Food Control 28 (2012) 1e6

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Food Control
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Determination of aatoxins and ochratoxin A in retail cereal products from Turkey by high performance liquid chromatography with uorescence detection
Bulent Kabak*
Hitit University, Department of Food Engineering, Faculty of Engineering, Cevre Yolu, 19030 Corum, Turkey

a r t i c l e i n f o
Article history: Received 22 December 2011 Received in revised form 15 April 2012 Accepted 28 April 2012 Keywords: Aatoxins Ochratoxin A Retail cereal products Occurrence Exposure

a b s t r a c t
In this study, a total of 110 retail cereal products from Turkey were analysed for aatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA). The mycotoxins were determined by liquidesolid extraction, immunoafnity column clean-up and high performance liquid chromatography with uorescence detection (HPLC-FD). Recoveries (83.9e92%) and both intra-day and inter-day repeatability (RSD < 12) of the method, meet the performance criteria set by EC regulation No. 401/2006 for the determination of the levels of mycotoxins in foodstuffs. OTA was the most prevalent toxin, with an incidence of 43.6% (range 0.066e1.125 mg kg1), but at levels below the European legislation limit of 3 mg kg1. Aatoxins were found in 27 of 110 analysed samples: twenty-seven samples with AFB1, fourteen samples with AFB2, seven samples with AFG1 and two samples with AFG2. The ranges for positive samples were 0.052 e0.233 mg AFB1 kg1, 0.022e0.044 mg AFB2 kg1, 0.053e0.149 mg AFG1 kg1 and 0.033e0.037 mg AFG2 kg1. The co-occurrence of AFB1 and OTA was observed in 14.6% of the samples. This is the rst study concerning the simultaneous occurrence of aatoxins and OTA in retail cereal products from Turkey. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Mycotoxins are toxic secondary fungal metabolites produced by many phytopathogenic and food spoilage fungi including Aspergillus, Penicillium and Fusarium species, which can cause a variety of adverse effects in humans, from allergic responses to cancer and death. While over 400 mycotoxins have been identied, a few of them have received increased attention and subjected to regulatory controls under national or European legislation on human foodstuffs and animal feeds. Among these common mycotoxins, aatoxins (AFs) are the most toxic and the strongest natural carcinogens that are produced primarily by two species of Aspergilli, Aspergillus avus and Aspergillus parasiticus. The AFs consist of about 20 similar compounds belonging to a group called the difuranocoumarins, but only four (AFB1, AFB2, AFG1 and AFG2) are naturally found in foodstuffs. AFB1 is the most toxic and amongst the most commonly found in foods (Sweeney & Dobson, 1998).

Abbreviations: AFB1, Aatoxin B1; AFB2, Aatoxin B2; AFG1, Aatoxin G1; AFG2, Aatoxin G2; ALARA, As Low As Reasonably Achievable; HPLC, High performance liquid chromatography; IAC, Immunoafnity column; LOD, Limit of detection; LOQ, Limit of quantication; OTA, Ochratoxin A; RSD, Relative standard deviation; TDI, Tolerable daily intake. * Tel.: 90 364 227 45 33; fax: 90 364 227 45 35. E-mail address: bulentkabak@hitit.edu.tr. 0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.04.043

Besides AFs, ochratoxin A (OTA) has received more attention within the scientic community, due to the hazard it poses to human health. OTA is produced mainly by Aspergillus ochraceus, Aspergillus carbonarius and Penicillium verrucosum (EFSA, 2006). It is a well-known nephrotoxic agent and has been associated with fatal human kidney disease, referred to as Balkan Endemic Nephropathy (BEN) and with an increased incidence of tumours of the upper urinary effect (JECFA, 2001). The International Agency for Research on Cancer (IARC) has dened AFB1 and naturally occurring mixtures of AFs as a human carcinogen (group 1), whilst OTA has been classied as possibly carcinogen (group 2B), based on inadequate evidence for carcinogenicity in humans and sufcient evidence in animals (IARC, 1993, pp. 489e521). Both groups of mycotoxins can be found in a variety of food commodities, including cereals, spices and dried fruits (gs, raisins etc.). Moreover, AFs have also been detected in oilseeds, peanuts and tree nuts (pistachios, almonds, pecans, walnuts etc.) (EFSA, 2004) and OTA has been found as a contaminant in pulses, wine, grape juice, beer, coffee beans, cocoa and a wide range of cereals including wheat, barley, oat, rye, maize, millet and rice. In the European diet, cereals such as barley, wheat, maize, oats and their by-products are considered the major source of OTA intake, corresponding to 50%, followed by wine (approximately 13%), coffee (approx. 12%), and spices (8%), others (6%), beer (5%), cocoa (4%), dried fruits (3%) and meat (1%) (European Commission, 2002a).

