Zearalenone

Gregor Kos and Rudolf Krska

Chemistry

Zearalenone (ZON) [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-beta-resorcyclic-acid-lactone] is a secondary fungal metabolite that is primarily produced by Fusarium graminearum and Fusarium culmorum (figure 1). The affected commodities include corn, barley, oats and wheat. The only naturally occurring ZON derivative is trans-a-zearalenol [6-(10-hydroxy-6-R-hydroxy-trans-1-undecenyl)-beta-resorcyclic-acid-lactone] (a-ZOL) (figure 2). Due to estrogenic activity and anabolic properties observed in swine, mice and rats, regular monitoring is required.

Tolerated levels of ZON in food range from 60 to 200 microgram/kg in Europe, so methods of analysis have to focus on the microgram/kg range. The SMT-ZONMAIZE project funded by the European Commission (SMT4-CT98-2228) is concerned with the development and production of a ZON certified reference material in maize and the production of ZON calibrants to make analyses more comparable, as differing calibrants and the method dependence of results account for the major problems in ZON determination.

Analytical procedures differ in extraction, clean up and determination steps, and the method of choice depends on available equipment and analytical requirements such as sensitivity and time of analysis. It is important to note that zearalenone is very sensitive to light exposure, especially in a solution, so special care should be taken in the laboratory to avoid degradation. Detailed reviews of available analytical methods have been given by Krska and Josephs [1], Steyn et al. [2], Betina [3], Frisvad and Thrane [4], Scott [5] and Lawrence and Scott [6].

Immunochemical Methods

Several immunochemical methods have been developed for the determination of ZON in cereals, milk and biological fluids. These include radio immunoassays (from porcine antibodies) as well as enzyme immunoassays using polyclonal (from rabbits) and monoclonal antibodies (from mice). Immunochromatography is also used. Commercially available immunoassays are produced and distributed by r-Biopharm (Darmstadt, Germany) and Neogen (Lansing, MI, USA). The former has an LOD of 50 microgram/kg and takes 15 min for testing. The latter has an LOD of 250 microgram/kg, which is above the guidelines established by most European countries. Both kits show cross-reactivities with the ZON derivatives a-ZOL (figure 2) and b-ZOL. Advantages include good accuracy, rapid analysis time and that kits come completely equipped making them easy to use in almost any analytical lab. High costs make them unsuitable for monitoring programmes though. An ELISA method for ZON in maize has been established as a First Action Screening Method by the AOAC (ZON concentration > 800 microgram/kg).

Quantitative Methods

Methods suitable for quantification usually include extraction and clean-up steps prior to determination of the analyte. Various methods can be employed, but all require varying degrees of experience in the field of analytical chemistry and considerable amounts of reagents. All methods feature high recoveries (70-110%) in the µg/kg range.

Extraction

Solvents used for initial extraction are usually a mixture of various organic liquids including ethyl acetate, methanol, acetonitrile and chloroform. The use of chlorinated solvents is steadily decreasing,  and acetonitrile in particular has become popular for extraction.

Clean-up

While a clean up is usually not necessary for immunoassays, extensive clean-up procedures are required before physicochemical methods can be employed. The main procedures are liquid-liquid partitioning, solid phase extraction (SPE), immunoaffinity columns (IAC) and multifunctional clean-up columns. Liquid-liquid partitioning involves the use of high amounts of (sometimes) chlorinated solvents, and when using chloroform and NaOH special care has to be taken to avoid hydrolysis of the ZON lactone ring under alkaline conditions. Additionally, recoveries of ZON are lower compared with IAC methods. The use of SPE requires a lot of experience to obtain reproducible and reliable results.

IACs and multifunctional clean-up columns are easy to use. The application of IACs for purification of ZON contaminated samples is already well investigated. The analyte molecules are bound to antibodies and subsequent to a washing step the toxin can be eluted. It features a higher recovery than standard liquid-liquid partitioning and is usually employed together with Reversed Phase High Performance Liquid Chromatograph (RP-HPLC). Additionally the use of chlorinated solvents can be avoided. IACs are commercially available form Rhone-Diagnostics (Glasgow, Scotland) and Vicam (Watertown, MA; USA). Both products perform equally well.

Another recent development in clean-up methods is the widely used commercial MycoSep™ column (Romer Labs Inc., Union, MT, USA, #224 for ZON). It allows quick sample purification (30 seconds) and no rinsing steps are required. Analytical interfering substances are retained while ZON is not adsorbed on the packing material. The method is not suitable for complex matrices such as mixed feed.

Separation and Detection Techniques

The most popular separation and detection method is RP-HPLC after a clean-up with IACs. Separation from interferences is good and the Limit of Detection (LOD) for samples analysed with RP-HPLC-FLD with prior clean-up using IAC has been reported to be 3-6 microgram/kg with a mean recovery of 98-100% in the 10-200 microgram/kg range.

A common extinction coefficient for the used mobile phase of the HPLC system is not available as different mixtures of acetonitrile and water are employed. Additionally not always the same band is used for calculations leading to different results for the same sample. Krska and Josephs recommended an extinction coefficient of 12623±111 at the 274 nm band, which could be measured most accurately (CV=1.2%) at a reference wavelength of 450 nm. The use of this common extinction coefficient, which was obtained during the mentioned SMT-ZONMAIZE project, will also contribute to an improved comparability of measured results.

With the availability of liquid chromatography systems with atmospheric pressure chemical ionisation and mass spectrometric detection (LC-APCI-MS), a new powerful method has been established for identification and quantification. High sensitivity can be achieved and even raw extracts can be measured without clean-up. After enzymatic digestion a simultaneous detection of ZON and its derivatives (alpha/beta-ZOL and zeranol) is possible. Disadvantages include the high costs and the need for highly trained staff. Systems are less robust and therefore require more servicing. If very low limits of detection are required fluorescence methods are still the method of choice, because of their extremely high sensitivity.

Another method features the direct detection of ZON with a fluorimeter after IAC clean-up and reaction of the analyte with aluminium chloride hexahydrate. Methods using TLC, GC and GC-MS have also been reported, but are of minor importance. TLC has shown less sensitivity, although the method is easy to apply.

Future developments will focus on the coupling of IAC columns to HPLC-FLD systems as well as the continued use of LC-MS(-MS) systems for identification and quantification, which is already the most important method today for the simultaneous detection of ZON and alpha-/beta-zearalenol.

References

[1] R. Krska, R. Josephs, The State-of-the-art in the Analysis of Estrogenic Mycotoxins in Cereals, Fresenius Journal of Analytical Chemistry, 369, 469-476 (2001)

[2] P.S. Steyn, P.G. Thiel, D.W. Trinder in J.E. Smith, R.S. Henderson (ed), Mycotoxins and Animal Foods, CRC Press Inc, Boca Raton, 165-221 (1991)

[3] V. Betina, in V. Betina (ed), Journal of Chromatography Library, 54, Elsevier, Amsterdam, 141-252 (1993)

[4] J.C. Frisvad, U. Thrane in V. Betina (ed), Journal of Chromatography Library, 54, Elsevier, Amsterdam, 253-372 (1993)

[5] P.M. Scott in V. Betina (ed), Journal of Chromatography Library, 54, Elsevier, Amsterdam, 373-426 (1993)

[6] J.F. Lawrence and P.M. Scott in D. Barcelo (ed), Environmental Analysis: Techniques, Applications and Quality Assurance, Elsevier, Amsterdam, 273-309 (1993)