Investigation of ochratoxin A in Syrian consumed baby foods

Food Control 62 (2016) 97e103

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Investigation of ochratoxin A in Syrian consumed baby foods Eman Darouj a, *, Laila Massouh a, Iyad Ghanem b a b

Faculty of Pharmacy, Damascus University, Damascus, Syria Department of Molecular Biology and Biotechnology, Atomic Energy Commisison of Syria, P.O. Box 6091 Damascus, Syria

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 June 2015 Received in revised form 10 October 2015 Accepted 17 October 2015 Available online 21 October 2015

An investigation on the occurrence of ochratoxin A (OTA) in baby food products consumed in Syria was conducted. A total of 42 samples of baby food formulae, including 30 cereal-based baby food and 12 fruitbased baby food formulae were analyzed using immunoaffinity columns (IAC) for sample clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). According to the results in the present study, the mean recoveries of OTA from spiked cereal-based baby food and fruitbased baby food samples were in the range of 92.85e107.4% and 83.3e87.86%, respectively. While the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.038 and 0.11 mg/kg for cerealbased baby foods and 0.05 and 0.17 mg/kg for fruit-based baby foods, respectively. Analytical results showed that 40.48% of the analyzed samples were found contaminated with OTA. Among the OTA contaminated samples, 43.33% samples of cereal-based baby food, and 33.33% samples of fruit-based baby food contained mean values of 0.094 and 0.093 mg/kg of OTA, respectively. Our findings show that contamination levels of OTA in all samples were lower than the permitted level set by European Commission Regulation. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Ochratoxin A Baby foods Immunoaffinity columns HPLC-FLD

1. Introduction Ochratoxin A (OTA) is a member of the mycotoxin secondary metabolites which was originally isolated and characterized by van der Merwe (1965) from maize based products contaminated with Aspergillus ochraceus (Van der Merwe, Steyn, & Fourie, 1965). But subsequent studies showed that the most relevant OTA-producing fungi species are Penicillium species, principally P. verrucosum, and several related Aspergillus species, especially A. ochraceus, Aspergillus carbonarius, and Aspergillus niger. The source of ochratoxin A in cool temperate regions is P. verrucosum and in the tropics and in subtropics is A. ochraceus and A. carbonarius (EFSA, 2004; JECFA, 2001). OTA has been a significant public health concern due to its range of toxicity as shown by results from a number of studies which suggested that OTA may be immunotoxic, genotoxic, carcinogenic and nephrotoxic in humans. It is suspected of being the main etiological agent responsible for human Balkan Endemic Nephropathy (BEN) and associated urinary tract tumours (Abid et al.,

* Corresponding author. E-mail addresses: [email protected] (E. Darouj), [email protected] (L. Massouh), [email protected] (I. Ghanem). http://dx.doi.org/10.1016/j.foodcont.2015.10.018 0956-7135/© 2015 Elsevier Ltd. All rights reserved.

€ nmez-Altuntas¸, Hamurcu, 2003; Castegnaro et al., 2006; Do lez-Arias Imamoǧlu, & Liman, 2003; Ehrlich et al., 2002; Gonza et al., 2014; Hassen, Abid, Achour, Creppy, & Bacha, 2004; Liu et al., 2012). Based on the available data, the International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B) (IARC, 1993). OTA is one of the most prevalent human contaminants in a wide variety of foodstuffs, OTA can also be found in processed products because of its resistance to technological processes (Bullerman & Bianchini, 2007), such as cereals (millet, maize, rice, peanuts, wheat, barley, and sorghum) (Sangare-Tigori et al., 2006; Soleimany, Jinap, Faridah, & Khatib, 2012), breakfast and baby s, Gonza lez-Pen  as, & Lo  pez de Cerain, food cereals, and beer (Aragua 2005), and grape derived products, dried fruits, and fruit juices (AlHazmi, 2010; Bircan, 2009; Rosa, Magnoli, Fraga, Dalcero, & Santana, 2004). To protect public health from this health risk, many countries have established the maximum permissible limit for OTA in the most frequently contaminated foods. According to the European Commission (Commission regulation EC No.1881/2006) and amending Regulation (Commission regulation EU No.105/2010) and (Commission regulation EU No.594/2012) the maximum limits of OTA in unprocessed cereals (5 mg/kg), all products derived from unprocessed cereals, including processed cereal products and