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These ve mycotoxins are heat-resistant within the conventional food processing temperatures so little or no reduction in overall toxin levels occurs as a result of normal cooking conditions (Kabak, 2009a). For example, roasting green coffee beans at 200  C for 10e20 min resulted in only a 0e12% reduction in OTA levels (Tsubouchi, Yamamato, Hisada, Sakabe & Udagawa, 1987). For this reason, both groups of mycotoxins can be found in processed foods and enter the human food chain through cereal-derived foods. The aim of this work was to evaluate presence of AFs and OTA in cereal-based foods commercialised in Turkey. A method based on immunoafnity column (IAC) clean-up followed by high performance liquid chromatography coupled with uorescence detection (HPLC-FD) was used to determine AFs and OTA in retail cereal products. 2. Materials and methods 2.1. Samples During the months of January and February 2011, a total of 110 cereal-based food samples from 16 most popular commercial brands were randomly purchased from different supermarkets located in Corum, Turkey. Cereal-based foods were categorised as biscuits (n 45), cookies (n 11), crackers (n 26), breadsticks (n 12), cereal bars (n 6) and wafers (n 10). The size of the packs was from 40 to 450 g. The minimum weight of the sample amount was 200 g. All of the samples were ground with Waring blender (Waring products Co., Connecticut, USA) to produce a homogeneous particle size and stored in a plastic container in a refrigerator until analysis. All samples were analysed within the shelf life of the product. 2.2. Chemicals and reagents Acetonitrile, methanol (both of HPLC grade), sodium chloride, potassium chloride and sodium hydroxide, were purchased from VWR (Leuven, Belgium). Monobasic potassium phosphate and sodium phosphate dibasic were supplied by SigmaeAldrich (St. Louis, MO, USA), while acetic acid, nitric acid and potassium bromide were from Merck (Darmstadt, Germany). Phosphatebuffered saline (PBS) was prepared in the laboratory by dissolving 0.2 g KCl, 0.2 g KH2PO4, 1.16 g anhydrous Na2HPO4 and 8 g NaCl in 1000 ml water and its pH was adjusted to 7.4 with 0.1 N NaOH. The immunoafnity columns (AaTest and OchraTest) were purchased from Vicam (Waterworn, MA, USA). Water, for the HPLC mobile phase and all analytical steps was produced in a Direct-Q 3 water purication system (Millipore, Molsheim, France). The mixed standards of AFB1, AFB2, AFG1 and AFG2 were provided from Supelco (Bellefonte, PA, USA) (Aatoxin Mix kit, catalogue no. 46304-U). The mixture in each ampul consists of 1 mg AFB1, 0.3 mg AFB2, 1 mg AFG1 and 0.3 mg AFG2 in one ml of methanol. The OTA standard in crystalline form was purchased from SigmaeAldrich (St. Louis, MO, USA). 2.3. Preparation of standard solutions Stock solution of aatoxin standard mix was diluted with methanol to obtain concentrations of 0.1 mg ml1 for AFB1 and AFG1, and 0.03 mg ml1 for AFB2 and AFG2. From this intermediate solution, a series of working standards from 0.5 to 20 ng ml1 for AFB1 and AFG1, and from 0.15 to 6 for AFB2 and AFG2 in LC mobile phase consisting of watereacetonitrileemethanol (6:2:3, v/v/v) were prepared. Stock standard solution of OTA (approximately 500 mg ml1) was prepared by solving 1 mg pure crystalline OTA in 2 ml tolueneeacetic acid (99:1, v/v). A series of working standards

