyclic aromatic hydrocarbons (PAHs) in different types of smoked meat products from Serbia. Meat Sci. 80:449–456. European Commission. 2005. Regulation (EC) No. 208/2005 of 4 February 2005 amending Regulation (EC) No. 466/ 2001 as regards polycyclic aromatic hydrocarbons. Off J Eur Comm. L 34:43–45. European Commission. 2006. Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Comm. L 364:5. European Commission. 2007. Regulation (EC) No. 333/2007 of 28 March 2007 laying down the methods of sampling and analysis for the official control of levels of benzo[a]pyrene in foodstuffs. Off J Eur Comm. L 88:29–38. European Union. 2002. Polycyclic aromatic hydrocarbons – occurrence in foods, dietary exposure and health effects. Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food. Document No. SCF/CS/CNTM/PAH/29 ADD1 Final. Available from: http://europa.eu.int/comm/food/ fs/sc/scf/index_en.html/ Falco´ G, Domingo JL, Llobet JM, Teixido´ A, Casas C, Muller L. 2003. Polycyclic aromatic hydrocarbons in foods: human exposure through the diet in Catalonia, Spain. J Food Protect. 12:2325–2331. Grimmer G, Bohnke H. 1975. Polycyclic aromatic hydrocarbon profile of high protein foods, oil and fats by gas chromatography. J AOAC. 58(4):724–733. Horwitz W, Kamps LR, Boyer KW. 1980. Quality assurance in the analyses of foods for trace constituents. J AOAC. 63(6):1344–1354. Inmetro. 2007. Orientac¸a˜o sobre validac¸a˜o de me´todos de ensaios quı´ micos DOQ-CGCRE-08. Review 2, June. Brası´ lia: Instituto Nacional de Metrologia, Normalizac¸a˜o e Qualidade Industrial. Available from: http://www.inmetro.gov.br/credenciamento/laboratorios/calibEnsaios.asp Instituto Brasileiro de Geografia e Estatı´ stica (IBGE). 2005. Pesquisa de orc¸amentos familiares 2002–2003: Consumo alimentar domiciliar per capita. Rio de Janeiro (RJ): Instituto Brasileiro de Geografia e Estatı´ stica. Kazerouni N, Sinha R, Hsu CH, Greenberg A, Rothman N. 2001. Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study. Food Chem Toxicol. 39:423–436. Lage-Yust MA, Davin˜a JLC. 2005. Supercritical fluid extraction and high performance liquid chromatography fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil. Food Contr. 16:59–64. Lin D, Youying T, Lizhong Z. 2005. Concentrations and health risk of polycyclic aromatic hydrocarbons in tea. Food Chem Toxicol. 43:41–48.
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Lodovici M, Dolara P, Casalini C, Ciappellano S, Testolini G. 1995. Polycyclic aromatic hydrocarbon contamination in the Italian diet. Food Addit Contam A. 12(5):703–713. Moret S, Conte LS. 2000. Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods. J Chromatogr A. 882:245–253. Moret S, Purcaro G, Conte LS. 2005. Polycyclic aromatic hydrocarbons in vegetable oils from canned foods. Eur J Lipid Sci Tech. 107:488–496. Nunes SP. 2007. Produc¸a˜o e consumo de o´leos de soja no Brasil. Departamento de estudos so´cio-econoˆmicos rurais (Deser), n. 159. Available from: http://www.deser.org.br/ documentos/doc/ Pandey MK, Mishra KK, Khanna SK, Das M. 2004. Detection of polycyclic aromatic hydrocarbons in commonly consumed edible oils and their likely intake in the Indian population. J Am Oil Chem Soc. 81(12): 1131–1136. Pupin AM, Toledo MCF. 1996. Benzo(a)pyrene in Brazilian vegetable oils. Food Addit Contam A. 13(6): 639–645. Purcaro G, Navas JA, Guardiola F, Conte LS, Moret S. 2006. Polycyclic aromatic hydrocarbons in frying oils and snacks. J Food Protect. 69(1):199–204. Rey-Salgueiro L, Garcia-Falco´n S, Carballo EM, SimalGa´ndara J. 2008. Effects of toasting procedures on the levels of polycyclic aromatic hydrocarbons in toasted bread. Food Chem. 108:607–615. Teixeira VH, Casal S, Oliveira MBPP. 2007. PAHs content in sunflower, soybean and virgin olive oils: Evaluation in commercial samples and during the refining process. Food Chem. 104(1):106–112. Tfouni SAV, Machado RMD, Camargo MCR, Vitorino SHV, Vicente E, Toledo MCF. 2007. Determination of polycyclic aromatic hydrocarbons in cachac¸a by HPLC with fluorescence detection. Food Chem. 101(1):334–338. Weibhaar R. 2002. Rapid determination of heavy polycyclic aromatic hydrocarbons in edible fats and oils. Eur J Lipid Sci Tech. 104:282–285. World Health Organization (WHO). 