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cereals intended for direct human consumption (3 mg/kg), processed cereal-based foods and baby foods for infants and young children (0.5 mg/kg), dried vine fruits (10 mg/kg), wine and grape juice (2 mg/kg), roasted coffee beans and ground roasted coffee (5 mg/kg), soluble coffee (10 mg/kg), spices (15 mg/kg), liquorice root (20 mg/kg), and liquorice extract (80 mg/kg). The European Union (EU) has not set a maximum allowable limit for OTA in beer; the appropriate level proposed for beer was 0.2 mg/l (Mateo, Medina, nez, 2007). Mateo, Mateo, & Jime Since there are no specific limits for OTA in such foods in Syria, the European Commission Regulation (Commission regulation EC No.1881/2006) was considered as the guide in this study. Based on toxicological and intake data the European Food Safety Authority (EFSA) has established the Provisional Tolerable Weekly Intake (PTWI) of 120 ng OTA/kg body weight (EFSA, 2006), whereas The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established the (PTWI) of OTA at 100 ng OTA/kg body weight (JECFA, 2008), which corresponds to a Provisional Tolerable Daily Intake (PTDI) of approximately 17 and 14 ng/kg bw/day, respectively. Several analytical methods are available for determination of OTA in various matrices, such as thin-layer chromatography (TLC) (Braicu, Puia, Bodoki, & Socaciu, 2008), enzyme-linked immunosorbent assay (ELISA) (Zhang et al., 2011), high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (Solfrizzo, Panzarini, & Visconti, 2008), or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) (Meng et al., 2014), and gas chromatography with mass spectrometry (Soleas, Yan, & Goldberg, 2001). Fluorescence-based methods are the most sensitive of methods due to the fact that OTA has a natural fluorescence (Hashemi & Alizadeh, 2009), so the technique most often used in the determination of OTA in food is HPLC-FLD. Generally, Clean-up procedures using immunoaffinity columns (IAC) have several advantages over other clean-up methods due to their higher selectivity and specificity, and lower detection limits  & Rauov (Belajova a, 2010; Scott & Trucksess, 1997). Special attention should be paid to infant and children in regard to safety of their food, because they are considered to be more susceptible to adverse effects of toxins than adults, due to their lower body weight, higher metabolic rate and lower ability to detoxify (Sherif, Salama, & Abdel-wahhab, 2009), addition they have a more limited diet and consume a higher proportion of their body weight than adults. While many studies have been carried out around the world in regard to the presence of OTA in baby food, there is no data on the occurrence of OTA in baby food consumed in Syria, so the main purpose of this study was the determination of OTA levels in cerealbased baby food and fruit-based baby food products consumed in Syria using high performance liquid chromatography with fluorescence detection (HPLC-FD), which is validated and sensitive enough to comply with the low maximum levels required by the European Legislation.

baby food products, respectively. Cereal-based baby foods consist of the following major ingredients, formulated individually or as a mixture: cereals (wheat, rice, barley, corn, and/or oat), skimmed milk or milk powder, sugar, vegetable oil, lecithin of soy, lysine, vitamins, minerals, fruits (natural or dried apple, apple juice, apricot, grapevine, orange, banana, and/or pineapple), baking agents, probiotic culture, malt extract, eggs, and/or flavorings. Fruit purees consist of the following major fruits, two or more: (peach, apple, banana, pear, and/or pineapple) with vitamins. Fruit juice consists of water, fruit juice (banana, orange juice, pineapple juice, pear, and apple juice), sugar and vitamins. All information about samples composition was obtained from their labels. Samples were classified into different groups, based on their label information, with regard to the components (cereal-based baby foods and fruit-based baby foods), and the type of product (local brands or imported brands).

2. Materials and methods

Vortex mixer (IKA-Werke, Germany), centrifuge (Jouan CR422, ST-Herblain, France), homogenizer standard unit, homogenizer Ultra-Turrax (T25basic, IKA-Werke, Germany), and turbo evaporator system (Caliper, USA) (water bath at 45
C under gentle nitrogen stream, nitrogen gas was of high purity 99.999%) were used during the extraction steps.

2.1. Samples A total of 42 samples of different local and imported brands of baby food products were purchased from supermarkets and pharmacies in Damascus (Syria). The collected samples included: 30 cereal-based baby foods and 12 fruit-based baby food products (9 purees and 3 juices) for infants and young children. The samples were stored in their original containers in the fridge at 4
C until analysis. The sample quantities ranged from 190 to 400 g for cerealbased baby foods and 130 g, 125 ml for purees and juice fruit-based

2.2. Chemicals and reagents OTA was purchased from Sigma-Aldrich (Steinheim, Germany) as a crystalline powder. All solvents used such as acetonitrile, acetic acid, and methanol were HPLC grade from Merck (Darmstadt, Germany). Highly purified water was generated by EASYpure Ro UV Ultra-Pure Water System from Barnsted (BI Barnsted, Dubuque, USA), and was used in the preparation of all aqueous solutions and HPLC mobile phase. All chromatographic solvents and water were filtered through a membrane filter (PTFE, 0.2 mm Ø 47 mm) (Whatman, Germany) under vacuum. Tween 20 and polyethylene glycol (PEG) 8000 were purchased from AppliChem (Darmstadt, Germany), hydrochloric acid (37%), sodium hydroxide, potassium chloride, sodium chloride, anhydrous disodium hydrogen-phosphate and ammonium acetate were obtained from Merck (Darmstadt, Germany), and potassium dihydrogen-phosphate, sodium hydrogen carbonate were pur€n (Seelze, Germany). chased from Riedel-de Hae Phosphate-buffered saline (PBS) was prepared by dissolving (0.2 g) potassium chloride KCl, (0.2 g) potassium dihydrogen phosphate KH2PO4, (1.16 g) anhydrous disodium hydrogen phosphate Na2HPO4, and (8.00 g) sodium chloride NaCl in 900 ml of ultrapure water. The pH of the PBS buffer was adjusted to 7.4 with 0.1 M HCl or 0.1 M NaOH as appropriate, and the final volume was made up to 1 L with ultrapure water. Ammonium acetate solution, 0.2 M was prepared by dissolving 15.416 g ammonium acetate in 1000 ml water. For clean-up step immunoaffinity columns (IAC) OchraStar™ COIAC2000 (Romer Labs, Tulln, Austria) were used. Immunoaffinity columns were kept under refrigeration at 2e8
C and were brought to room temperature before analysis. 2.3. Equipments

