FACT SHEET - www.micotoxinas.com.br
Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KT22 7RY, England
Patulin is a mycotoxin produced by certain species of Penicillium, Aspergillus, and Byssochylamys moulds, which may grow on a variety of foods (1). Its chemical name is 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one; CAS No. 149-29-1, and it is an unsaturated heterocyclic lactone with the structure described in Fig. 1. It has been identified in rotten apples contaminated by fungi and in commercial sweet apple cider at levels up to 45 mg/litre. Patulin has also been identified in numerous other mouldy fruits, vegetables, cereals, and animal feeds; it is stable in apple and grape juices and in dry corn. In almost all cases, however, patulin contamination has been limited to rotting or mouldy foods. Patulin is a colourless, crystalline compound with a melting point of 110 °C. Maximum UV absorption is at 276 nm (e 14,450). The EI mass spectrum shows prominent ions at m/z 154 (M+), 136, 126, 110, 97, 82, 71, 69, 55 and 53. Regulation levels range from 30 to 50 µg/litre in several countries.
Extraction and Clean-up
Several methods for extraction and clean-up of samples, (mainly fruit juices such asapple juice and pear juice), have been established in recent years. Extraction with polar solvents such as ethyl-acetate or acetone are widespread. The following clean-up can be performed with column chromatography using silica gel 60, florisil or celite. Another method includes the use of sodium carbonate solution as a washing agent. Mycosep Columns (#224) can also used, but as a quick and easy clean-up. The column is inserted into the test tube and because of a rubber seal, the extract is forced through the column. After 10-30 s, the extract appears on top of the column (2).
Separation and Detection
One- and two-dimensional thin-layer chromatography (TLC) was among the first and most popular methods to be introduced for patulin analysis as a cost-effective and easy to use method. Silica gel has proved to be most effective for separation and, because patulin is fluorescent, detection can be performed without derivatisation. Nevertheless, derivatisation for UV detection is possible and detection limits of 20-30 µg/litre have been achieved. Recovery rates varied between 85 and 119 %. Vero et al. have reported using a toluene/ethylacetate/formic acid mixture (6+3+1) as a mobile phase. The detection wavelength was 412 nm, when MBTH (3-methyl-2-benzothiazolylzone) was used as a detection reagent.
High-performance liquid chromatography (HPLC) has become increasingly popular, whereas the use of TLC is decreasing. Reversed-phase (RP) columns are used for patulin, and detection depends on the detector chosen by the operator. Simple UV detectors result in detection limits from 2 to 10 µg/litre (3), whereas fluorescence detectors reach detection limits as low as 10 5 µg/litre. An HPLC-MS/MS method is described by Sewram et al. using an atmospheric pressure chemical ionisation device. HPLC separation is achieved with a mobile phase of acetonitrile and water (10:90), resulting in a detection limit of 4 µg/litre for apple juice samples. Advantages are the high performance of these techniques, their reliability and the fact that it can be easily automated. Especially high operating costs and the need for highly trained staff contribute to the disadvantages (4).
Gas chromatography (AC) is among the techniques that find less use in patulin analysis, as HPLC is a much more reliable method. Furthermore, for AC analysis, it is necessary to derivatise patulin with tri-methylsilylether or hepta-fluorobutyrat (5) to make it volatile. The use of GC-MS can lower the detection limit to 1 µg/litre. A limit of quantification of 10 µg/litre was achieved by employing GC/MS after diphasic dialysis extraction with in situ acylation.
HPLC and Gas chromatographic are the methods of choice in routine analysis of patulin. All other methods of analysis described here, are subject of ongoing research and are still being developed. No development of an ELISA method for patulin has been reported recently. There is, however, ongoing research on the production of antibodies of patulin derivatives (6, 7)
Tsao and Zhou have reported using micellar electrokinetic capillary chromatography (MECC) for the determination of patulin in cider. This method features low reagent consumption and needed 2 ml of sample for the procedure. Detection was performed with a photodiode array detector at 273 nm with a linear range from 24 µg/litre to 75 µg/litre. A limit of detection of 3.8 µg/litre was achieved.
Semiquantitative methods of analysis include polarography, which has a detection limit of 25-30 µg/litre (8) and column chromatography. The latter consists of a glass column packed with silica gel and calcium sulfate, which is used for separation. Detection takes place under UV light. The method is cheap and simple to use with detection limits in the range of 30-50 µg/litre (9).
An analytical method for patulin in cloudy and clear apple juices has been validated recently as part of the SMT project funded by European Union.
The test portion of clear apple juice was directly extracted with ethyl acetate; cloudy apple juice and apple purée, were treated with the pectinase enzyme before extraction. After back-extraction into sodium carbonate, the extract was dried and concentrated. Patulin was determined by liquid chromatography (LC) with UV detection. Recoveries of patulin ranged from 80 to 92%. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) ranged from 8 to 35% and 11 to 36%, respectively. RSDr values of 610% and RSDR values of 1125% were obtained for clear and cloudy apple juice spiked above 50 ng/g, which is either the statutory limit or the advisory level for patulin contamination in apple juices in many countries.
This method is also on the list of future CEN Standards and will be published in the Official Methods of Analysis book of AOAC (10).
(1) U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods and Beverages, Patulin in Apple Juice, Apple Juice Concentrates and Apple Juice Products, http://vm.cfsan.fda.gov/~dms/patuguid.html, (2000)
 Malone BR, Humphrey CW, Fleetwood KD, Romer T, Rapid Low-Solvent Methods for Patulin in Apple Juice and Apple Juice Concentrate, presented at Mycotox 98, Toulouse, France (1998)
 Anonymous, Apple Juice, Apple Juice Concentrates and Drinks Containing Apple Juice-Determination of Patulin Content, ISO Method, 8128-1 (1993)
 Tarter EJ, Scott PM, Determination of Patulin by Capillary Gas Chromatography of the Heptafluorobutyrate Derivative, J. Chromatography, 538, 441-441 (1991)
 Sheu F, Lee O, Shyu YT, The synthesis of antigens and the product of antibodies against patulin derivatives, J Food Drug Analysis 7 (1), 65-72 (1999)
 Sewram V, Nair JJ, Nieuwoudt TW, et al., Determination of Patulin in Apple Juice by High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry, J Chromatography A 897, (1-2), 365-374 (2000)
 McElroy LJ, Weiss CM, The Production of Polyclonal Antibodies against the Mycotoxin Derivative Patulin Hemiglutarate, Can J Microbiology, 39 (9), 861-863 (1993)
 Wasicki P, Scholz F, Schleinitz KD, Polarographic Determination of Patulin, Pharmazie, 41(12), 843-844 (1986).
 Brause AR, Trucksess MW, Frederick TS, Page SW, Determination of Patulin in Apple Juice by Liquid chromatography, J of AOAC, 79(2), 451-455 (1996)
 MacDonald S., Long M., Gilbert J. Liquid chromatographic method for determination of patulin in clear and cloudy apple juices and apple puree: collaborative study. Journal of AOAC International, 83 (6), 1387-1394, (2000)