Ochratoxin A

Gregor Kos and Rudolf KrskaChemistryOchratoxin A (OTA; C20H18ClNO6, Mw=403.82 g/mol) is a colourless, crystalline compound and its chemical name is L-phenylalanineN-[5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H2-benzopyran-7-yl]carbonyl-(R)-isocoumarin, CAS No. [303-47-9]. The main producers arePenicllium. verrucosumand severalAspergillusspecies.The ultraviolet absorption spectrum, which varies with pH and solvent polarity, shows maxima at 213 nm and 332 nm in ethanol (e = 36,800 and 6,400, respectively). A maximum in the fluorescence emission spectrum can be observed at 428 nm.This fact sheet focuses on the occurrence of OTA in agricultural commodities, where it is frequently found: These are: Cereals Coffee Wine

Fig. 1: Structural formula of ochratoxin AObserved concentrations are 0.3-1.6 g/kg in cereals, 0.8 g/kg in coffee and 0.01-0.1 g/kg in wine (1). Validated methods have been developed for the analysis of ochratoxin A in maize, barley, rye, wheat, wheat bran, wheat whole meal, roasted coffee, wine and beer and are based on high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The limit of quantification of these methods is 0.03 g/kg for wine and beer, and about 0.3-0.6 g/kg for other commodities. Two certified reference materials are available at the Institute for Reference Materials and Measurements, which improve quality assurance in laboratories. Screening methods based on thin-layer chromatography are available but have been used in only a few laboratories (2).The analytical methodology for OTA usually includes extraction, clean-up, separation, detection and quantification.ExtractionExtraction is usually performed with a mixture of water and organic solvents, depending on the type of matrix. A IUPAC/AOAC method for the determination of OTA in barley uses a mixture of CHCl3and H3PO4(1); for green coffee, CHCl3is only employed (3). For determination in wheat, a number of extraction solvents are used, including mixtures of toluene/HCl/MgCl2, CHCl3/ethanol/acetic acid and dichloromethane/H3PO4.There is a shift to non-chlorinated solvents, because of the environmental hazards involved. Burdaspal has proposed tert-butyl methyl ether as an extraction solvent for OTA in baby food (4). Other non-chlorinated solvent mixtures used for wheat samples include methanol/water and acetonitrile/water. Two methods for the determination of OTA in barley and roasted coffee have recently been validated: Extraction from barley was carried out with acetonitrile/water, and the extract was diluted with phosphate buffered saline solution (PBS) before a clean-up with immunoaffinity columns (5).A proficiency study for OTA in roasted coffee samples mainly employed a mixture of methanol with a 3% aqueous sodium bicarbonate solution (50:50) for extraction (6).Zimmerli and Dick (1995) have reported a method for the determination of OTA in red wine employing chloroform for extraction.Clean-upImmunoaffinity columns (IAC) represent the state-of-the-art in the clean up of OTA. The extract is forced through the column and ochratoxins are bound to the antibody. The analyte is eluted with an appropriate solvent (e.g. acetonitrile). The immunological reaction is specific for OTA and therefore IAC columns represent a reliable tool for sample clean-up.The analyte is eluted from the column after a rinsing step.Visconti et al. (1999) have been using IAC columns for OTA in wine and beer after diluting the sample with a water solution containing polyethylene glycol and NaHCO3. For roasted coffee samples, a phenyl silane SPE clean-up prior to the IAC stage was introduced, to avoid possible deleterious effects of caffeine. There are also commercial columns available for the simultaneous determination of OTA and zearalenone (ZON).The use of silica gel cartridges has also been reported. After loading the column, a washing step is followed by elution with a toluene/acetone/formic acid mixture. For wheat, mixtures of toluene and acetic acid are used for elution from silica columns and methanol for immunoaffinity columns.Separation and DetectionAfter clean up with immunoaffinity columns, separation of the ochratoxins and detection take place with HPLC-FLD. Determination is possible in the g/kg range.Fluorescence detection at pH 7.5 has been reported using ion-pair HPLC (7). Other scientists determining ochratoxin A in wheat have used a reversed phase HPLC approach with a C18column and an acidic buffer (acetic acid) in an acetonitrile/water mixture as a mobile phase. Recoveries of 70-100% were observed.Reinhard and Zimmerli (1999) have tested a number of reversed-phase materials for HPLC and the influence of several parameters (e.g. pH, temperature) on the selectivity of OTA separation.A collaborative study following the AOAC/IUPAC/NMKL method for OTA in barley, wheat bran and rye established that the method was suitable for an OTA content of