2.4. HPLC apparatus and chromatographic conditions Chromatographic analyses were performed using an HPLC instrument, Jasco LC-NetІІ/ADC HPLC system (Jasco, Tokyo, Japan). The system consisted of a quaternary gradient inert pump equipped

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with a degasser (Jasco PU-2089i plus) connected to an autosampler (Jasco AS-2050 plus), column thermostat (Jasco CO-2060 plus), and a Shimadzu fluorescence detector (RF-10A XL) (Shimadzu, Kyoto, Japan) was connected to the HPLC. The analytical column was an Agilent Eclipse XDB- C18 (3.5 mm, 150  4.6 mm i.d.) (Agilent, USA). The instrument was controlled by Chrom NAV software (Jasco, Tokyo, Japan). A mobile phase was used in isocratic conditions: 50% of solution A (acetonitrile) and 50% of solution B (2% acetic acid in water) and the flow rate was set at 0.6 ml/min, running time was for 15 min. The column temperature was kept at 42
C. The injection volume was 50 ml for both standard solution and sample extracts. The fluorescence detector was operated at an excitation and emission wavelengths of 247 and 480 nm, respectively. 2.5. Preparation of standard solutions A stock solution of OTA (1 mg/ml) was prepared by dissolving 1 mg of pure crystalline OTA in 1 ml of HPLC grade methanol. The intermediate solution (10 mg/ml) and the working solution (100 ng/ ml) were prepared by appropriate dilution of the stock solution in HPLC grade methanol. All standard solutions were prepared in amber vials (Agilent, USA), stored at 8
C in the dark, and maintained at room temperature and in darkness before each use. 2.6. Sample extraction and immunoaffinity clean-up Samples were extracted and cleaned using OchraStar™ immunoaffinity columns according to the manufacturer's instructions with some modifications. Modifications were introduced in order to decrease the quantitation limit of the method. For extraction of cereal-based baby foods, 25 g of ground sample was added to 100 ml of acetonitrile-water (60:40, v/v), and the mixture was blended with a standard unit homogenizer (IKAWerke, Germany) at 2400 rpm for 4 min. The mixture was centrifuged at 3500 rpm for 15 min at 20
C. The supernatant was filtered through Whatman No. 4 filter paper (Whatman, Germany). An aliquot of the filtrate (8 ml ¼ 2 g sample) was transferred to a tube and carefully evaporated to near dryness using a turbo evaporator system (water bath at 45 C under gentle nitrogen stream) and then it was diluted with 10 ml PBS solution. For extraction of fruit-based baby food products, 10 g of sample and 10 ml of extraction solution (5% NaHCO3 with 1% PEG 8000) were mixed using an Ultra-Turrax homogenizer (T 25 basic, IKAWerke, Germany) at 2400 rpm for 4 min, followed by centrifugation at 3500 rpm for 15 min at 20
C. The supernatant was then filtered through Whatman No. 4 filter paper. An aliquot of 5 ml (5 ml ¼ 2.5 g sample) of this filtrate was then diluted with 20 ml PBS solution. For clean up step pH of the diluted extracts was adjusted to 7.4 using either 0.1 M NaOH or 0.1 N HCl solutions depending on the initial pH of each extract. Clean up was performed using immunoaffinity columns (IAC) OchraStar™ fitted on a vacuum manifold (Millipore Corporation, Bedford, MA, USA). Extracts were passed through immunoaffinity columns which were then washed with 20 ml (2  10 ml) of 0.2 M ammonium acetate solution, (For the cereal-based baby foods an additional rinse was added with 25 ml of 0.025% Tween-20 in PBS followed by 5 ml of 0.2 M ammonium acetate solution), any remaining liquid was removed from the column by applying slight vacuum at the end of the washing step. OTA was then eluted by passing 3 ml (3  1 ml) of methanol/acetic acid (98:2, v/v) through the column. To ensure complete elution of the bound toxin from the antibody, the solvent was maintained in contact with the column for a few seconds before starting the elution. The flow rate was 1e3 ml/min for all application procedure.

