**1. Introduction**

Mycotoxins are the most common contaminants in agricultural crops produced by several species of mold and fungi. During growth, maturity, harvest, storage and processing of food and animal feed products, the fungus produces mycotoxins and other secondary metabolites [1]. These mycotoxin-contaminated food and feed threaten human and animal health even at very low concentration [2]. Various degrees of toxicity in food commodities can cause acute or chronic diseases such as immune suppression, cancer, pathological lesions and growth problems [3,4]. Moreover, the presence of mycotoxins in consuming animal products such as milk and meat are a significant safety concern as well [5,6]. Hence, creating an accurate and fast analytical method to quantify the contamination levels of mycotoxins plays a vital role in food and feed safety assessment risks.

Among hundreds of mycotoxins, a few have been recognized as a food safety concern including aflatoxins, fumonisins, ochratoxin A, zearalenone, deoxynivalenol, nivalenol, diacetoxyscirpenol, T-2 and HT-2. After two decades of research, simultaneous quantitative determination of mycotoxins and their derivatives in one analysis is challenging due to the wide polarity, solubility and physicochemical properties of these compounds.

Food crops and feed materials can be easily exposed to moisture that is needed for the growth of molds and fungi. High level of contamination by these mycotoxins has been frequently reported in food and feed. This attracted much attention in recent years due to high risk of contamination and consumption of these commodities all over the world [7,8].

Among a wide variety of mycotoxin sample preparations, accelerated solvent extraction [9], ultrasonic extraction [10], liquid-liquid extraction [11], immunoaffinity column [12] and solid-phase extraction [13] have been intensively researched. Most of the existing methods suffer from poor recovery, insufficient sensitivity and non-reproducibility. This makes these methods unsuitable for simultaneous determination of multi-mycotoxin. Chemical diversity, polarity and solubility of the mycotoxins are important characteristics that can significantly affect extraction efficiency.

Despite all these significant features, co-eluting of matrix components is the most challenging issue. Co-eluting matrix components creates enhancement or suppression of an analyte ionization affecting quantitation [14,15]. Matrix-matched calibration, standard addition and isotopic internal standards are the common solutions for compensation of this problem [16]. Matrix-matched calibration standards were used to reduce the interferences from the extraction process and improve the quantitation results. Therefore, developing an extraction technique to overcome all these challenges is daunting.

A variety of analytical instrumentations have been reported for mycotoxins such as TLC, GC-MS, LC-MS, HPLC-FLD, HPLC-UV and LC-MS/MS [17–21]. Liquid chromatography coupled with triple mass spectroscopy is the most recognized analytical instrumentation for the wide range of chemical contaminants such as mycotoxins in agricultural commodities. LC-MS/MS is known as a sensitive, selective, specific and efficient technique because of its versatility and reliability [22].

The objective of this work was to develop a robust and reliable extraction and clean up technique for multi-mycotoxin in a wide range of agricultural commodities by using LC-MS/MS and validate it through different approaches. The results illustrated here show that this sample preparation method can be applied in many laboratories analyzing feed materials.