from 0.5 to 25 ng OTA ml1 was prepared in LC mobile phase consisting of acetonitrileewatereacetic acid (47:51:2, v/v/v). The working standards of AFs and OTA were renewed every two weeks. They were used to calibrate the LC detector response and recovery studies. 2.4. Extraction and clean-up with IACs The method used in the extraction of AFs from retail cereal products and IAC clean-up was according to the modied procedure of AOAC Ofcial Method 999.07 (Stroka, Anklam, Jorissen, & Gilbert, 2000). Fifty grams of ground cereal-based food samples were extracted with 100 ml methanolewater (8:2, v/v) using a Waring Blender (Waring Products Co., Connecticut, USA) at high speed for 1 min. The extract was ltered through Whatman No. 4 lter paper. A 10 ml aliquot of the ltrate was diluted with 40 ml PBS, shaken vigorously and then ltered once more through a glass microber lter (Whatman GF/A, 125 mm, England). The nal ltrate was passed through an AaTest IAC attached onto a vacuum manifold (Agilent Technologies, Santa Clara, CA, USA). The column was washed with 20 ml (2 10 ml) water and then dried with air. AFs were eluted by passing twice 0.8 ml of methanol through the column at a ow rate of 2e3 ml min1 and collected in HPLC vials (SupelcoTM, Bellefonte, PA, USA). The eluate was then evaporated to dryness at 45  C under N2 stream and the residue was re-dissolved with 1 ml of LC mobile phase solution. The method used for OTA extraction from cereal-based foods was based on AOAC Ofcial method 2000.03 (Entwisle, Williams, Mann, Slack, & Gilbert, 2000). A portion of 50 g of grinded cerealbased foods was extracted with 100 ml of acetonitrileewater (6:4, v/v) by a Waring blender at high speed for 1 min. The extract was ltered through lter paper, and then the ltrate was collected. Ten millilitres of ltrate was mixed with 40 ml PBS and again ltered through a glass microber lter (Whatman GF/A, 125 mm, England). The homogenised solution was applied to an OchraTest column. The IAC was washed twice with 10 ml of water and then dried with air. OTA was eluted two times with 0.8 ml of methanol. The combined elutes were evaporated to dryness at 45  C under N2 stream and the residue was re-dissolved in 1 ml of LC mobile phase. 2.5. HPLC equipment and chromatographic conditions The HPLC system consisted of a LC-20AD pump, a DGU-20A3 online degasser, a SIL-20AHT autosampler and a uorescence detector model RF-20AXL (Shimadzu, Tokyo, Japan). Data acquisition and handling were done by a system control CBM-20Alite with Shimadzu software LC solution. Chromatographic separations were performed on a reversed phase intersil ODS-3 analytical column (250 4.6 mm, 5 mm, GL Sciences Inc, Tokyo, Japan). The column temperatures were maintained at 35  C for AFs and 40  C for OTA. For both HPLC analyses, the injection volume into HPLC system for both standard and sample was 100 ml. For AFs analysis the mobile phase was the mixed solution of watereacetonitrileemethanol (6:2:3, v/v/v) containing 0.12 g l1 potassium bromide and 350 ml l1 nitric acid (4 M), and the ow rate was 1 ml min1. To enhance the uorescent responses of AFB1 and AFG1, an online and postcolumn derivatisation was carried out with bromine generated in a Cobra cell with the electrochemical reaction current of 100 mA (Coring System Diagnostics GmbH, Gernsheim, Germany). The postcolumn reaction coil consists of a 340 mm 0.5 mm i.d. PTFE tubing. The uorescence detector was set to an excitation and emission wavelengths of 360 and 440 nm, respectively. The run time for one cycle was 20 min, and the retention times of AFB1, AFB2, AFG1 and AFG2 under these conditions were approximately, 15.2, 12.6, 11.2 and 9.5 min, respectively.