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The eluate was then evaporated to dryness using a turbo evaporator system (water bath at 45 C under gentle nitrogen stream) and the residue was redissolved in 600 ml of methanol and filtered through a 0.45-mm PTFE syringe filter (Pall, Germany) into amber vials (Agilent, USA) before the injection (50 ml) into the HPLC system for analysis. Two injections were performed for each sample.

2.7. Method validation Validation of the method for ochratoxin A analysis was performed on two different matrices and was based on the following criteria: selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), and recovery. Linearity of the method was estimated from the calibration curves generated by analysis of five standard solutions (0.1, 0.2, 0.5, 1and 5 ng/ml). Standards were prepared by appropriate dilution of known volumes of working solution (100 ng/ml) with methanol. Each calibration curve was constructed by plotting OTA peak areas versus the corresponding concentrations of standard solutions. Calibration curves were generated for each sequence of analysis. Selectivity of the method was evaluated by analyzing extracts of blank and spiked samples (cereal-based baby foods and fruit-based baby foods) at OTA levels close to the regulated level fixed by EU (0.5 mg/kg for OTA) (Commission regulation EC No.1881/2006). Sensitivity of the method was characterized by its limit of detection (LOD) and limit of quantitation (LOQ) values. For their determination, calibration curves were prepared using blank sample extracts (matrix-matched sample). After extraction matrix extracts were evaporated to dryness under gentle nitrogen stream at 40 C, residues were then redissolved in methanolic OTA standard solutions at the levels (0.05, 0.1, 0.2, 0.5, 1, 2 and 3 ng/ml). standard curves for the two matrices were generated. . The LOD and LOQ were calculated using the following equations:

LOD ¼ 3:3  s= S LOQ ¼ 10  s= S Where s ¼ the residual standard deviation of the response S ¼ the slope of the calibration curve Recoveries of the extraction method were determined from spiked blank samples of each matrix, for this purpose, OTA was spiked at three levels of 0.2, 0.5 and 3 mg/kg into cereal-based baby food blank samples and of 0.5 and 1 mg/kg into fruit-based baby food blank samples by adding adequate volumes of working standard solutions to obtain the desired concentrations. Spiked samples were stored overnight in the fridge at 4
C. Spiking was carried out in triplicates on three different days for each level; extraction and analysis of the spiked samples were carried out using the previously indicated procedure. Quantitation of OTA concentration in each sample was performed by measuring its peak area relying on the calibration curve of the standard solutions. Calculation of the equivalent concentrations of OTA in mg/kg was achieved using the following expression:

Csampleðmg=kgÞ ¼ ðC analyte  V1  V2Þ=V3  sample weight Where: Canalyte (ng/ml) ¼ concentration of analyte determined from the standard calibration curve, V1 (ml) ¼ final volume after reconstituting the evaporated sample, V2 (ml) ¼ total volume of

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extraction solution, V3 (ml) ¼ equivalent of undiluted extract volume taken for cleanup, and sample weight (g) ¼ weight of sample taken for analysis. For the cereal-based baby foods

Csample ðmg=kgÞ ¼ ðCanalyte  0:6  100Þ=8  25 ¼ C analyte  0:3 For the fruit-based baby food products

Csample ðmg=kgÞ ¼ ðC analyte  0:6  20Þ=5  10 ¼ C analyte  0:24 Results were not corrected for recovery. 2.8. Statistical analysis Statistical analysis was performed using SPSS 20 software. The study has taken into account all OTA levels between the LOD and LOQ. For samples having no detectable levels of ochratoxin A, half of LOD value was assigned. Significant differences between sample groups were evaluated by nonparametric ManneWhitney U test for any two independent sample groups, while the nonparametric KruskaleWallis test was used for k independent samples. A probability value of 0.05 was used to determinate the statistical significance. 3. Results and discussion 3.1. Method validation The retention time obtained in this study (RT ca. 8.7 min) was shorter than that obtained in other studies (longer than 10 min) (Biffi et al., 2004; Ozden, Akdeniz, & Alpertunga, 2012; Zinedine et al., 2010), but was similar to that revealed by (Iqbal, Rabbani, Asi, & Jinap, 2014). The results have shown good linear response, with correlation coefficients (R2) higher than 0.999 in all experiments that were performed on OTA standard solutions and on matrix-matched standards for determination of LOD and LOQ. The method was found to be highly specific with no interferences from matrix components were observed at the same retention time of OTA peak. Fig. 1 shows the chromatographic profile of OTA standard solution, Figs. 2 and 3 show the chromatographic profiles of OTA blank and spiked samples (cerealbased baby food and fruit-based baby food). The limit of detection (LOD) and limit of quantitation (LOQ) of