2005. Summary and conclusions of the Sixty-fourth Meeting of the Joint FAO/ WHO Expert Committee on Food Additives. Rome (Italy): FAO. p. 47. World Health Organization (WHO). 2008. Proposed draft code of pratice for the reduction of contamination of food with polycyclic aromatic hydrocarbons (PAH) from smoking and direct drying processes. Summary and conclusions of the thirty-first Joint FAO/WHO Food Standards Programme, Codex Committee on Contaminants in foods. Rome (Italy): FAO CX/CF 08/2/9.
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VIEW DATASET Polycyclic aromatic hydrocarbons in Brazilian commercial soybean oils and dietary exposure M.C. Rojo Camargo*, P.R. Antoniolli, E. Vicente and S.A.V. Tfouni Food Science and Quality Center, Institute of Food Technology – ITAL, Av. Brasil, 2880, 13070-178 – Campinas-SP, Brazil (Received 12 October 2010; final version received 28 April 2011) In this study the 13 polycyclic aromatic hydrocarbons (PAHs) identified as being genotoxic and carcinogenic by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) were determined in different brands of soybean oils available on the Brazilian market, totalling 42 samples. A solid-phase extraction (SPE) method for sample clean-up with a C18 cartridge, followed by reversed-phase HPLC with fluorescence detection, was used for determination. The method showed good recoveries for most PAHs studied with values between 74% and 111%. Good intra- and inter-day precisions (0.55RSD511.9) and high correlation coefficients (r240.999) were obtained. The presence of PAHs was detected in all 42 samples with mean summed PAH levels ranging from 10.4 to 112.0 mg kg1. The mean and maximum dietary exposures for total PAHs were estimated as 12.4 and 19.1 ng kg bw1 day1, respectively. Keywords: oils and fats; processed foods; PAH; process contaminants – PAHs
Introduction Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemical compounds that are formed during incomplete combustion of organic matter and have been found as contaminants, predominantly from environmental pollution and food processing, in a variety of foodstuffs, including vegetables, fruit, cereals, oils and fats, smoked fish and meat, coffee and tea (Weibhaar 2002; Camargo and Toledo 2003; Bishnoi et al. 2005; Lin et al. 2005; Moret et al. 2005; Purcaro et al. 2006; Tfouni et al. 2007; Djinovic et al. 2008; Rey-Salgueiro et al. 2008). Ever since PAHs were first recognised as being carcinogenic, the presence of these compounds in food has been a matter of concern worldwide. Studies carried out in different countries have identified oils and fats as important sources of PAHs in the diet (Lodovici et al. 1995; Pupin and Toledo 1996; Moret and Conte 2000; Camargo and Toledo 2002; Falco´ et al. 2003; Barranco et al. 2004; Lage-Yust and Davina˜ 2005; Purcaro et al. 2006). The presence of PAHs in vegetable oils is mainly attributed to direct drying of the oil seeds with combustion gases before oil extraction during processing, resulting in relatively high and variable levels of these contaminants in the final product. In view of the potential health effects, the necessity of establishing maximum limits for PAHs has been recognised by some countries in different opportunities. As consequence, the Codex Committee on Food *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2011.585244 http://www.informaworld.com
