Revue Méd. Vét., 2007, 158, 10, 504-508 Introduction Fusariotoxins which contaminate foods and feed worldwi- de, are metabolites from several species of Fusarium genus, soil fungi producing probably the most important quantity of toxin in temperate north countries [16]. Zearalenone (ZEA), one of the most widely distributed fusariotoxins, is common in maize and maize products but also in soybeans and various cereals grains, which represent a major part of human food and animal feed. Crop contamination is often inevitable and constitutes a potential risk for human and ani- mal health [3]. Human and animal exposure comes mainly from chronic contaminated food ingestion, but human expo- sure can be direct via corn and cereals or indirect via animal products. ZEA causes oestrogenic syndrome in animals, resulting in reproductive disorders and decreased fertility in a great num- ber of species, with high variations of sensitivity. Female pigs are considered as the most sensitive animal species while poultry and ruminants show a lower responsiveness to ZEA [7]. Absorption and metabolism are implicated in these variations between species [6]. In particular, following oral exposure in mammals, ZEA is metabolized in various tis- sues, particularly in the liver, to the main metabolites identified in vitro and in vivo: alpha zearalenol, α-ZOL and beta zeara- lenol, ß-ZOL [13,14,25]. The rate of conversion and the ratio α-ZOL/ß-ZOL shows species variations, and may account for the species differences in the sensitivity to ZEA, as α-ZOL shows a several-fold higher oestrogenic activity compared to the parent compound ZEA whereas ß-ZOL has approximately the same activity as ZEA [5,24]. Both these ZEA derivatives have been described in naturally contaminated cereal grains [10]. Further reduced forms, zearananone and zearalanols (Figure 1) have also been identified in mammal urine [12,27]. In poultry species, only little information is available concerning ZEA metabolism whereas exposure is likely to occur via feedstuffs, specially during force-feeding, corres- ponding to a final average feed intake of approximately 10 kg of maize per duck [22]. SUMMARY HPLC analysis of zearalenone (ZEA), zearalenols (α-ZOL and ß-ZOL) and zearalanols (α-ZAL and ß-ZAL) was developed, in order to obtain a sensitive and reproducible method to quantify ZEA and its reduced metabo- lites in subcellular fractions of animal livers (S9 samples). Optimal in vitro metabolism was observed by incubating 5 mg S9 proteins with 0.016 μmol. ZEA. Acetonitrile and diethylether/chloroform mixture were compared for extraction, as well as different mobile phases and two detection modes in HPLC analysis. Extracted samples were eluted with water/acetonitrile (55:45, v/v) at a flow-rate of 1.0 ml/min -1 , resulting in well separated peaks between ZEA and the metabolites. The limits of detection ranged from 0.5 to 2 ng/mg S9 proteins using UV, and from 0.04 to 4 ng/mg S9 proteins, using fluorescence detection. Fluorescence showed a ten-fold higher sensitivity than UV detection for ZEA and α-ZOL. Repeatability (10 assays) was 2.7% to 6.99% for zearalenols. Day-by-day coefficients of variation for zearalenone and zeranols with UV detection were 3.3 to 8.5 %, and 2.5 to 4.3 %, respectively. This analysis applied to S9 samples from ducks after 30 min of ZEA incu- bation allowed to demonstrate that α-ZOL is the main reduced metabolite in the duck. The present method is particularly adapted for studying in vitro metabolism of ZEA and inter-species variations. Keywords : Zearalenone, zearalenols, S9 liver fractions, duck, metabolism, HPLC. RÉSUMÉ Analyse HPLC de la zéaralénone et de ses métabolites réduits dans des fractions S9 de foie de e canard L’analyse HPLC de la zéaralénone (ZEA), des zéaralénols (α-ZOL et ß-ZOL) et zéaralanols (α-ZAL et ß-ZAL) a été mise au point pour disposer d’une méthode sensible et reproductible pour quantifier la ZEA et ses métabolites réduits dans des fractions hépatiques subcellulaires. Le métabolisme in vitro optimal a été observé après incubation de 5 mg de protéines de S9 avec 0.016 μmol de ZEA. L’acétonitrile et des mélanges diéthy- léther/chlorofome ont été comparés pour l’extraction, ainsi que différentes pha- ses mobiles et deux détecteurs pour l’analyse HPLC. Les échantillons extraits élués avec un mélange eau/acétonitrile (55:45, v/v) à un débit de 1.0 ml/min -1 , ont conduit à des pics bien séparés entre ZEA et ses métabolites réduits. Les limi- tes de détection ont été comprises entre 0.5 et 2 ng/mg de protéines avec détec- tion UV, et entre 0.04 et 4 ng/mg de protéines avec détection fluorimétrique. Cette dernière a montré une sensibilité dix fois supérieure à l’UV pour la ZEA et l’α-ZOL. La répétabilité (10 répétitions) a été de 2.7% à 6.99% pour les zéara- lénols. Les coefficients de variation jour-à-jour mesurés en détection UV ont été de 3.3% à 8.5% et de 2.5% à 4.3% pour la zéaralénone et les zéranols, respecti- vement. Cette méthode appliquée à des S9 de canard incubés 30 minutes avec de la ZEA a permis de montrer que l’α-ZOL constitue le principal métabolite réduit chez le canard. La méthode décrite est particulièrement adaptée à l’étude du métabolisme in vitro et aux variations interspécifiques. Mots-clés : Zearalénone, zéaralénols, fraction S9 hépa- tique, canard, métabolisme, HPLC. HPLC assay of zearalenone and reduced metabolites in S9 fractions of duck liver M. KOLF-CLAUW*, F. AYOUNI, D. TARDIEU, P. GUERRE Unité de Mycotoxicologie, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, BP87214, 31076 Toulouse Cedex FRANCE * Corresponding author: E-mail: m.kolf-clauw@envt.fr Revue 10 REMANIEE 18/10/07 9:42 Page 504