the method were 0.038 and 0.11 mg/kg for cereal-based baby foods and 0.05and 0.17 mg/kg for fruit-based baby foods, respectively. The LOD and LOQ for cereal-based baby foods are similar to those of Kabak (2009), and for the fruit-based baby foods they were similar ~ es (2012). to that revealed by Rubert, Soler, and Man Table 1 presents the mean recoveries with standard deviation (SD) and relative standard deviation (RSD) for the two kinds of samples at different spiking levels. The mean recoveries of OTA were in the range of 92.85e107.4% (RSD ¼ 1.86e3.91%) and 83.3e87.86% (RSD ¼ 1.81e3.44%) for cereal-based baby food and fruit-based baby food samples, respectively. It was a good recovery values, corresponding to the levels established by legislation of European Commission Directive for OTA determination methods (Commission regulation EC No.401/2006). In the EU legislation, it is stated that, at <1 mg/kg, recoveries are acceptable in the range 50e120%. The recoveries in this study were higher than those indicated by Beretta et al. (2002) s et al. (2005), but were similar to those indicated by and Aragua Coronel, Marín, Cano-Sancho, Ramos, and Sanchis (2012) for cerealbased baby foods.

3.2. OTA in baby food products Results of occurrence of OTA in analyzed samples are presented in Tables 2 and 4. In the present study, the concentrations were considered positive if they were in samples extracts higher than LOD. As shown, OTA was present in 17 samples out of 42 (40.48%) total analyzed samples. Results obtained in the present study showed also that OTA was detected in 13 out of 30 (43.33%) cerealbased baby food samples, and in 4 out of the 12 (33.33%) samples of fruit-based baby food at levels ranging from 0.02 to 0.329 and 0.019e0.156 mg/kg with mean value of 0.094 and 0.093 mg/kg, respectively. Several studies have measure OTA in baby food. In Spain, s et al. (2005) found levels of OTA in 14 out of 20 (70%) Aragua cereal-based baby food samples at a mean concentration of n et al. (2011) 0.187 mg/kg with levels below 0.740 mg/kg; Beltra analyzed 14 Spanish baby food samples, two samples (14.28%) were OTA positive and contained lower than maximum permitted levels (0.5 mg/kg). In a later study Rubert et al. (2012) showed that 2 out of 35 (5.71%) baby food samples were contaminated with OTA at a range of (0.35e0.5 mg/kg). In Turkey Kabak (2009) observed that 4 out of 24 (17%) cereal-based baby foods contained OTA ranging from 0.122 to 0.374 mg/kg, with an overall mean of 0.221 mg/ kg; more recently, Ozden et al. (2012) detected OTA in 4 out of the 21 (19.05%) analyzed cereal-based baby foods at a range of

Fig. 1. Chromatogram of 0.5 mg/kg OTA standard solution.

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Fig. 2. Chromatograms of cereal-based baby food samples: blank sample (d), spiked sample with 0.5 mg/kg OTA (d).

Fig. 3. Chromatograms of fruit-based baby food samples: blank sample (d), spiked sample with 0.5 mg/kg OTA (d).

Table 1 Recovery data for ochratoxin A of three replicates for two kinds of baby food samples at various spiking levels. Spiked sample

Spiking level (ng/g)

Recovery (%mean ± SD)

Cereal-based baby foods

0.2 0.5 3 0.5 1

92.85 107.4 104.11 83.3 87.86

Fruit-based baby foods

± ± ± ± ±

RSD (%)

1.73 4.2 2.17 1.51 3.02

1.86 3.91 2.08 1.81 3.44

SD, Standard deviation. RSD, Relative standard deviation.

Table 2 Occurrence of OTA in the cereal-based baby food samples based on information provided in their label. Cereal-based baby foods

No. sample

Positive (%)

OTA concentration (mg/kg) Mean ± SD of positive sample

Range of positive sample

Mean ± SD of total sample

Median of total sample

Fruit-free cereals Cereals with fruits Total

21 9 30

8 (38.09%) 5 (55.55%) 13 (43.33%)

0.065 ± 0.06 0.142 ± 0.135 0.094 ± 0.099

0.02e0.201 0.026e0.329 0.02e0.329

0.036 ± 0.042 0.087 ± 0.115 0.052 ± 0.074

0.019 0.026 0.019

ManneWhitney test (U ¼ 68.500; Significance ¼ 0.244).

Table 3 Occurrence of OTA in the cereal-based baby food samples based on the type of brand product. Cereal-based baby foods

Local brand Imported brand

No. sample

15 15

Positive (%)

10 (66.67%) 3 (20%)

ManneWhitney test (U ¼ 57.500; Significance ¼ 0.021).

OTA concentration (mg/kg) Mean ± SD of positive sample

Range of positive sample

Mean ± SD of total sample

Median of total sample

0.109 ± 0.109 0.047 ± 0.007

0.02e0.329 0.04e0.055

0.079 ± 0.098 0.025 ± 0.012

0.026 0.019

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Table 4 Occurrence of OTA in the fruit-based baby food samples. Fruit-based baby foods

Puree (peach& apple) Puree (banana& apple) Puree (fruit cocktail) Fruit juice Total

No. sample

3 3 3 3 12

Positive (%)

2 (66.67%)
OTA concentration (mg/kg) Mean ± SD of positive sample

Range of positive sample

Mean ± SD of total sample

0.099 ± 0.029
0.079e0.12
0.075 0.025 0.067 0.025 0.048

± ± ± ± ±

0.048 0.000 0.077 0.000 0.046

Median of total sample 0.079 0.025 0.025 0.025 0.025

KruskaleWallis test (Chi-Square ¼ 2.469; Significance ¼ 0.481).