Additives and Contaminants (CCFAC) placed these contaminants in the priority list for the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) evaluation. During its 64th meeting the JECFA, based on the documents from European Union Scientific Committee on Food (SCF) and from International Programme on Chemical Safety (IPCS), concluded that 13 PAHs (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ j]fluoranthene, benzo[a]pyrene, dibenzo[a, h]anthracene, dibenzo[a, e]pyrene, dibenzo[a, h]pyrene, dibenzo[a, i]pyrene, dibenzo[a, l]pyrene, indeno[1,2,3-cd]pyrene and 5-methylchrysene) were clearly carcinogenic and genotoxic, even in low exposure doses (European Commission 2005; WHO 2005). Due to the genotoxicity of PAHs, it is not possible to assume a threshold mechanism and consequently a provisional tolerable weekly intake (PTWI) cannot be established (WHO 2008). Thus, in 2006, the CCFAC at its 38th session agreed to elaborate a proposed draft Code of Practice for the reduction of contamination of food with PAHs from smoking and direct drying processes (WHO 2008). Furthermore, the European Commission established maximum levels for benzo[a]pyrene in different food groups. For oils and fats the maximum limit has been restricted to 2.0 mg kg1 (European Commission 2006). In Brazil, the yearly consumption of vegetable oils account for roughly 3.72 million tons, 86% of which correspond to soybean oil (Nunes 2007). A preliminary
Food Additives and Contaminants: Part B study conducted by the present authors pointed relatively high and variable levels of PAHs in the most consumed brands of soybean oils. In this manner, considering the importance of this product in the Brazilian diet and the lack of information about possible sources of contamination, the results showed the necessity of extending the investigation to other brands of soybean oil available on the national market, once the Brazilian regulation for oils and fats does not establish a maximum level for any PAH. Therefore, focusing on all 13 PAHs identified as being carcinogenic and genotoxic by the JECFA, the present study had three purposes: (1) to validate a SPE-HPLC-FLD method for the simultaneous determination of these 13 compounds, (2) to determine the levels of PAHs in different brands of soybean oils commercially available in Brazil, and (3) to estimate the Brazilians dietary exposure to these contaminants through oil consumption.
Materials and methods Standards and reagents PAHs standards were purchased from Supelco Inc. (St. Louis, MO, USA) (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a] pyrene, dibenzo[ah]anthracene and indeno[1,2,3cd]pyrene), Fluka (Munich, Germany) (benzo[ j]fluoranthene, dibenzo[al]pyrene, dibenzo[ae]pyrene and dibenzo[ah]pyrene), Cambridge Isotope Laboratories Inc. (Andover, MA, USA) (5-methylchrysene) and ChemService Inc. (West Chester, PA, USA) (dibenzo[ai]pyrene). Hexane, methanol and N,N-dimethylformamide (HPLC grade) were acquired from Tedia Brazil Ltda (Brazil). Acetonitrile (HPLC grade) was supplied by J.T. Baker (Mexico City, Mexico). Water was purified on a Milli-Q system, Millipore Corp. (Bedford, MA, USA). The SPE cartridges AccuBondII (500 mg, 3 ml) were from Agillent Technologies Inc. (Allentown, PA, USA). Stock standard solutions were prepared by diluting the PAHs standards in acetonitrile.
Samples Soybean oils commercially available in Brazil were purchased at supermarkets in the region of Campinas, SP, between 2008 (11 different brands, three batches each) and 2009 (three different brands, three batches each), totalising 42 samples. All were analysed in duplicate for benzo[a]anthracene (B[a]P), chrysene (Chy), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[ j]fluoranthene (B[ j]F), benzo[a]pyrene (B[a]P), dibenzo[a, h]anthracene (D[ah]A), dibenzo[a, e]pyrene (D[ae]P), dibenzo[a, h]pyrene (D[ah]P), dibenzo[a, i]pyrene (D[ai]P), dibenzo[a, l]pyrene
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(D[al]P), indeno[1,2,3-cd]pyrene (Indeno) and 5methylchrysene (5MeChy).