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For OTA analysis, the mobile phase was acetonitrileewatereacetic acid (47:51:2, v/v/v) at 1 ml min1 and the uorescence detector was set at excitation wavelength of 333 nm and emission wavelength of 460 nm. The run time for one cycle was 20 min, and the retention time of OTA was 16.3 min. 2.6. Analytical quality parameters and validation procedures The following performance characteristics were evaluated to ensure the method quality: linearity, accuracy, precision, limits of detection (LOD) and quantication (LOQ). Linearity of the method was estimated by injecting triplicate mycotoxins standard solutions at concentrations of 0.5, 1, 2, 5, 7.5, 10 and 20 ng ml1 for AFB1 and AFG1, 0.15, 0.3, 0.6, 1.5, 2.25, 3 and 6 ng ml1 for AFB2 and AFG2, and 0.5, 1, 2, 5, 10, 20 and 25 ng ml1 for OTA. The linearity was evaluated by linear regression analysis using the least squares method. For recovery experiment non-infected cereal-based food samples were spiked at three concentration levels (1, 2 and 5 mg kg1 for AFB1 and AFG1, 0.3, 0.6 and 1.5 mg kg1 for AFB2 and AFG2, and 0.75, 1.5 and 3 mg kg1 for OTA). Spiking was carried out in six replicates. Both the spiked and unspiked samples were extracted, cleaned-up and subjected to the HPLC analysis by the same operator according to the previously described. The precision of global method was calculated in terms of intra-day and inter-day repeatability with relative standard deviation (RSD) by sixreplicated analysis of the samples. For the determination of intraday repeatability, six-replicated samples spiked with AFs and OTA at three concentration levels were analysed on the same day, while the inter-day repeatability was estimated repeating the analyses on ve consecutive days. The LODs and LOQs of the AFs and OTA were estimated for a signal-to-noise ratio of 3 and 10, respectively. 3. Results and discussion 3.1. Analytical method performance The range of linearity for each mycotoxin, linear regression equation, the determination coefcient (R2), and LOD and LOQ for each compound are shown in Table 1. The calibration curves were linear with determination coefcients of 0.9998, 0.9996, 0.9997, 0.9994 and 0.9998 for AFB1, AFB2, AFG1, AFG2 and OTA, respectively. Based on the signal-to-noise ratio of 3 and 10, the LODs and LOQs of analytical method were ranged from 0.021 to 0.055 mg kg1 and 0.070 to 0.183 mg kg1 for studied mycotoxins, respectively. These values were very close to those obtained previously for AFs and OTA using HPLC-FD method (Fu, Huang, & Min, 2008; Ibez-Vea, Martnez, Gonzlez-Peas, Lizarraga, & Lpez de Cerain, 2011). The results of recovery test and precision (intra-day and inter-day repeatability, as relative standard deviation, RSD) of the analytical method are summarised in Table 2. The mean recoveries for AFB1,
Table 1 Linearity, correlation coefcients, calibration curve, LOD and LOQ for AFs and OTA by HPLC method with uorescence detection. R2 Compound Linearity range (mg l1) AFB1 AFB2 AFG1 AFG2 OTA 0.5e20 0.15e6 0.5e20 0.15e6 0.5e25 0.9998 0.9996 0.9997 0.9994 0.9998 LOQb (mg kg1) Linear regression LODa (mg kg1) equation y y y y y 332.4x 687.3x 286.5x 312.8x 538.5x 35.7 47.1 53.4 17.3 10.4 0.034 0.021 0.046 0.029 0.055 0.113 0.070 0.153 0.096 0.183

Table 2 The recoveries and precision (intra-day and inter-day repeatability) of the cerealbased food by HPLC-FD method. Compound Spiked concentration (mg kg1) (n 6) 1 2 5 0.3 0.6 1.5 1 2 5 0.3 0.6 1.5 0.75 1.5 3 Mean recovery (%) 89.9 90.4 90.7 86.1 87.9 88.2 88.6 90.3 89.5 85.0 83.9 86.3 90.7 91.3 92.0 Intra-day repeatabilitya RSD (%) 5.8 4.8 3.8 6.2 8.6 5.8 4.4 3.6 4.3 5.9 5.6 5.7 6.6 6.3 4.7 Inter-day repeatabilityb RSD (%) 8.6 8.3 9.7 7.5 11.8 7.2 7.8 6.2 6.6 8.7 9.8 8.2 11.5 10.4 7.7

AFB1

AFB2

AFG1

AFG2

OTA

a Intra-day repeatability was estimated by three concentration level of each toxin on the b Inter-day repeatability was estimated by three concentration level of each toxin on the

analysis of six replicate samples at same day. analysis of six replicate samples at consecutive days.

R2: Determination coefcient. a LOD, limit of detection of the chromatographic method (S/N 3). b LOQ, limit of quantication of the chromatographic method (S/N 10).

