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Journal of Chromatography A, 1167 (2007) 95–101

Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high-performance liquid chromatography with fluorescence detection
Victoria Bascar´ n, Alma Hern´ ndez de Rojas, Paloma Chouci˜ o, Teresa Delgado ∗ a a n
Instituto de Productos L´ cteos de Asturias (IPLA-CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias,Spain a Received 13 April 2007; received in revised form 14 August 2007; accepted 16 August 2007 Available online 21 August 2007

Abstract The present work describes a new analytical method for direct immunoaffinity column clean-up of ochratoxin A (OTA) in milk samples followed by determination of the toxin using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Twodifferent immunoaffinity cartridges (IAC) were investigated, and Ochraprep columns were chosen because they showed the best results. An average recovery of 89.8% and a mean RSD of 5.8% for artificially contaminated cow’s milk in the range of 5–100 ng/L were attained. The calculated limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.5 and 5 ng/L, respectively. This new easy andfast method avoids a previous liquid–liquid extraction step and therefore the use of toxic chlorinated solvents. Chromatograms of the final extracts were clean and OTA could be easily detected at a retention time of 8.4 min without interferences. To assess the presence of the toxin in cow’s milk eight samples of skimmed and four samples of whole milk were analysed and OTA was not detected over theestablished detection limit. © 2007 Elsevier B.V. All rights reserved.
Keywords: Ochratoxin A; Immunoaffinity columns; Milk analysis; High-performance liquid chromatography; Fluorescence detection

1. Introduction Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus (A. carbonarius) and Penicillium (P. verrucosum) moulds. It has potent nephrotoxic, carcinogenic,teratogenic and immunosuppressive effects in several animal species, and is suspected to be a possible cause of a chronic kidney disease known as “Balkan endemic nephropathy” occurring in south-eastern Europe. Consequently, the International Agency for Research of Cancer (IARC) has classified OTA in group 2B, as a possible human carcinogen (Fig. 1). This mycotoxin occurs in a large variety of foods(cereals, beans, groundnuts, spices, dried fruits, grapes, wine, beer, coffee, cocoa, etc.), and consequently, human exposure to OTA is of great concern. In fact, investigations carried out on human biological fluids have shown that OTA contamination is widespread in several European and developed countries [1–9]. OTA was also detected in human milk in Norway, Sweden, Germany, Italy,

∗Corresponding author. Tel.: +34 98 589 33 52; fax: +34 98 589 22 33. E-mail address: (T. Delgado).

Australia, and Brazil, indicating a possible risk for infants fed with contaminated breast milk [10–17]. The Joint FAO/WHO Expert Committee on Food Additives (JEFCA) has established a provisional tolerable weekly intake of 100 ng kg−1 body weight [18], and regulatory limits for OTAhave been settled by the European Union [19] with maximum allowable concentrations ranging from 2 to 10 ppb in some commodities (cereals and derived products, grapes, dried grapes, grape juice, must, wine and coffee). Consumption of cow’s milk has not been postulated as a possible source of OTA intake, because transmission studies of the toxin showed a low biotransfer when OTA-contaminated feedswere administrated to milking cows. This mycotoxin is metabolised by rumen fluid microorganisms, producing a less toxic metabolite, ochratoxin , which exhibits no definite toxicity [20,21]. However, residues of this toxin have been reported in cow’s milk samples from Sweden and Norway at levels of 10–40 ng/L [10,22], using a very sensitive HPLC method with a detection limit of 10 ng/L. Ochratoxin A...
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