Extraction and clean-up Soybean oils (0.5 g) were weighed in an erlenmeyer flask and 5 ml of hexane were added. The mixture was transferred into a 60 ml separating funnel and PAHs were extracted twice with 5 ml of N,Ndimethylformamide–water (DMF-H2O) (9:1, v/v) (Grimmer and Bohnke 1975; Barranco et al. 2003). The combined extract was concentrated under a flow of nitrogen until it reached approximately 50% of its initial volume. Then, the resulting solution was diluted with 5 ml of water before solid-phase extraction (SPE) clean-up. The SPE cartridges were prepared by prewashing with 5 ml of methanol, followed by 5 ml of water using a Vacuum Manifold from SupelcoÕ (Bellefonte, PA, USA). Then the sample solution was applied and the column was washed with 10 ml of N,Ndimethylformamide–water (1:1, v/v), followed by 10 ml of water, all eluates being discarded. The cartridges were dried under vacuum for 20 min. PAHs were eluted with 10 ml hexane at a flow rate of 2 ml min1 and the eluate was taken to dryness under a nitrogen stream. The residue was diluted in 0.5 ml acetonitrile, filtered through a 0.45 mm filter (PVDF, Millex-HV, Millipore) into a HPLC vial and capped for the HPLC analysis.
HPLC analysis The analysis was carried out in a Shimadzu LC-20 A Prominence HPLC (Japan) equipped with quaternary pumps (model LC-10AT), on-line degasser, a model SIL-20 A autosampler and a model RF-10AXL fluorescence detector. A C18 Vydac 201 TP column (5 mm, 250 4.6 mm) at 30 C and a mobile phase composed of acetonitrile and water, at a flow rate of 1 ml min1, was used to separate the PAHs. The gradient elution programme started with a linear gradient from 70% to 75% acetonitrile in 20 min, followed by a 15 min linear gradient from 75% to 100% acetonitrile and maintained 100% acetonitrile isocratic until 55 min, when finally returned to the initial conditions and the column was re-equilibrated with the initial mobile phase composition for 15 min. The injection volume was set to 30 ml. The following excitation (ex) and emission (em) wavelength programme was used to determine the PAHs: 0.01 min (268/398 nm) for B[a]A, Chy, 5MeChy; 16.70 min (312/507 nm) for B[ j]; 18.20 min (290/430 nm) for B[b]F, B[k]F, B[a]P, D[al]P, D[ah]A; 32.40 min (300/500 nm) for Indeno; 34.90 min (297/403 nm) for D[ae]P and 45 min (304/ 457 nm) for D[ai]P, D[ah]P. Data were acquired and processed with LC solution software.
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Identification and quantification of PAHs The identification of PAHs was performed by comparison of their retention times with those obtained by injecting standards in the same conditions. Peak identity was also confirmed by spiking the extracts with pure standards (standard addition method). The compounds were quantified using the external standard plot method. A mixed standard stock solution with PAHs was prepared in acetonitrile and stored at 4 C for 3 months. From this solution, seven working ones, ranging from 0.5 to 250 ng ml1, were used to construct linear regression lines (peak area ratios versus PAH concentration).
Validation study The following parameters were used for method validation: accuracy, precision (inter- and intraday repeteability), linearity, specificity, limit of detection (LOD) and limit of quantification (LOQ), according to Institute of Metrology, Standardisation and Industrial Quality (Inmetro) guidelines, under ISO 17025 criteria (Inmetro 2007). The accuracy and repeatability of the method were evaluated by performing recovery tests. Recovery (accuracy) was determined by spiking a blank control sample of soybean oil with the PAHs studied at 0.5, 1.0, 1.5 and 5.0 mg kg1 (n ¼ 3 replicates for each level) and the values were reported as average per cent recovery. Inter- and intraday repeatabilities, expressed as the percentage of relative standard deviation (% RSD), were checked by analysing the same sample control spiked with PAHs standard, in triplicate, during the same day and in 3 different days. In addition, the precision of the chromatographic system was carried out by injecting the same oil sample extract, fortified with a working standard PAHs solution (1.2 mg kg1) five times (n ¼ 5), during three consecutive days (n ¼ 3) and then checking the RSD of retention times and peak areas. Linearity was tested by the square correlation coefficients (r2) of the calibration curves. Method LOD and LOQ were determined using spiked matrices. For this purpose, seven independent soybean oil sample (blank) spiked with PAHs at a level of 0.5 mg kg1 were analysed. LOD and LOQ were calculated as the analyte concentration corresponding to, respectively, mean sample blank þ 3 s and mean sample blank þ 5 s. Finally, specificity was confirmed by analysis of the blank oil sample control, which was produced with soybeans dried naturally in order to avoid any kind of contamination.