AFB2, AFG1 and AFG2 were in the range of 89.9e90.7%, 86.1e88.2%, 88.6e90.3%, and 83.9e86.3%, respectively. Recovery averages for OTA in cereal-based foods were 90.7% at 0.75 mg kg1, 91.3% at 1.5 mg kg1 and 92% at 3 mg kg1. All recovery values, ranging between 83.9 and 92%, are in agreement with the requirements of the Commission Regulation (EC) No. 401/2006 (European Commission, 2006b) for these compounds that states that recovery rate of 70e110% at the concentration range of 1e10 mg kg1 and 50e120% at the concentration below 1 mg kg1 is acceptable for both AFB1 (and sum of AFB1 AFB2 AFG1 AFG2) and OTA. The results of the present study are in agreement with those of previous studies on other food materials. Stroka et al. (2000) conducted a collaborative study for determination of aatoxins with HPLC-FD using postcolumn bromination in peanut butter, pistachio paste, g paste, and paprika powder. Recoveries were ranged from 71 to 92% at spiking levels of 2.4 and 9.6 mg kg1 for total aatoxins and 82e109% at spiking levels of 1 and 4 mg kg1 for AFB1. Similarly, mean recoveries of AFs were in the range of 83.6e89.9%, 80.5e88.5%, and 85.7e89.6% for peanut, pistachio and walnut samples spiked with 5 mg kg1, respectively (Juan, Zinedine, Molt, Idrissi, & Maes, 2008). In another study, Fu et al. (2008) determined AFs in peanut and maize samples with HPLCeUV system without derivatisation of AFs. The recoveries of AFs were found in the range of 83.4e94.7% at spiking levels of 0.22e5 mg kg1. With regard to OTA, Park, Chung, and Kim (2005) found that the average recoveries of OTA obtained from IAC and HPLC-FD were more than 80% for polished rice, beer, barley and wheat our. Hernndez Hierro, GarciaVillanova, Torrero, and Fonseca (2008)reported recoveries of 61.4e99.7% and 63.9e75.7% for AFs and OTA in paprika, respectively, at two spiking levels of 1 and 5 mg kg1 with HPLC-FD technique. More recently, Campone, Piccinelli, Celano, and Rastrelli (2011) determined AFB1, AFB2, AFG1 and AFG2 in cereals with liquideliquid microextraction, IAC, HPLC-FD and reported recoveries of 67e92% for AFs (spiking levels of 0.2, 0.5, 2 and 5 mg kg1 for AFB1 and AFG1, and 0.05, 0.13, 0.5 and 1.3 mg kg1 for AFB2 and AFG2). With regard to precision of the method, the repeatability obtained for intra-day and inter-day variations ranged from 3.6 to 8.6% and from 6.2 to 11.8%, respectively, for AFs at three concentration levels. The intra-day and inter-day repeatability for OTA was found in the range of 4.7e6.6% and 7.7e11.5%, respectively. In light of this, both intra-day and inter-day repeatability of the employed

4 Table 3 Occurrence of AFs and OTA in retail cereal products from Turkey. Mycotoxin Positive n (%) Frequency distribution (mg kg1)

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Contamination (mg kg1) Averagec 0.083 0.029 0.093 0.035 0.286

<LODa LOD e 0.5 0.5e1 >1 Rangeb AFB1 AFB2 AFG1 AFG2 OTA 27 14 7 2 48 (24.5) (12.7) (6.4) (1.8) (43.6) 83 96 103 108 62 27 14 7 2 38 e e e e 9 e e e e 1 0.052e0.233 0.022e0.044 0.053e0.149 0.033e0.037 0.066e1.125

a Limit of detection (0.034 mg kg1 for AFB1, 0.021 mg kg1 for AFB2, 0.046 mg kg1 for AFG1, 0.029 mg kg1 for AFG2 and 0.055 mg kg1 for OTA). b Minemax. c Mean of positive samples.

