Solid-phase extraction and HPLC determination of Ochratoxin A in cereals products on Chilean market

Food Control 20 (2009) 631–634

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Solid-phase extraction and HPLC determination of Ochratoxin A in cereals products on Chilean market Mario Vega a,*, Katherine Muñoz a, Carolina Sepúlveda a, Mario Aranda a, Victor Campos a, Ricardo Villegas a, Orialis Villarroel b,1 a b

Department of Food Science, Nutrition and Dietetic, Faculty of Pharmacy, University of Concepción, Edmundo Larenas s/n casilla 237, Correo 3, Concepción, Chile Food Chemistry Unit, Institute of Public Health, Av. Marathon 1000, Ñuñoa, Santigo, Chile

a r t i c l e

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Article history: Received 11 June 2007 Received in revised form 22 September 2008 Accepted 26 September 2008

Keywords: Ochratoxin A Cereals Mycotoxin Chile and HPLC

a b s t r a c t Ochratoxin A (OTA) is a mycotoxin produced by different species of Aspergillus and Penicillium fungi. The presence of this mycotoxin in cereals-based products has relation with manufacturing practices, especially with storage conditions. An extraction procedure for OTA from wheat-based products was implemented in this study. The method uses an alkaline extraction with NaHCO3, purification with Sep-PakÒ RP-18 cartridges; and quantitative analysis by high performance liquid chromatography with fluorescence detection. The presence of OTA was confirmed by the formation of Ochratoxin A methyl ester. The method shows good validation parameters with a rate of recovery rate over 95%, limits of detection and quantification of 0.6 and 2.1 lg kg 1, respectively. Once the method was validated; 31 samples including, flour, corn starches and rice were analyzed. About 70% of flour samples, 50% of rice and 63% of corn starch samples resulted positives for OTA. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Ochratoxin A (OTA) is a mycotoxin produced by those called ‘‘store” fungi, such as Aspergillus ochraceus (Harris & Mantle, 2001), Aspergillus níger (Blumenthal, 2004), Penicillium verrucosum, etc. OTA has a worldwide occurrence and has been found in different foods and feeds, especially in cereals (Solfrizzo, Avantaggiato, & Visconti, 1998) and derivates products (Beretta et al., 2002; Visconti, Pascale, & Centonze, 2000). Through the food chain OTA can appear in human fluids, such as plasma (Muñoz, Vega, Rios, Muñoz, & Madariaga, 2006), urine (Domijan, Peraica, Miletic-Medved, Lucic, & Fuchs, 2003) and milk (Skaug, Helland, Solvoll, & Saugstad, 2001) and also in animal tissues (Monaci, Tantillo, & Palmisano, 2004). OTA is recognized as a contaminant with a strong nephrotoxic activity (Schaaf et al., 2002) as shown in studies performed in animals (Golinski et al., 1984). The International Agency for Research on Cancer has classified OTA as possibly carcinogenic for humans (group 2B) (IARC, 1993), also teratogenic and carcinogenic effects have been described in some animal species (Lioi, Santoro, Barbieri, Salzano, & Ursini, 2004; Veselá, Vesely´, & Jelïnek, 1983). Chemically OTA is a molecule composed by an iso-coumarin ring linked to phenylalanine molecule (Li, Marquardt, & Frohlich, * Corresponding author. Tel.: +56 41 2204544; fax: +56 41 2210568. E-mail address: [email protected] (M. Vega). 1 Tel.: +56 2 3507477; fax: +56 2 3507578. 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.09.014

2000). Based on its acidic properties, several methods for extraction have been developed. These methods generally involve a liquid extraction with different acid or alkaline solvents and further purifications with immunoaffinity columns (Sugita-Konishi et al., 2006), solid phase extraction or liquid–liquid clean up procedures (Solfrizzo et al., 1998). For quantitative analysis, high performance liquid chromatography (HPLC) with fluorescent detection and HPLC–MS are the most used. The presence of this mycotoxin has been related to deficient water activity and temperature control (Lindblad, Johnsson, Jonsson, Lindqvist, & Olsen, 2004). Different prevention strategies for OTA contamination have been developed, such as control and monitoring of environmental parameters (Olsen et al., 2003), improving drying and storage conditions and the use of natural and chemical agents, irradiation, detoxification, among others (Belajová, Rauová, & Dasko, 2007; Kabak, Dobson, & Var, 2006). Several countries have reported the presence of OTA in cereal products. Specially in Europe there is a continuous monitoring of OTA levels in food, feeds and biological tissues (Rizzo, Eskola, & Atroshi, et al., 2002). Considering that cereals contribute to 50% with the OTA intake (Directorate General-Health and Consumer Protection, 2002), several countries have established maximum allowed limits. The European Community has established a maximum of 5 lg kg 1 for raw cereal grains and 3 lg kg 1 for all derived products from cereal (EC 472/2002). Chile does not have regulation concerning OTA because the lack of studies concerning this

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2.4. Sample quantification

Table 1 Results of OTA content in cereal-based products at the Chilean market.