Statistical analysis The software Statistica for Windows 5.5 (StatSoft Inc., Tulsa, OK, USA) was used to perform the analysis of variance (ANOVA). PAHs contamination levels in
different periods of time were compared by Tukey test (95% confidence).
Dietary exposure Intakes were calculated using the mean and maximum PAHs levels determined in the samples, considering all brands available on the market, during a year of production. Food consumption data were obtained from a National Household Survey, conducted by the Instituto Brasileiro de Geografia e Estatı´ stica (IBGE; Brazilian Institute of Geography and Statistics 2005) among 48,470 selected households covering the urban and rural areas of all 27 Brazilian states, during 12 months (July 2002 to June 2003). Considering only the south-east of the country, where the region from which samples were collected is located, a daily soybean oil consumption of 24.9 g per person per day was assumed. Individual daily intakes of PAHs (ng kg bw1 day1) were estimated by multiplying the average daily intake (g day1) of the selected food item by the individual PAH concentration (mg kg1) determined in this product. Also, a worst-case scenario was drafted using the maximum PAH concentrations determined in the analysed oils. A consumer average body weight of 60 kg was considered in the calcultations.
Results and discussion Analytical procedure One objective of this study was to optimise and validate a methodology by SPE-HPLC-FLD to determine 13 PAHs in soybean oils with minimum of interferences. For this purpose, different variables involved in the extraction procedure and HPLC analysis were studied. The effectiveness of different types of silica-based C18 cartridges and different amounts of sample was assessed with regard to the quantity of co-extractives remaining after evaporation of the solvent that affected positive or negatively in the recovery. Satisfactory results were obtained using 500 mg of C18 sorbent and 0.5 g of oil sample. Among 500 mg cartridges tested, the best performance was achieved with the higher carbon loading, i.e. using the AccuBondII 500 mg, 3 ml. Once defined the SPE sorbent, the solvents (volume and flow rate) used during conditioning, rinsing and elution steps were evaluated. Best results were obtained using 5 ml of methanol, followed by 5 ml of water for pre-washing the cartridges and 10 ml of N,N-dimethylformamide–water (1:1, v/v), followed by 10 ml of water for washing after the sample was applied. To determine the ideal rinsing conditions, a series of washes varying from 10% to 50% methanol at 10% increments was applied. It was observed that an increase in the
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Figure 1. HPLC chromatogram of soybean oil spiked with a standard mixture of PAHs. Table 1. Analytical parameters of the method used for PAHs analysis in soybean oils.
PAHa B[a]A Chy 5MeCri B[ j]F B[b]F B[k]F B[a]P D[al]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P
LOD (mg kg1)b
LOQ (mg kg1)b
Linear range (mg kg1)
0.03 0.02 0.16 0.39 0.20 0.16 0.16 0.12 0.15 0.54 0.07 0.10 0.76
0.05 0.03 0.27 0.65 0.35 0.25 0.25 0.20 0.24 0.90 0.12 0.25 0.96
0.5–50 0.5–50 0.5–50 1.0–250 0.5–50 0.5–50 0.5–50 0.5–50 0.5–50 1.0–250 0.5–50 0.5–50 0.5–50
r2
Mean recovery RSD (%)c
Interday precision RSD (%)d
0.9994 0.9994 0.9998 0.9999 0.9993 0.9990 0.9998 0.9993 0.9984 0.9997 0.9999 0.9967 0.9993
93 3.7 85 7.4 111 5.6 85 4.4 84 3.5 82 3.0 101 4.5 82 11.9 93 10.7 74 6.5 94 4.9 64 5.0 94 3.8
2.7 2.9 2.9 6.1 4.1 4.9 0.5 1.1 2.2 4.5 3.4 5.8 2.5
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeCri, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b LOD, limit of detection; LOQ, limit of quantification. c n ¼ 9 determinations in the same day relative standard deviation (RSD, %), expressed as a percentage of the recovery for a sample blank spiked at three different concentrations according to PAH. d n ¼ 9 determinations in three different days. r2, regression coefficient.