method provided RSD values lower than 12%, showing good precision for ve compounds at three concentration levels. 3.2. Analysis of real samples The liquidesolid extraction, IAC clean-up followed by HPLCeFD determination was applied to assess the incidence and concentration of AFs and OTA in a total of 110 retail cereal products. The results of the analysis are presented in Table 3. The most prevalent mycotoxin was OTA, which was present in 43.6% (48/110) of tested samples, with an average contamination of 0.286 mg kg1 (range of 0.066e1.125 mg kg1). However, none of the 48 samples reached the maximum tolerable limit of 3 mg kg1 set by EU regulations for all products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption (European Commission, 2006a). It has been found that OTA contamination was 51.1% in biscuits, 45.5% in cookies, 50% crackers, 25% breadsticks, 16.7% in cereal bars and 30% in wafers (Table 4). The highest concentration (1.125 mg kg1) was detected in a sample of biscuit containing wheat our, bran and oat. This is the rst survey on the AFs and OTA contents of retail cereal products, so that comparison of results is not possible. However, The Rapid Alert System for Food and Feed (RASFF) which is a network established on the basis of Regulation (EC) No 178/2002 by the EU Member States (European Commission, 2002b), reported that honey cookies originating from Ukraine were found to be contaminated with OTA at levels ranging from 5 to 10.2 mg kg1 in 2007. Similarly, in 2009 a notication on OTA in vanilla avoured biscuits (contaminated with 6.4 mg OTA kg1) from
Table 4 Occurrence of AFs and OTA in retail cereal products, based on type of product. Type of product Biscuits (n 45) Parameter % Positive samples Mean of positive samples Rangea (mg kg1) % Positive samples Mean of positive samples Range (mg kg1) % Positive samples Mean of positive samples Range (mg kg1) % Positive samples Mean of positive samples Range (mg kg1) % Positive samples Mean of positive samples Range (mg kg1) % Positive samples Mean of positive samples Range (mg kg1) (mg kg1) AFB1

Moldova has been noted and rejected at the external borders of EU (http://webgate.ec.europa.eu/rasff-window/portal/). In this study, it was clear that contamination of retail cereal products by OTA is mostly due to wheat-based cereals but also to those containing maize and oats. Natural occurrence of OTA in cereals and their derived products is well reported in scientic literature. Cereals based foods are the major contributors to human exposure to OTA, accounting for 50% to the mean European dietary intake (European Commission, 2002a). In the United States of America, Trucksess, Giler, Young, White, and Page (1999) found OTA levels higher than 0.03 mg kg1 in 56 out of 383 wheat samples.Juan, Molt, Lino, and Maes (2008) detected OTA ranging from 0.2 to 27.1 mg kg1 in 18 out of 83 cereal samples (22%) in Spain and Portugal, while OTA was present in 7 out of 48 cereal samples in Jordan, with ranging from 2.04 to 5.86 mg kg1 (Salem & Ahmad, 2010). In France, 45 breakfast cereal samples were analysed, and 31 of them contained OTA in the range of 0.2e8.8 mg kg1 (Molini, Faucet, Castegnaro, & Pfohl-Leszkowicz, 2005). More recently, Kabak (2009b) analysed 24 breakfast cereal samples from Turkey, and the occurrence of OTA was found in 38% of breakfast cereals at levels ranging from 0.172 to 1.84 mg kg1. AFs were detected in 27 samples (24.5%) at concentrations between 0.052 and 0.459 mg kg1, with a mean level of 0.124 mg kg1. AFB1 and AFB2 have been detected more frequently than AFG1 and AFG2 in cereal-based food samples. This might be due to the invasion of cereals or other susceptible ingredients used in the manufacturing of cereal-based foods by A. avus rather than A. parasiticus. It should be noted that AFB1 was detected in all 27 aatoxin-positive samples in the range of 0.052 and 0.233 mg kg1, with a mean value of 0.083 mg kg1. Besides AFB1, AFB2 was simultaneously detected in 14 samples (12.7%), always with levels below the LOQ (0.070 mg kg1). The AFB2 contamination ranged from 0.022 to 0.044 mg kg1 (mean 0.029 mg kg1). Seven samples (6.4%) were also positive for AFG1 at levels between 0.053 and 0.149 mg kg1 (mean 0.093 mg kg1), while only 2 samples (1.8%) were contaminated with AFG2, ranging from 0.033 to 0.037 mg kg1 (mean 0.035 mg kg1). It is interesting to note that two samples contained higher concentrations of AFG1 (0.148 and 0.127 mg kg1) than AFB1 (0.138 and 0.118 mg kg1). According to these results, none of the samples analysed exceeded maximum level of 2 mg kg1 for AFB1 and 4 mg kg1 for sum of AFB1, AFB2, AFG1 and AFG2 established by EU regulations for all cereals and products derived from cereals, including processed cereal products.

