Ochratoxin A

Gregor Kos and Rudolf Krska

Chemistry

Ochratoxin A (OTA; C20H18ClNO6, Mw=403.82 g/mol) is a colourless, crystalline compound and its chem
name is L-phenylalanine N-[5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H2-benzopyran-7-yl]carbo
(R)-isocoumarin, CAS No. [303-47-9]. The main producers are Penicllium. verrucosum
several Aspergillus species.

The ultraviolet absorption spectrum, which varies with pH and solvent polarity, shows maxima at 213 nm
332 nm in ethanol (e = 36,800 and 6,400, respectively). A maximum in the fluorescence emission spectrum
be observed at 428 nm.

This fact sheet focuses on the occurrence of OTA in agricultural commodities, where it is frequently found: T
are:

Cereals

Coffee

Wine

Fig. 1: Structural formula of ochratoxin A

Observed 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
Validated methods have been developed for the analysis of ochratoxin A in maize, barley, rye, wheat, wheat b
wheat whole meal, roasted coffee, wine and beer and are based on high-performance liquid chromatography
fluorescence detection (HPLC-FLD). The limit of quantification of these methods is 0.03 g/kg for wine
beer, and about 0.3-0.6 g/kg for other commodities. Two certified reference materials are available at
Institute for Reference Materials and Measurements, which improve quality assurance in laboratories. Scree
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

quantification.
Extraction

Extraction is usually performed with a mixture of water and organic solvents, depending on the type of matri
IUPAC/AOAC method for the determination of OTA in barley uses a mixture of CHCl 3 and H3PO4 (1); for g
coffee, CHCl3is only employed (3). For determination in wheat, a number of extraction solvents are u
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
proposed tert-butyl methyl ether as an extraction solvent for OTA in baby food (4). Other non-chlorinated sol
mixtures used for wheat samples include methanol/water and acetonitrile/water. Two methods for
determination of OTA in barley and roasted coffee have recently been validated: Extraction from barley
carried out with acetonitrile/water, and the extract was diluted with phosphate buffered saline solution (P
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
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 emplo
chloroform for extraction.
Clean-up

Immunoaffinity columns (IAC) represent the state-of-the-art in the clean up of OTA. The extract is fo
through the column and ochratoxins are bound to the antibody. The analyte is eluted with an appropriate sol
(e.g. acetonitrile). The immunological reaction is specific for OTA and therefore IAC columns represe
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 wi
water solution containing polyethylene glycol and NaHCO 3. For roasted coffee samples, a phenyl silane
clean-up prior to the IAC stage was introduced, to avoid possible deleterious effects of caffeine. There are
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 followe
elution with a toluene/acetone/formic acid mixture. For wheat, mixtures of toluene and acetic acid are used
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 HP
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 determi
ochratoxin A in wheat have used a reversed phase HPLC approach with a C 18 column and an acidic buffer (a
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 influenc

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
established that the method was suitable for an OTA content of <10 g/kg sample. (8, 9).
For determination of OTA in baby food, a limit of quantification of 0.008 g/kg was reported using HPLC
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 (1
reported a method for OTA in flour using LC/MS/MS after derivatisation of OTA to its methyl ester. A
separation with HPLC, the sample was introduced into the MS. Samples were quantified with ochratoxi
methyl(d3)ester as internal standard.

TLC and ELISA tests are also used for separation and detection. Both techniques are easy to use and ELIS
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 w
mixture of toluene/methanol/acetic acid. Under long wavelength UV light OTA will appear blue-green
retention value of 0.6. Schneider et al. (1995) developed an ELISA method using test strips coating
immunoaffinity membrane with antibodies. Samples were extracted with methanol and, after filtration
dilution with PBS (phosphate buffered saline), the assay was performed. The detection limit for visual inspec
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 O
in cereals, use extraction with toluene after adding HCl and MgCl 2 solution. Clean-up is performed with a
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 Ac
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
biosensor assay, and an ELISA system for OTA in cereals, using molecular imprinted polymers, and an EL
system together with PCR based molecular detection for OTA-producing fungi, will be among the tasks of
project, which is coordinated by Monica Olsen at the National Food Administration of Sweden.
References

(1) Battalglia R., Hatzold T., Kroes R., Guest Editorial: Conclusions form the Workshop on Ochratoxin in F
(ILSI Europe, Aix-en-Provence, 10-12 January 1996), Food Additives and Contaminants, 13, Supplement,
(1996)

(2) Joint FAO/WHO Expert Committee on Food Additives (Jecfa), 56th meeting, Geneva, 6-15 February 2001
(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 col
cleanup for determination of ochratoxin A in barley: Collaborative study, Journal of AOAC International, 83
1377-1383 (2000)

(6) Krutler O., Delami C., Wiedermann G., Proficiency Study Ochratoxin A in Coffee Samples, Federal Inst
for Food Control and Research, Vienna, September 2000

(7) Breitholz A., Olsen M., Dahlbck A., Hult K., Plasma Ochratoxin A Levels in Three Swedish Populat
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., Mller T., Liquid Chromatographic Determination of Ochratoxin A in Barley, Wheat Bran
Rye by the AOAC/IUPC/NMKL Method: NMKL collaborative study, Journal of AOAC International, 79
1102-1105 (1996)