percentage of methanol provided unsatisfactory results. Differently, the use of dimethylformamide– water (1:1, v/v), according to Barranco et al. (2003), followed by pure water improved the method recovery and reproducibility. Then chromatographic conditions using different mobile phase composition, flow rate, column temperature, gradient elution and wavelength programme were applied. Mobile phase composition and gradient elution were optimised in order to achieve a good separation of the organic compounds in the shortest time analysis. As can be observed in Figure 1, the chromatographic conditions established were satisfactory when separating the 13 PAHs studied. Fluorescence detection was optimised with wavelength
programming for excitation and emission. Based on each PAH fluorescence spectra the most appropriate wavelengths for each group of compounds during the chromatographic run were selected in order to detect all of them and keep the gradient programme applied. After establishing the extraction procedure and cromatographic conditions the validation of the analytical method was certified according to the figures of merit described above. Table 1 summarises the proposed method performance. The average recoveries obtained for the compounds (64–111%) were satisfactory for determinations at the mg kg1 level and were in agreement with the criteria for methods of analysis for benzo(a)pyrene provided by Regulation EC No. 333
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(Horwitz et al. 1980; European Commission 2007). Intra- and interday repeatability of the extraction procedure, expressed as the percentage of relative standard deviation (% RSD), ranged from 3.0% to 11.9% and from 0.5% to 6.1%, respectively. For the chromatographic system, the RSD of the detector response was less than 1.5% (including intra- and interday repeatability) and the RSD of the retention times was lower than 0.09% for each compound. The PAHs responses were linear over the concentration range studied, as showed in Table 1. The LOD and LOQ for the target compounds varied from 0.02 to 0.76 mg kg1 and from 0.03 to 0.96 mg kg1, respectively.
PAHs in Brazilian commercial soybean oils Table 2 presents the mean and maximum levels of PAHs determined in three different batches of eleven brands (A–K) of soybean oils available in the Brazilian market. Variable levels of contamination were found within different brands of oils and within different batches of the same brand. Only B[ j]F was not detected in three samples analysed. The mean levels of individual PAHs ranged from 0.2 to 26.1 mg kg1. Brand K showed the highest PAH contamination (112.0 mg kg1). Brand B also presented high levels of PAHs, with the maximum levels (61.4 mg kg1) almost three times higher than the mean content of summed PAHs (26.9 mg kg1). This should be a motive of concern since brand B is the same one identified as the most contaminated in preliminary studies and, additionally, is the second brand most consumed among Brazilians. Besides, five soybean oils (B, D, I, J and K) presented levels of B[a]P higher than those established by the European Union (2.0 mg kg1) (European Commission 2005). In oil K, the most contaminated one, the mean and maximum B[a]P concentration was almost eight times (15.8 mg kg1) the maximum permitted limit. A comparison with other studies showed that the present levels are higher than those reported by other authors for the same kind of oil, whose values ranged between 0.09 and 6.1 mg kg1 (Pupin and Toledo 1996; Pandey et al. 2004; Teixeira et al. 2007). A year later, the three most consumed brands (A–C) were re-evaluated. Figure 2 compares the results obtained for these selected oils in both years (2008 and 2009). As can be observed, there was a significant variation in summed PAHs levels among different brands and batches, indicating the seasonality of oils contamination by PAHs (Tukey, p50.05). In 2009, most samples evaluated showed higher levels of PAHs than the respective ones in 2008. These variability could be related to different soybean seed
drying processes used prior to oil extraction (Camargo and Toledo 1998; WHO 2008).
