## 1 Introduction  
Zeralenone (ZEN) is a naturally occurring mycotoxin produced by several species of Fusariumolds [1; 2]. Many crops can be easily infested by these molds and thereby contaminated by ZEN. Hence, humans and animals could be at high risk of being exposed to ZEN through the intake of contaminated food or feed. ZEN showed adverse effects on reproductive systems of mammalian species [2; 3; 4; 5], despite its relatively low acute toxicity. According to some reports regarding blood parameters, ZEN was also evidenced to be haematotoxic and hepatotoxic [3; 6; 7]. After ingestion, ZEN is primarily metabolized to two hydroxyl isomers \(\alpha\)-zearalenol (\(\alpha\)-ZOL) and \(\beta\)-zearalenol (\(\beta\)-ZOL) [1,8,9], which can be subsequently reduced to \(\alpha\)-zearalanol (\(\alpha\)-ZAL) and \(\beta\)-zearalanol (\(\beta\)-ZAL), respectively [10,11]. A small portion of the \(\alpha\)-ZAL is also found to be converted to \(\beta\)-ZAL and zearalanone (ZAN) [12]. Besides, these metabolites as well as ZEN itself can be partially conjugated with glucuronic acid and substantially excreted in bile. The glucuronide of ZEN may also be re-absorbed and metabolized further by intestinal mucosal cells, ultimately entering the liver and the systemic circulation via the portal blood supply [13]. Humans are easily exposed to ZEN through the food consumption [14]. For the past decades, exposure assessment of mycotoxins was conventionally performed by combining the occurrence data with the food consumption data [15, 16, 17, 18]. However, due to the heterogeneous distribution of mycotoxins in food and variability of individual toxicokinetics, this approach is considered to be not quite reliable. Hence, an internal exposure strategy directly detecting mycotoxin biomarkers in biological fluids could be more accurate and reliable for assessment purpose [3]. It seems important to develop a method to satisfy the current need. A number of approaches to quantify ZEN and its metabolites have been proposed for biological samples in last decade, which are commonly performed with gas or liquid chromatography coupled to mass or tandem mass spectrometry [19, 20, 21, 22, 23, 24, 25, 26, 27]. Among these methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS) gradually became a prior technique for mycotoxin analysis in biological samples for its high selectivity and sensitivity. However, since the scarcity of human serum or plasma samples, some of the methods was applied to analyze animal serum or plasma [28,29]. And some of the applications limited their detection to ZEN and one (ZAN) [30] or two (\(\alpha\)-ZOL, \(\beta\)-ZOL) [31] metabolites. Furthermore, ZEN and its metabolites were easy to form conjugates, such as ZEN-14-glucuron acid (ZEN-14-GlcNAc), ZEN-74-GlcNAc and \(\alpha/\beta\)-ZOL-14-GlcNAc in vivo. Some works published previously [20,27] failed to monitor the conjugated forms. A strategy involving enzymatic hydrolysis was then developed and applied to chicken and pig serum [24]. However, the defects of labor-intensive and time-consuming operation (liquid-liquid extractions, solid phase extraction, evaporation and reconstitution) restricted its further application in large-scale sample analysis. Recently, a method has been successfully established to determine the total (free + conjugated) amount of ZEN and its metabolites in human serum, involving enzymatic digestion, sample cleanup and UPLC-MS/MS analysis [32]. This method achieved higher sensitivity but still had drawback of time-consuming due to the step of evaporation and reconstitution. To address such an issue, a material (96-well \(\upmu\)Elution plate) for high-throughput sample preparation was employed in this work, allowing for the simultaneous preparation of multiple samples, avoiding evaporation step and meanwhile reducing the time and labor consumed.