FACT SHEET - www.micotoxinas.com.br
Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KT22 7RY, England
Ochratoxin A (OTA; C20H18ClNO6, Mw=403.82 g/mol) is a colourless, crystalline compound and its chemical name is L-phenylalanine N-[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 are Penicllium verrucosum and several Aspergillus species.
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:
Fig. 1: Structural formula of ochratoxin A
Observed concentrations are 0.3-1.6 µg/kg in cereals, 0.8 µg/kg in coffee and 0.01-0.1 µg/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 µg/kg for wine and beer, and about 0.3-0.6 µg/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.
Extraction 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 CHCl3 and H3PO4 (1); for green coffee, CHCl3 is 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.
Immunoaffinity 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 Detection
After clean up with immunoaffinity columns, separation of the ochratoxins and detection take place with HPLC-FLD. Determination is possible in the µg/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 C18 column 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 <10 µg/kg sample. (8, 9).
For determination of OTA in baby food, a limit of quantification of 0.008 µg/kg was reported using HPLC with enhanced fluorescence detection and post-column reaction with ammonia (4).
LC-MS is a new technique that can be used for identification and quantification. Jorgensen and Val (1999) reported a method for OTA in flour using LC/MS/MS after derivatisation of OTA to its methyl ester. After separation with HPLC, the sample was introduced into the MS. Samples were quantified with ochratoxin A methyl(d3)ester as internal standard.
TLC and ELISA tests are also used for separation and detection. Both techniques are easy to use and ELISA in particular has become popular for rapid screening of samples.
OTA detection by TLC can be performed spotting samples and spikes on a SG-60 plate and development with a mixture of toluene/methanol/acetic acid. Under long wavelength UV light OTA will appear blue-green at a retention value of 0.6. Schneider et al. (1995) developed an ELISA method using test strips coating an immunoaffinity membrane with antibodies. Samples were extracted with methanol and, after filtration and dilution with PBS (phosphate buffered saline), the assay was performed. The detection limit for visual inspection was found to be 100 ng/g for OTA.
Another HPLC method, which has been adopted as CEN Standard (prEN 15141-1) for the determination of OTA in cereals, use extraction with toluene after adding HCl and MgCl2 solution. Clean-up is performed with a mini silica gel column, followed by determination with RP-HPLC with fluorometric detection.
Methods for OTA in barley and roasted coffee (5) have been accepted by the AOAC International as First Action Method and are under active consideration by CEN for adoption as a Standard.
The OTA PREV project (QLK1-1999-00433) will develop rapid monitoring methods. The development of a biosensor assay, and an ELISA system for OTA in cereals, using molecular imprinted polymers, and an ELISA system together with PCR based molecular detection for OTA-producing fungi, will be among the tasks of the project, which is coordinated by Monica Olsen at the National Food Administration of Sweden.
(1) Battalglia R., Hatzold T., Kroes R., Guest Editorial: Conclusions form the Workshop on Ochratoxin in Food (ILSI Europe, Aix-en-Provence, 10-12 January 1996), Food Additives and Contaminants, 13, Supplement, 1-3 (1996)
(2) Joint FAO/WHO Expert Committee on Food Additives (Jecfa), 56th meeting, Geneva, 6-15 February 2001, 21
(3) AOAC, Ochratoxin A in Green Coffee 975.38 (1990)
(4) Burdaspal P., Determination of Ochratoxin A in Baby Food, CEN/TC 275/WG5, N 219
Additives and Contaminants, 14, 3, 237-248 (1997)
(5) Entwisle A.C., Williams A.C., Mann P.J., et al., Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley: Collaborative study, Journal of AOAC International, 83 (6), 1377-1383 (2000)
(6) Kräutler O., Delami C., Wiedermann G., Proficiency Study Ochratoxin A in Coffee Samples, Federal Institute for Food Control and Research, Vienna, September 2000
(7) Breitholz A., Olsen M., Dahlbäck A., Hult K., Plasma Ochratoxin A Levels in Three Swedish Populations Surveyed Using a Ion-pair HPLC Technique, Food Additives and Contaminants 8, 183-192, (1991)
(8) AOAC Official Methods of Analysis, 991.44 (1992)
(9) Larsson K., Möller T., Liquid Chromatographic Determination of Ochratoxin A in Barley, Wheat Bran and Rye by the AOAC/IUPC/NMKL Method: NMKL collaborative study, Journal of AOAC International, 79, 5, 1102-1105 (1996).