PAHs dietary intake through soybean oil consumption in the region of Campinas, SP, Brazil Table 3 shows the estimated PAHs exposure through soybean oil consumption. As can be observed, the dietary intake of a single PAH ranged between 0.2 and 2.8 ng kg bw1 day1, while the mean exposure calculated for the sum of all of them was 12.4 ng kg bw1 day1. When the worst case scenario was considered, the total daily intake raised to 19.1 ng kg bw1 day1. For comparison purposes, with regard to B[a]P intakes, the mean and maximum estimated values were 1.2 and 1.7 ng kg bw1 day1, respectively, which can be considered relatively higher than the daily intake from total diets described by two US (0.8– 2.3 ng kg bw1 day1) (Kazerouni et al. 2001) and six European Union (0.8–4.8 ng kg bw1 day1) (European Union 2002) surveys, once in this study only one region and only one product were taken into account. When the margin of exposure (MOE) was considered for risk assessment purposes, values of 83.333 and 58.823 were obtained for mean and high level intakes, respectively. These values were calculated using a BMDL10 of 0.1 mg B[a]P kg bw1 day1 established by the JECFA (WHO 2005). According to the Committee, MOEs of 25,000 and 10,000 for mean and high level intakes, respectively, have been suggested to be of low concern for human health. Although the soybean oil represents a small contribution of the average diet, vegetable oils generally show higher levels of PAHs than other kind of foods. Consequently, in the Brazilian diet, along with many countries, despite differences in dietary pattern, the oils are expected to be the major contributors to PAHs intake (Dennis et al. 1983; De Vos et al. 1990; Kazerouni et al. 2001; Camargo and Toledo 2002; European Union 2002). In this manner and considering that PAHs are both carcinogenic and genotoxic, their levels in edible oils, likewise their intake, should be reduced to as low as possible in order to reduce consumer exposure to these compounds.
Conclusion Various sources are responsible for the presence of PAHs in vegetable oils resulting in a great variability of the contamination levels. Although the use of active carbon in the refining process is highly efficient to reduce the PAHs in oils, especially the ‘heavy fraction’ (four to five rings), oil refineries in Brazil do not use this adsorbent. Thus, it is recommended that a rigid
4.0 (9.3) 4.4 (11.0) 1.3 (2.5) 5LQ (4.5) 2.1 (8.5) 0.8 (2.7) 1.4 (5.5) 5LQ (0.3) 1.8 (4.1) 5LQ (5.2) 0.6 (1.3) 0.8 (2.6) 5LQ 17.2 (57.5)
A
4.2 (10.6) 5.9 (10.6) 1.7 (1.0) 1.7 (3.5) 2.3 (6.0) 1.1 (2.6) 2.1 (7.0) 0.2 (0.4) 3.4 (8.5) 3.1 (6.4) 0.5 (1.0) 0.7 (3.8) 5LQ 26.9 (61.4)
B 1.7 (4.9) 2.5 (4.4) 1.3 (5.5) 5LQ 0.6 (1.5) 0.4 (0.6) 0.5 (1.0) 5LQ (0.9) 0.9 (1.2) 2.0 (2.4) 0.5 (1.2) 5LQ (0.7) 5LQ 10.4 (24.3)
C 7.1 (13.0) 7.9 (11.3) 2.0 (2.4) 5LQ 2.6 (3.5) 0.8 (1.2) 2.2 (2.4) 0.6 (0.2) 1.2 (1.4) 2.2 (2.3) 0.7 (0.8) 1.0 (0.7) 1.7 (4.8) 30.0 (44.0)