Positive samples (%) Median (lg kg 1) Range (lg kg 1)

Wheat flour

Rice

Corn starch

21/30 (70) 0.15 ND–2.1

13/31 (42) 0 ND–12.5

12/30 (40) 0 ND–1.2a

LOD = 0.6 lg kg 1. LOQ = 2.1 lg kg 1. a An estimation since is below the LOQ.

mycotoxin, for this reason the objective of this study was to determine the OTA presence in cereal of the Chilean market that will contribute to establish regulation regarding this xenobiotics (see Table 1).

For quantification, a nine-point calibration curve (0.3–30 ng/ml) was used, covering the range of interest for the test sample. The OTA content was quantified by interpolation in the calibration curve. 2.5. Statistical analysis STATSÒ software was used for the statistical analysis. Statistical evaluation was developed using parameters as precision, repeatability, among others, as recommended by AOAC (Horwitz, 2000).

3. Results and discussion 3.1. Validation parameters

2. Experimental 2.1. Standard, reagents and samples OTA standard was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The crystalline powder was dissolved in methanol and measured spectrophotometrically at 333 nm using the extinction coefficient 6400 M 1 cm 1 (Pohland, Nesheim, & Friedman, 1992). The spiking (0.1 and 1 lg/ml) and calibration solutions were prepared by dilution with methanol. Methanol, acetonitrile, ortho-phosphoric acid 85% (HPLC grade) and the other analytical grade reagents were purchased from Merck KGaA (Darmstadt, Germany). The phosphate-buffered saline (PBS) solution was prepared as described by Scott et al. (1998). Sep-PakÒ RP-18 columns, were purchased from Waters (Milford, USA). Samples were obtained directly from supermarkets in two different Chilean cities (Santiago and Valdivia). 2.2. Clean-up with SPE, RP-18 column Ten grams of sample were weighed accurately in a 250-ml amber flask and extracted with 200 ml of sodium hydrogen carbonate 1% w/v by shaking for 30 min in an orbital shaker. After standing by 10 min, the solution was filtered through filter paper Whatman No. 1 and 10 ml of the filtrate were mixed with 10 ml of buffer phosphate saline solution and passed through the Sep-PakÒ column, previously conditioned with 10 ml of buffer saline solution. The column was washed with 5 ml distilled water and the sample was eluted with 3 ml of methanol into a conical amber 4 ml tube. The eluate was evaporated to dryness under nitrogen at 40 °C. 2.3. HPLC conditions The purified extracts were analyzed by HPLC using a MerckHitachi (Tokyo, Japan) system consisting in: quaternary pump Lachrom L-7000, Rheodyne injector with a 20 ll loop, fluorescence detector L-7485 and data acquisition system Varian Star 4.0 Software. Mobile phase: acetonitrile, methanol and o-phosphoric acid 0.15 mol/L; (1+1+1 v/v/v) at a flow rate 0.8 ml/min. Column Waters Symmetry C18, 3.9  150 mm 5 lm. Previous to the chromatographic analysis, the residue was dissolved in 500 ll of mobile phase and filtered by a 0.45 lm pore size PTFE syringe filter. Fluorescence detection was done using 333 nm as excitation wavelength and 470 nm as emission wavelength. Confirmation of OTA in positive samples was done following the method of Zimmerli and Dick (1995) by formation of the methyl ester derivative. The retention time was between 5 and 5.5 min for OTA and between 8.5 and 9 min for OTA methyl ester using the same chromatographic conditions. TM

