DETERMINATION OF OCHRATOXIN A IN CEREAL-BASED FEED BY A HIGH-PERFORMANCE CHROMATOGRAPHIC METHOD
ANA CIŞMILEANU, GEORGETA VOICU, VIVIANA CIUCĂ, MINODORA IONESCU Pasteur Institute, Bucharest, Romania
Summary For routine testing of ochratoxin A (OTA) in cereal feed samples, a reverse-phase high-performance liquid chromatographic(HPLC) method, with immunoaffinity column cleanup was developed. The method is suitable for the investigation of OTA content in the toxicologically significant range corresponding to1.5-30 ppb.
Mycotoxins in cereals and feedstuffs are a potential hazard to the health of people and the performance of farm animals. Ochratoxin A (OTA), one of the most studied mycotoxins, is produced by Penicilliumverrucosum and by Aspergillus ochraceus and a few isolates of Aspergillus niger. These three groups differ in their ecological niches, in the commodities they affect, and in the frequency of occurrence in different geographical regions. Current methods used for OTA determination include thin layer chromatography, ELISA methods, liquid chromatography coupled or not with mass spectrometry.Advantages and disadvantages of each method depend on its capability to separate impurities from the analytes, the time of sample preparation and economic aspects. The high-performance liquid chromatography (HPLC) seems to be a quick and simple method for OTA determination. In this paper is presented a reverse-phase HPLC method, with immunoaffinity column clean-up. The method will be validated inconformity with ISO 17025 requirements. Materials and methods Materials and Reagents The ochratoxin A standard and the immunoaffinity columns for ochratoxin A were supplied by R-Biopharm Rhone, Scotland. The analytical grade solvents were obtained from Sigma, USA, and the pure water was produced by SG Water (Germany) device. Instruments The liquid chromatograph model Alliance (Waters, USA) equipped withquaternary pump, autoinjector and fluorescence and UV detectors was used with a stainless steel reverse phase column ZORBAX SB-C18 (250x4.6 mm, 5 µm particle size) (Agilent, USA). 570
LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLI, 2008, TIMIŞOARA
Analytical Procedures In the HPLC method the essential steps are: extraction of the analyte (mycotoxin) from sample, clean-up (or purification)and the chromatographic analysis. Extraction. 50g of ground sample were placed into ultraturrax and then 200 ml of 60% acetonitrile/water (v/v) were added. The mixture was stirred for 2 min. at high speed. The extract was filtered through a Whatman No 3 filter paper and then through a microfibre filter. Clean-up by immunoaffinity chromatography. We used immunoaffinity columns (OCHRAPREP fromR-Biopharm Rhone) and the following procedure: 4 ml of the final extract, corresponding to 1 g of the original material was diluted with 44 ml of phosphate buffered saline (PBS, pH 7.4, R-Biopharm Rhone) to give a solvent concentration of 2.5% or less (in order to protect the antibodies in immunoaffinity columns). The mixture was allowed to pass through column by gravity or at a flow rate of 5 ml/min.The column contains monoclonal antibodies to ochratoxin A bound to a solid support. By passing the diluted extract through column any ochratoxin A present in the sample is bound to the antibody within the column. The column was then washed with 20 ml of PBS. The elution of ochratoxin A was done with 1.5 ml of acetic acid: methanol mixture (2:98) and 1.5 ml of pure water. HPLC chromatography. 100µl of the samples were injected into the O HPLC column heated to 40 C. The mobile phase was an acetonitrile: water: acetic acid solution (51:47:2, v/v). The flow rate was 1 ml/min. For fluorescent detection of ochratoxin A the excitation wavelength was 333 nm, and the emission wavelength was 443 nm. For creating calibration curve a OCHRATOXIN A STANDARD (R-Biopharm Rhone) was used. It is a...