D 1.2 (2.4) 2.0 (3.0) 0.7 (1.2) n.d. 1.3 (1.6) 0.5 (0.5) 1.0 (1.3) 0.7 (1.9) 1.6 (2.1) 2.0 (2.4) 0.5 (0.6) 0.3 (0.4) 5LQ 11.8 (17.4)
E 3.7 (4.6) 4.0 (4.7) 1.2 (2.1) n.d. 2.8 (3.7) 0.6 (0.8) 0.7 (0.8) 0.3 (0.7) 1.4 (2.3) 2.5 (3.9) 0.7 (0.9) 0.5 (0.9) 5LQ 18.4 (25.4)
F 3.0 (5.9) 3.6 (5.2) 1.2 (2.3) n.d. 1.6 (2.3) 0.6 (0.7) 0.9 (1.4) 0.2 (0.2) 1.1 (1.2) 5LQ (2.2) 0.7 (0.8) 0.3 (0.4) 1.4 (2.5) 14.6 (25.1)
G 5.2 (7.9) 5.0 (7.0) 1.0 (1.5) 5LQ 2.9 (5.2) 0.7 (1.1) 1.2 (1.8) 5LQ (0.2) 0.9 (1.1) 2.0 (2.8) 0.4 (0.5) 0.3 (0.3) 5LQ 19.6 (29.4)
H
5.7 (12.6) 8.2 (10.4) 0.9 (1.1) 2.6 (3.8) 4.5 (6.0) 1.9 (2.6) 4.8 (5.0) 5LQ (0.3) 4.4 (5.6) 5.5 (9.4) 1.0 (1.6) 5LQ (0.3) 5LQ 39.5 (58.7)
I 4.2 3.8 0.7 1.7 2.6 1.0 2.5 0.4 4.0 3.9 0.6 0.4 5LQ 25.8
(4.5) (4.3) (0.7) (1.9) (2.7) (1.3) (3.0) (1.0) (5.8) (6.1) (0.7) (0.9) (1.1) (34.0)
J 13.6 26.1 1.1 10.3 11.1 6.5 15.8 0.6 22.9 5LQ 1.2 1.2 1.6 112.0
(14.3) (27.3) (1.1) (11.8) (11.2) (6.8) (16.2) (0.8) (26.2) (2.1) (1.2) (1.2) (2.1) (122.3)
K
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeCri, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b Mean of three samples (in duplicate). n.d., not detected; 5LOQ, lower than the limit of quantification given in Table 1.
B[a]A Cri 5MeCri B[ j]F B[b]F B[k]F B[a]P D[al ]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P HPAs
PAHa
Soybean oils, mean (maximum) levels (mg kg1)b
Table 2. Mean and maximum PAHs levels in commercial soybean oils.
Food Additives and Contaminants: Part B 157
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Figure 2. Mean summed PAHs levels in commercial soybean oils (brands A–C) during 2008 and 2009.
Acknowledgements Table 3. PAHs dietary intake from soybean oils in Campinas, SP, Brazil.
Financial support from FAPESP (Proc. 05/59974-8) and scholarship from CNPq are gratefully acknowledged.
Intake (ng kg bw1 day1) PAHa B[a]A Chy 5MeChy B[ j]F B[b]F B[k]F B[a]P D[al ]P D[ah]A Indeno D[ae]P D[ai]P D[ah]P HPAs
Means
Maximumsb
2.0 2.8 0.5 0.6 1.3 0.6 1.2 0.1 1.7 0.9 0.3 0.2 0.2 12.4
3.4 3.7 0.8 1.1 2.0 0.8 1.7 0.2 2.2 1.7 0.4 0.5 0.6 19.1
Notes: aB[a]A, benzo[a]anthracene; Chy, chrysene; 5MeChy, 5-methylchrysene; B[ j]F, benzo[ j]fluoranthene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P, benzo[a]pyrene; D[al]P, dibenzo[al]pyrene; D[ah]A, dibenzo[ah]anthracene; Indeno, indeno[1,2,3-cd]pyrene; D[ae]P, dibenzo[ae]pyrene; D[ai]P, dibenzo[ai]pyrene; D[ah]P, dibenzo[ah]pyrene. b Worst-case scenario: ingestion of foods with the maximum PAH concentration and taking values5LOD to be equal to the LOD.
monitoring programme for PAHs in soybean oil be initiated by the refining industries and a maximum or guideline level for B[a]P in vegetable oils established in the national standards. Additionally, a more comprehensive study should be adressed in order to evaluate the possible influence of each step of the refining process on the decrease of PAH contamination.
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