AFB2 17.8 0.028 0.022e0.044 27.3 0.026 0.025e0.027 3.9 0.032 0.032 e <LOD <LOD e <LOD <LOD 20 0.033 0.023e0.042

AFG1 11.1 0.089 0.053e0.149 e <LODb <LOD 3.9 0.055 0.055 e <LOD <LOD e <LOD <LOD 10 0.148 0.148

AFG2 2.2 0.033 0.033 e <LOD <LOD e <LOD <LOD e <LOD <LOD e <LOD <LOD 10 0.037 0.037

OTA 51.1 0.288 0.066e1.125 45.5 0.181 0.100e0.306 50 0.326 0.080e0.871 25 0.460 0.087e0.741 16.7 0.071 0.071 30 0.162 0.096e0.156

Cookies (n 11)

(mg kg1)

Crackers (n 26)

(mg kg1)

Breadsticks (n 12)

(mg kg1)

Cereal bars (n 6)

(mg kg1)

Wafers (n 10)

(mg kg1)

31.1 0.090 0.053e0.233 36.4 0.074 0.052e0.085 15.4 0.066 0.052e0.094 8.3 0.063 0.063 e <LOD <LOD 40 0.087 0.063e0.138

a b

Minemax. Limit of detection (0.034 mg kg1 for AFB1, 0.021 mg kg1 for AFB2, 0.046 mg kg1 for AFG1, 0.029 mg kg1 for AFG2 and 0.055 mg kg1 for OTA).

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The incidence of AFB1 was 31.1% in biscuits, 36.4% in cookies, 15.4% in crackers, 8.3% in breadsticks and 40% in wafers, while all cereal bars analysed were free of aatoxin contamination (Table 4). The highest AFs level was found in a sample of biscuit containing wheat our and maize starch (0.233 and 0.459 mg kg1 for AFB1 and total AFs, respectively) followed by a sample of wafer containing maize starch, hazelnut cream (68%) and hazelnut puree (10%) (0.138 and 0.365 mg kg1 for AFB1 and total AFs, respectively). According to present study, AFs were mainly found in maize-based cereal products. This result conrms the preliminary observation by Tam et al. (2006), who reported higher coincidence and levels in maize-based breakfast cereals than in wheat-based samples. More recently, Ibez-Vea et al. (2011) reported that AFB1 appeared only in the maize-based breakfast cereals, whereas zearalenone and OTA showed the highest contamination rates in the samples containing wheat and wheat and rice, respectively. In 2009, the Rapid Alert System for Food and Feed (RASFF) received a total of 669 notications on mycotoxins, of which as many as 638 concerned AFs. There were 13 notications on AFs in cereal and bakery products of which 8 in rice, 4 in maize meal/our and 1 in cookies. According to the RASFF notication, the contamination levels in chocochip almond and hazelnut cookies from France were 13 and 44 mg kg1 for AFB1 and sum of AFB1, AFB2, AFG1 and AFG2, respectively (http://webgate.ec.europa.eu/rasff-window/ portal/). Another parameter to take into account is the other ingredients such as chocolate, cocoa and tree nuts (hazelnuts, almonds, walnuts etc.) used as avouring agents in the manufacture of cereal-based foods. Tree nuts are susceptible to aatoxin contamination. Thus, the retail cereal products with tree nuts would be more prone to have AFs. In this survey, AFs occurred most frequently in cerealbased foods containing hazelnuts (51.9% of positive samples). Turkish people consume high amounts of nuts directly or as an ingredient in prepared foods such as special sweets, cookies/ biscuits. Turkey, the worlds second largest tree nut producer, leads the world in output of hazelnuts and also produces signicant quantities of pistachios, walnuts, almonds and other tree nuts (Johnson, 1997). In 2009, RASFF reported a total of 518 notications on AFs in nuts, nut products and seeds of which 111 notications concern products originating from Turkey. The notications on AFs in the category nuts, nuts products and seeds from Turkey relate to mainly hazelnuts (61 notications) and pistachios (35 notications) (http://ec.europa.eu/food/food/rapidalert/docs/report2009_ en.pdf). Co-occurrence of AFB1 and OTA was detected in 14.6% (16 samples) of the cereal-based food samples, while simultaneous contamination with ve mycotoxins (AFB1, AFB2, AFG1, AFG2 and OTA) was found in only two of 110 samples analysed. The cooccurrence of mycotoxins in cereal and cereal-derived products has been reported previously (Ibez-Vea et al., 2011). It is known that co-occurrence of mycotoxins, in general, lead to combined additive or synergistic effects. This may be of concern for organ toxicity and also carcinogenesis, especially considering AFB1 and OTA, respectively, are classied in group 1 and 2B by IARC (SangareTigori et al., 2006). Based on the data obtained in the present study, it has been also developed a theoretical exposure scenario for cereal-based foods both for adults and children. It has been estimated that the average annual per capita consumption of biscuits and wafers varies from 0.5 kg in Ireland to 18.7 kg in the Netherlands with an EU mean of 8.0 kg, while their consumption is about 4 kg in Turkey (http:// www.argemar.com/biskuvi.htm). This is equivalent to an average daily per capita consumption of 0.011 kg. There is no indication of upper intake amounts in the Argemar information, but referring to the Concise European Food Consumption Database at the 95th

