**1. Introduction**

Aromatic and medicinal plants (AMPs) are known to contain various compounds that can be valorized for several purposes, including preservative, therapeutic, and organoleptic proprieties, most of which are precursors for chemo-pharmaceutical semi-synthesis [1,2]. AMPs have been widely used to treat and/or prevent diseases and promote health since ancient times. However, AMPs are frequently exposed to various fungi responsible for the decrease in market quality, as their growth allows the presence of mycotoxins. Such fungi respond to contamination in soil, or during harvesting, drying, transport, manipulation, or storage [3,4].

**Citation:** El Jai, A.; Zinedine, A.; Juan-García, A.; Mañes, J.; Etahiri, S.; Juan, C. Occurrence of Free and Conjugated Mycotoxins in Aromatic and Medicinal Plants and Dietary Exposure Assessment in the Moroccan Population. *Toxins* **2021**, *13*, 125. https://doi.org/10.3390/ toxins13020125

Received: 8 January 2021 Accepted: 4 February 2021 Published: 8 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

E-46100 Valencia, Spain; ana.juan@uv.es (A.J.-G.); jordi.manes@uv.es (J.M.); cristina.juan@uv.es (C.J.)

Nowadays, several studies have reported the presence of mycotoxins in herbal plants and derivative products, considering that this contamination is a global issue, particularly in developing countries [5]. In fact, previous investigations reported that, under specific conditions, toxigenic fungal species from *Aspergillus, Penicillium, Fusarium,* and *Alternaria* genus can generate mycotoxins and contaminate herbal medicines. Indeed, these reports described mycotoxin contamination of medicinal herbs and related products, showing that mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), fumonisins, and trichothecenes are the most commonly present [6]. Another important point is related to the metabolization that can partially suffer from some mycotoxins, such as ZEN and deoxynivalenol (DON), by the fungus producer and by the infected host plant. The Phase I and II reactions in the metabolism process aim to eliminate these compounds, and this is often accomplished by the attachment of hydrophilic groups. The most indicated enzymatic system in the literature is the UDP-glucosyltransferase (UGT), which is capable of converting ZEN into ZEN-4-glucoside (ZEN-4-Glc) [7–9], or in Zearalenone-14-Glucoside (ZEN-14-Glc) and Zearalenone-16-Glucoside (ZEN-16-Glc) [10]. Furthermore, α-zearalenol ( α-ZEL) and β-zearalenol (β-ZEL) suffer glycosylation as part of the host plant's metabolism, leading to α–ZEL-14-glucoside ( α-ZEL-14-Glc) and β–ZEL-14-glucoside (β-ZEL-14-Glc), respectively [11]. This happens with other mycotoxins, such as DON, which is converted to DON-3-glucoside (DON-3-Glc) [12,13]. Moreover, during fungal metabolism, the sulfate form of ZEN, which is partially converted to Zearalenone-4-sulfate (ZEN-4-Sulf), has been found [14,15]. Despite their chemical alteration, there is evidence that metabolites have a similar toxic potential to those of their precursors when it is ingested, as attached functional groups like glycosylic or sulfate residues are likely to be enzymatically cleaved during digestion [14,16]. In most of the analytical approaches published, the main target of study has been the parental molecules forgetting the detection of their metabolites, resulting in an underestimation of the inherent toxicity of a contaminated sample. Altered metabolites of mycotoxins are also referred to as masked mycotoxins [16], thus, it is important to perform a non-target analysis to study the formation, determination, and significance of masked and other conjugated mycotoxins present in foodstuffs [17].

There is legislation established for mycotoxins in some spices and plants to avoid hazardous effects associated with their presence in herbal material [18]. The European Commission has established maximum limits (MLs) for mycotoxins, such as AFB1 (5 ng/g) and AFs (10 ng/g) in various spices (*Capsicum spp., Piper spp., Myristica fragrans, Zingiber officinale,* and *Curcuma longa*); however, no MLs have been established for aromatic plants [19]. In the European Pharmacopoeia (2016) [20], MLs have been set for AFs as 2 ng/g for AFB1 in herbal drugs and 4 ng/g for the sum of AFs [21]; while in the US Pharmacopeia (USP), ML of 5 ng/g for AFB1 and 20 ng/g for AFs have been implemented for certain types of raw medicinal herb materials, as well as their by-products (in powder and/or dry extract) [6].

In Morocco, several reports have evaluated the presence of mycotoxins in spices and herbal materials [22–24]. Recent Moroccan regulations have set MLs for certain mycotoxins in food products. For example, the ML is set at 5 ng/g for AFB1, 10 ng/g for the sum of AFs, and 15 ng/g for OTA in selected spices, such as *Capsicum spp., Piper spp., Myristica fragrans, Zingiber officinale,* and *Curcuma longa* [25]. However, no information is available regarding the possible co-occurrence of mycotoxins in AMP samples consumed in the country. Thus, the aims of the present study were: (i) to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to determinate 15 mycotoxins in AMPs from Morocco; (ii) to develop a liquid chromatography coupled with a time-of-flight mass spectrometry (LC-QTOF-MS) method as the screening tool to obtain not only confirmation, but also the detection of possible co-occurrence non-target mycotoxins, including masked mycotoxins; and (iii) to apply the developed method in the most consumed Moroccan AMP varieties supplied from the Moroccan market to investigating their co-occurrence. Data obtained from contamination levels will permit the assessment of the risk of dietary exposure for the Moroccan population to these mycotoxins through AMP intake.

#### **2. Results and Discussion**