0.08e0.20 mg/kg with a mean value of 0.14 mg/kg. In Russia Aksenov, Eller, and Tutel'ian (2006) demonstrated that OTA occurred in 9 out of 40 (22.5%) baby food samples with a mean concentration of 0.31 mg/kg. ~ es, and Ritieni (2014) reported that 15 In Italy, Juan, Raiola, Man out of 25 (60%) cereal-based baby food samples contained OTA at levels ranging from 0.05 to 0.120 mg/kg. Our study is the first report on the presence of OTA in baby foods commercialized in Syria. In this study none of the baby food samples were above the EU maximum limit of OTA in baby foods for infant and young children (0.5 mg/kg). The results were in agreement with the above cited literature on OTA occurrence with the s et al. (2005) study which reported levels of exception of Aragua OTA higher than the EU limits in 2 out of 20 cereal-based baby food samples. In Morocco, Zinedine et al. (2010), analyzed cereal-based baby foods and found that all samples (20 samples) were free of OTA contamination. Also, OTA was not detected in any baby food samples analyzed (10 samples) by Wu, Tan, Wang, and Xu (2012) in China. Statistical study of the samples analyzed in the present study showed no significant differences between OTA levels found in the two types of baby food samples, i.e. cereal and fruit based baby foods (U of ManneWhitney ¼ 119.500; p ¼ 0.092). The cereal-based baby food samples were classified according to the information provided on the label of each sample into two groups: fruit-free cereal and cereal with fruit (see Table 2). Statistical analysis showed no significant differences between those two groups of samples (U of ManneWhitney ¼ 68.500; p ¼ 0.244). Nevertheless, a higher occurrence for OTA was observed in cereal with fruit samples (38.09 and 55.55% for fruit-free cereal, and cereal with fruit samples, respectively), with a higher mean values and maximum level of OTA contamination. Regarding fruit-free cereal group of the samples, the highest contamination rates of OTA were found in the samples containing several cereals, which consisted, mainly, of wheat, rice, barley, and corn with skimmed milk. Also, cereal samples were classified according to the type of brand into two groups, namely local brand and imported brand (see Table 3). The Mann Whitney U test has shown significant differences between the two groups (U ¼ 57.500; P value ¼ 0.021). A higher levels and occurrence of OTA were observed in local brand samples. These differences could be attributed to the origin of raw materials used in preparing the local brand of cereals, the climatic conditions and agricultural and storage practices, which can affect the content of OTA. The KruskaleWallis test showed that the level of contamination of OTA in different types of fruit based baby food was not statistically different (p ¼ 0.481), OTA appeared only in four puree samples (peach and apple, fruit cocktail Puree samples) (66.67%) (See Table 4), with the maximum level of contamination appearing in peach and apple samples. It is worth mentioning that all fruit based baby foods surveyed