Table 5 Estimated daily intakes of AFB1 and OTA from consumption of contaminated retail cereal products. Mean toxin concentrationa (mg kg1) Dietary exposure ng kg1 b.w. day1 60 kg adults Mean 0.033 AFB1 0.176 OTA
a b c b

20 kg child
c

95th percentile 0.012 0.065

Meanb 0.018 0.097

95th percentilec 0.036 0.194

0.006 0.032

Samples below the LOD were taken as LOD/2. Mean daily consumption of 0.011 kg. 95th percentile (heavy consumers) daily consumption of 0.022 kg.

percentile level (high consumers) have a consumption close to double that of average consumers or, if applying this proportion to the Argemar information, an estimated daily consumption of 0.022 kg. The estimated dietary intakes of AFB1 and OTA from the consumption of retail cereal-based products by the average and 95th percentile (heavy consumers) consumers are shown in Table 5. Calculations are presented for adults at 60 kg b.w. and children at 20 kg b.w. The estimated mean AFB1 intake from cereal-based foods is 0.006 ng kg1 b.w. day1 for adults and 0.018 ng kg1 b.w. day1 for children. The 95th percentile exposure is 0.012 ng kg1 b.w. day1 for adults and 0.036 ng kg1 b.w. day1 for children. AFs are considered to be genotoxic carcinogens. In the risk management of genotoxic carcinogens, no threshold is presumed and the FAO/WHO Joint Expert Committee on Food Additives (JECFA) and European Commissions Scientic Committee on Food (SCF) recommended that AFs concentrations in food should be reduced to As Low As Reasonably Achievable (ALARA), because it is not possible to identify an intake without risk. International expert committees did not specify a numerical tolerable daily intake (TDI) for AFs and concluded that even very low levels of exposure to AFs, i.e. 1 ng kg1 b.w. day1 still contribute to the risk of liver cancer (EFSA, 2007). The mean OTA intake of the Turkish population is 0.032 ng kg1 b.w. day1 for adults and 0.097 ng kg1 b.w. day1 for children. The 95th percentile exposure is 0.065 ng kg1 b.w. day1 (equivalent TDI of 0.46e1.3%, JECFA-SCF) in adults and 0.194 ng kg1 b.w. day1 (equivalent TDI of 1.39e3.88%, JECFA-SCF) in children. The present values are largely below the TDI established by JECFA (14 ng kg1 b.w. day1) and SCF (5 ng kg1 b.w. day1). For this reason, the estimated exposure to OTA from retail cereal products does not represent a series health risk both for adults and children in Turkey. 4. Conclusions The method validation parameters such as linearity, accuracy, precision, LOD and LOQ for all of the 5 toxins were within the acceptable range to assess the occurrence of mycotoxins in cereal-based foods. The method was applied to 110 retail cereal products from Turkey. This is the rst report on AFs and OTA presence in retail cereal products (biscuits, cookies, wafer etc.) from Turkey. OTA was the most prevalent mycotoxin in cerealbased foods, even if contamination levels do not seem to be a serious health hazard according to current scientic ndings. Similarly, mean contamination level of AFs in samples should be classied as a no serious human health hazard. On the other hand, surveillance must be continuous and widespread, since the quality of the raw cereals and other ingredients such as hazelnuts, pistachios, almonds etc. of retail cereal products may differ from year to year.

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Conict of interest The author has declared no conict of interest. Acknowledgements The author is grateful to the Scientic and Technological Research Council of Turkey (Project no: TUBITAK-TOVAG 108O502) for nancial support. References
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