The validation parameters were determined using a wheat flour matrix without OTA contamination. The calibration curve showed a linear behavior in the range of 0.3–30 ng/ml, with a determination coefficient (r2) of 0.9997. Sensitivity was estimated by the standard error method (Miller & Miller, 2002) and the limit of detection (LOD) and limit of quantification (LOQ), being 0.6 and 2.1 lg kg 1, respectively. The recovery was determined at three levels: 1.5, 3.0 and 4.5 lg kg 1; each level with three replicates and the recoveries resulted of 85.0 ± 2.8%, 99.8 ± 2.4% and 89.3 ± 0.7%, respectively, with a relative standard deviation (RSD) less than 5%. Repeatability (Intra-laboratory) and reproducibility (Inter-Laboratories) studies were performed with five replicates at a spiking level of 3 lg kg 1 and presented a relative standard deviations of 1.9%, respectively (Chan, Lee, lam, & Zhang, 2004; Horwitz, 2000). Alternatively, the method was tested for other matrixes: corn starch and rice, the results were similar to wheat flour. For corn starch the validation parameters were tested in the same way than for wheat flour. As recovery, the results were 95.5 ± 0.9%, 101.0 ± 1.9% and 89.7 ± 0.5% for each level of spiking. The repeatability and reproducibility showed an RSD of 1.9 and 1.5%, respectively. In the case of rice, the matrix was considered as raw grain, reason why the spiking level was considered as 2.5, 5.0 and 7.5 lg kg 1. The recovery test showed results of 96.8 ± 0.7%, 86.9 ± 2.1% and 104.6 ± 1.0% with an RSD less than 2.5% for all levels. Repeatability was performed by quintuplicate at a spiking level of 5 lg kg 1. The RSD was 2.4%. The chromatographic conditions used were adequate for the analysis of OTA and OTA methyl ester in wheat flour, corn starch and rice. Under the frame of the Regulation 401/2006 of the European Union (2006), this method is appropriated for the Ochratoxin A analysis in the matrixes before mentioned. The chromatograms show a clean base line, without interference close to OTA for wheat flour (see Fig. 1). 3.2. Samples analysis at the Chilean market A total of 91 samples were analyzed. The samples were collected at the local market along the country, during the first semester of 2006 and analyzed as soon the samples arrived to the laboratory. The median of the samples was 0.15 lg kg 1 for flour, meaning that most of samples had very low values for OTA contamination. In the case of rice and corn starch, the value for the median was lower than LOQ, that it means that more than 50% of the samples were negative to OTA. In the same way, all flour and corn starch samples showed results below 3.0 lg kg 1, which correspond to the European Community limit for cereal derived products (EC 472/2002). In the case of rice, just one sample was be-

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Fig. 1. Chromatogram of a blank wheat flour sample (a) and a OTA naturally contaminated sampled of 2.1 lg kg

yond of the limit with a result of 12.5 lg kg 1. All samples were confirmed by methyl ester formation, no false positives were found. 4. Conclusions The alkaline extraction and reversed-phase HPLC fluorometric detection was very useful for the OTA analysis in different cereal matrixes. The low costs, fastness, accuracy and repeatability for the analysis, makes of this method an important analytical tool for OTA contamination-control in different cereals. The fact of no false positive samples was found, could mean that the alkaline extraction coupled with solid phase clean-up increase the selectivity of the method. In relation with the samples at the Chilean market, no significant results were found. Almost all samples were below the limit established for the European Community. Except one rice sample, that represents 1.1% of the samples. These results are similar to those reported by Magnoli et al. (2006), in Argentina, where 50 corn samples for human consumption were negative (values below 1 lg kg 1) to OTA. In the case of Brazil, the research has been focused mainly to the OTA contamination in coffee. In a summary made by Rodríguez-Amaya and Sabino (2002) about the mycotoxin research in Brazil, in nine studies was investigated the presence of OTA in cereals products. Three of these studies informed positive values for OTA with means over 10.0 lg kg 1. In six studies OTA was informed as nondetected in analysis done by thin layer chromatography (TLC). Other South American countries, such as Uruguay, have also informed a similar situation (Pineiro, Dawson, & Costarrica, 1996). Concern to the regulatory aspect, Chile does not have regulation for OTA. This situation should be considered due that around 20% of the cereals consumption in Chile comes from abroad. As summary of this research no significant OTA contamination was found in the samples obtained from the Chilean market. Acknowledgement This work is part of the Mycotoxin Project (Contract No. ICA4CT-2002-10043) funded by the European Commission (INCO-DEV program). References Belajová, E., Rauová, D., & Dasko, L. (2007). Retention of ochratoxin A and fumonisin B1 and B2 from beer on solid surfaces: Comparison of efficiency of adsorbents with different origin. European Food Research and Technology, 224, 301–308.

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