in this investigation were of imported brands. Occurrence of ochratoxin A in cereal and fruit based baby foods reported in the present study is probably related to the fact that cereals and fruits are prone to OTA contamination as was reported in several research studies (Al-Hazmi, 2010; Akdeniz Ozden, & Alpertunga, 2013; Zaied et al., 2009). 4. Conclusions The occurrence of OTA has been determined in 13 (43.33%) cereal-based baby foods and 4 (33.33%) fruit based baby foods consumed in Syria. None of the products tested contained OTA at levels above the recommended level by EU for safe consumption. To our knowledge, this is the first report on OTA contamination in baby food products from Syria. Therefore, it is of at most importance to continue monitoring for OTA contamination in baby food in Syria especially that the country has not established its own maximum permissible limit of OTA in foods. References Abid, S., Hassen, W., Achour, A., Skhiri, H., Maaroufi, K., Ellouz, F., et al. (2003). Ochratoxin A and human chronic nephropathy in Tunisia: is the situation endemic? Human and Experimental Toxicology, 22, 77e84. Akdeniz, A. S., Ozden, S., & Alpertunga, B. (2013). Ochratoxin A in dried grapes and grape-derived products in Turkey. Food Additives Contaminants: Part B Surveillance, 37e41. http://dx.doi.org/10.1080/19393210.2013.814719. Aksenov, I. V., Eller, K. I., & Tutel'ian, V. A. (2006). Ochratoxin A content in baby food. Voprosy Pitaniia, 75(5), 66e69. Al-Hazmi, N. A. (2010). Determination of patulin and ochratoxin A using HPLC in apple juice samples in Saudi Arabia. Saudi Journal of Biological Sciences, 17(4), 353e359. s, C., Gonza lez-Pen  as, E., & Lo pez de Cerain, A. (2005). Study on ochratoxin A Aragua in cereal-derived products from Spain. Food Chemistry, 92, 459e464. , E., & Rauova , D. (2010). Comparison of two clean up techniques in isolation Belajova of ochratoxin A from red wine. Czech Journal of Food Sciences, 28(3), 233e241.  Yus n, E., Ib ~ ez, M., Sancho, J. V., Corte s, M.A., ndez, F. (2011). Beltra an a, V., & Herna UHPLC e MS/MS highly sensitive determination of aflatoxins, the aflatoxin metabolite M1 and ochratoxin A in baby food and milk. Food Chemistry, 126, 737e744. Beretta, B., De Domenico, R., Gaiaschi, A., Ballabio, C., Galli, C. L., Gigliotti, C., et al. (2002). Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation. Food Additives and Contaminants, 19(1), 70e75. Biffi, R., Munari, M., Dioguardi, L., Ballabio, C., Cattaneo, A., Galli, C. L., et al. (2004). Ochratoxin A in conventional and organic cereal derivatives: a survey of the Italian market, 2001e02. Food Additives and Contaminants, 21(6), 586e591. Bircan, C. (2009). Incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs. Food and Chemical Toxicology, 47(8), 1996e2001. Braicu, C., Puia, C., Bodoki, E., & Socaciu, C. (2008). Screening and quantification of aflatoxins and ochratoxin a in different cereals cultivated in Romania using thin-layer chromatography-densitometry. Journal of Food Quality, 31, 108e120. Bullerman, L. B., & Bianchini, A. (2007). Stability of mycotoxins during food processing. International Journal of Food Microbiology, 119, 140e146. Castegnaro, M., Canadas, D., Vrabcheva, T., Petkova-Bocharova, T., Chernozemsky, I. N., & Pfohl-Leszkowicz, A. (2006). Balkan endemic nephropathy: role of ochratoxins A through biomarkers. Molecular Nutrition and Food Research, 50(6), 519e529. Commission regulation (EC) No.1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L364, (2006), 5. Commission regulation (EC) No.401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L70, (2006), 12.

E. Darouj et al. / Food Control 62 (2016) 97e103 Commission regulation (EU) No.105/2010 of 5 February 2010 amending commission regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. Official Journal of the European Union, L35, (2010), 7e8. Commission regulation (EU) No.594/2012 of 5 July 2012 amending commission regulation (EC) No. 1881/2006 as regards the maximum levels of the contaminants ochratoxin A, non dioxin-like PCBs and melamine in foodstuffs. Official Journal of the European Union, L176, (2012), 43e45. Coronel, M. B., Marín, S., Cano-Sancho, G., Ramos, A. J., & Sanchis, V. (2012). Exposure assessment to ochratoxin A in Catalonia (Spain) based on the consumption of cereals, nuts, coffee, wine, and beer. Food Additives & Contaminants: Part A, 29(6), 979e993. € nmez-Altuntas¸, H., Hamurcu, Z., Imamoǧlu, N., & Liman, B. C. (2003). Effects of Do ochratoxin A on micronucleus frequency in human lymphocytes. Nahrung e Food, 47(1), 33e35. EFSA. (2004). Opinion of the scientific panel on contaminants in food chain on a request from the commission related to ochratoxin A ( OTA ) as undesirable substance in animal feed. The EFSA Journal, 101, 1e36. EFSA. (2006). Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. The EFSA Journal, 365, 1e56. Ehrlich, V., Darroudi, F., Uhl, M., Steinkellner, H., Gann, M., Majer, B. J., et al. (2002). Genotoxic effects of ochratoxin A in human-derived hepatoma (HepG2) cells. Food and Chemical Toxicology, 40, 1085e1090. lez-Arias, C. A., Benitez-Trinidad, A. B., Sordo, M., Robledo-Marenco, L., Gonza n-Vivanco, B. S., et al. (2014). Low doses of ochratoxin A Medina-Díaz, I. M., Barro induce micronucleus formation and delay DNA repair in human lymphocytes. Food and Chemical Toxicology, 74, 249e254. Hashemi, J., & Alizadeh, N. (2009). Investigation of solvent effect and cyclodextrins on fluorescence properties of ochratoxin A. Spectrochimica Acta e Part A: Molecular and Biomolecular Spectroscopy, 73(1), 121e126. Hassen, W., Abid, S., Achour, A., Creppy, E., & Bacha, H. (2004). Ochratoxin A and b₂microglobulinuria in healthy individuals and in chronic interstitial nephropathy patients in the centre of Tunisia: a hot spot of ochratoxin A exposure. Toxicology, 199, 185e193. IARC. (1993). Ochratoxin A. IARC monographs on the evaluation of carcinogenic risk of chemicals to humans. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins (Vol. 56, pp. 489e521). IARC Scientific Publication. Iqbal, S. Z., Rabbani, T., Asi, M. R., & Jinap, S. (2014). Assessment of aflatoxins, ochratoxin A and zearalenone in breakfast cereals. Food Chemistry, 157, 257e262. JECFA. (2001). In Joint FAO/WHO Expert Committee of food additives fifty-sixth meeting Geneva, 6e15 February 2001. JECFA. (2008). Joint FAO/WHO Expert Committee of food additives sixty-eighth meeting Geneva. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series:, 59. ~ es, J., & Ritieni, A. (2014). Presence of mycotoxin in comJuan, C., Raiola, A., Man mercial infant formulas and baby foods from Italian market. Food Control, 39, 227e236. Kabak, B. (2009). Ochratoxin A in cereal-derived products in Turkey: occurrence and exposure assessment. Food and Chemical Toxicology, 47(2), 348e352. Liu, J., Wang, Y., Cui, J., Xing, L., Shen, H., Wu, S., et al. (2012). Ochratoxin A induces

103

oxidative DNA damage and G1 phase arrest in human peripheral blood mononuclear cells in vitro. Toxicology Letters, 211(2), 164e171.  Mateo, E. M., Mateo, F., & Jime nez, M. (2007). An overview of Mateo, R., Medina, A., ochratoxin A in beer and wine. International Journal of Food Microbiology, 119(1e2), 79e83. Meng, H., Wang, Z., De Saeger, S., Wang, Y., Wen, K., Zhang, S., et al. (2014). Determination of ochratoxin A in cereals and feeds by ultra-performance liquid chromatography coupled to tandem mass spectrometry with immunoaffinity column clean-up. Food Analytical Methods, 7, 854e864. Ozden, S., Akdeniz, A. S., & Alpertunga, B. (2012). Occurrence of ochratoxin A in cereal-derived food products commonly consumed in Turkey. Food Control, 25(1), 69e74. Rosa, C. A. R., Magnoli, C. E., Fraga, M. E., Dalcero, A. M., & Santana, D. M. N. (2004). Occurrence of ochratoxin A in wine and grape juice marketed in Rio de Janeiro, Brazil. Food Additives and Contaminants, 21(4), 358e364. ~ es, J. (2012). Application of an HPLC-MS/MS method for Rubert, J., Soler, C., & Man mycotoxin analysis in commercial baby foods. Food Chemistry, 133(1), 176e183. Sangare-Tigori, B., Dem, A. A., Kouadio, H. J., Betbeder, A. M., Dano, D. S., Moukha, S., et al. (2006). Preliminary survey of ochratoxin A in millet, maize, rice and ^ te d'Ivoire from 1998 to 2002. Human and Experimental Toxicology, peanuts in Co 25, 211e216. Scott, P. M., & Trucksess, M. W. (1997). Application of immunoaffinity columns to mycotoxin analysis. Journal of AOAC International, 80, 941e949. Sherif, S. O., Salama, E. E., & Abdel-wahhab, M. A. (2009). Mycotoxins and child health: the need for health risk assessment. International Journal of Hygiene and Environmental Health, 212, 347e368. Soleas, G. J., Yan, J., & Goldberg, D. M. (2001). Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection. Journal of Agricultural and Food Chemistry, 49(6), 2733e2740. Soleimany, F., Jinap, S., Faridah, A., & Khatib, A. (2012). A UPLC-MS/MS for simultaneous determination of aflatoxins, ochratoxin A, zearalenone, DON, fumonisins, T-2 toxin and HT-2 toxin, in cereals. Food Control, 25(2), 647e653. Solfrizzo, M., Panzarini, G., & Visconti, A. (2008). Determination of ochratoxin A in grapes, dried vine fruits, and winery byproducts by high-performance liquid chromatography with fluorometric detection (HPLC-FLD) and immunoaffinity cleanup. Journal of Agricultural and Food Chemistry, 56(23), 11081e11086. Van der Merwe, K. J., Steyn, P. S., & Fourie, L. (1965). Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus. Nature, 205, 1112e1113. Wu, J., Tan, Y., Wang, Y., & Xu, R. (2012). Occurrence of ochratoxin A in grain and manufactured food products in China detected by HPLC with fluorescence detection and confirmed by LCeESIeMS/MS. Mycopathologia, 173, 199e205. Zaied, C., Abid, S., Zorgui, L., Bouaziz, C., Chouchane, S., Jomaa, M., et al. (2009). Natural occurrence of ochratoxin A in Tunisian cereals. Food Control, 20(3), 218e222. Zhang, A., Ma, Y., Feng, L., Wang, Y., He, C., Wang, X., et al. (2011). Development of a sensitive competitive indirect ELISA method for determination of ochratoxin A levels in cereals originating from Nanjing, China. Food Control, 22(11), 1723e1728. ~ es, J. (2010). Zinedine, A., Blesa, J., Mahnine, N., El Abidi, A., Montesano, D., & Man Pressurized liquid extraction coupled to liquid chromatography for the analysis of ochratoxin A in breakfast and infants cereals from Morocco. Food Control, 21(2), 132e135.