**3. Conclusions**

Mycotoxin analysis showed that 90% of Moroccan AMP samples were positive and 52% presented co-occurrence. Besides the high incidence in samples, the concentration ranged from 0.35 ng/g (ENA1) to 309 ng/g (AOH). The most detected were AOH (85%) and ZEN (27.5 %), while ZEN + AOH was the most frequent co-occurrent mycotoxins (20% of positive samples). AFs were present in 25% of samples (*Lavandula intermedia*, *Myrtus communis,* and *Rosmarinus officinalis),* seven of which exceeded the European and Moroccan recommended levels [19,20,32].

In the present study, a sensitive, rapid, robust, and reliable LC–MS/MS method was validated for the simultaneous determination of 15 target mycotoxins in five different species of AMP: *Origanum vulgare, Rosmarinus officinalis, Matricaria chamomilla, Myrtus communis,* and *Verveine officinale.* A new LC–QTOF–MS method was applied for the simultaneous screening of non-target mycotoxins and conjugated mycotoxins in positive AMP samples. ZEN-14-Glc (11%) and ZEN-14-Sulf (9%) conjugated mycotoxins were detected in AMP samples. The strategy of combining QTOF–MS and MS/MS detectors with LC is a powerful approach for the routine monitoring of mycotoxin and conjugated mycotoxins in contaminated AMPs and other foodstuffs, providing quality and safety to the food industry and consumers.

#### **4. Material and Methods**

#### *4.1. Chemicals and Reagents*

Standards of mycotoxins (four aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), beauvericin (BEA), four enniatins (ENA, ENA1, ENB, and ENB1), zearalenone (ZEN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), alternariol (AOH), tentoxin (TENT), T-2, and HT-2 toxins) were purchased from Sigma Aldrich (St. Louis, MO, USA). Individual stock solutions containing a concentration of 1000 μg/mL were prepared in methanol. Working solutions were prepared starting from the appropriate individual stock solutions. All solutions were prepared and stored in the dark at −20 ◦C. Methanol and acetonitrile (≥ 99.9% purity) liquid chromatography tandem mass spectrometry grades (LC-MS/MS) were supplied by VWR international Eurolab (Barcelona, Spain). Formic acid (≥ 98%) was obtained from Sigma Aldrich (St. Louis, MO, USA ). Ammonium formate (≥ 99.995%) and chloroform (CHCl3) (99%) were obtained from Merck KGaA (Darmstadt, Germany). Ethyl acetate (EtOAc) (HPLC-grade, > 99.5%) was purchased from Alfa Aesar (Karlsruhe, Germany). The water used was purified (≤ 10 MΩ cm<sup>−</sup><sup>1</sup> resistivity) in the laboratory using a Milli-Q SP® Reagent water system (Millipore, Bedford, MA, USA).

### *4.2. Plant Sampling*

A total of forty AMP samples were randomly collected in 2019 from local markets (retailers and supermarkets) in three different areas of Rabat (Morocco): Témara, Bitat, and Kamra. All samples belonged to eight varieties of AMP plants (*Origanum vulgare* (*n* = 12), *Rosmarinus officinalis* (*n* = 7), *Myrtus communis* (*n* = 5), *Matricaria chamomilla* (*n* = 5), *Verveine officinale* (*n* = 4), *Mentha spicata* (*n* = 2), *Lavandula Intermedia* (*n* = 3), and *Artemisia absinthium* (*n* = 2)). The selection of these plants was based on the criteria that they are among the most traditionally used plants and consumed by the Moroccan population for their aromatic and/or therapeutic properties [46]. The amount of each AMP sample was at least 50 g, packed in bags and stored in a dark and dry place until analysis.

#### *4.3. Mycotoxin Extraction Procedure*

The sensitive and accurate analysis of mycotoxins in complicated matrices (e.g., herbs) typically involves challenging sample pretreatment procedures and an efficient detection instrument. A modified DLLME method [47] was applied to extract the studied mycotoxins. Firstly, 2 g of AMP samples were boiled with 200 mL of water for 5 min in a glass container. Next, 10 mL of the aqueous solution tea filtrated with Whatman filter paper was placed in a conical polytetrafluoroethyl (PTFE) centrifuge tube (15 mL), and 2 g of NaCl was added. Then, 1.9 mL of acetonitrile (dispersion solvent) and 1.24 mL of ethyl acetate (extraction solvent) was added and vortexed for 1 min. It was centrifuged for 5 min at 4000 rev/min at 5 ◦C, using Eppendorf centrifuge 5810R (Eppendorf, Hamburg, Germany), and a cloudy solution of the three phases was formed. The organic phase at the top (Tube 1) was recovered and placed in a second PTFE centrifuge tube (15 mL, Tube 2), while the remaining residue (Tube 1) was saved for a second extraction with 3.2 mL of a mixture of methanol and chloroform (60:40, *v*/*v*). Then, Tube 1 was vortexed for 1 min, and, with a

centrifugation at 4000 rev/min for 5 min at 5 ◦C, the separated organic phase was recovered and added to the collected organic phase in Tube 2.

Both separated organic phases in PTFE centrifuge Tube 2 were evaporated to dryness under a nitrogen stream using a TurboVap LV evaporator (Zymark, Hopkinton, MA). The dried residue was reconstituted with 1 mL of methanol and water (70:30, *v*/*v*), and filtered through a 13 mm/0.22 μm nylon filter (Membrane Solutions, Plano, TX, USA). Next, 20 μL of the filtrate was injected into the LC-MS/MS analysis.

#### *4.4. Analysis of Mycotoxins by LC-MS/MS*

Analysis of the mycotoxins was performed using an LC Agilent 1200 with a binary pump and an automatic injector, and coupled to a 3200 QTRAP ®ABSCIEX (Applied Biosystems, Foster City, CA, USA) equipped with a Turbo-VTM source (ESI) interface. The chromatographic separation of the analytes was conducted at 25 ◦C with a reverse phase analytical column Gemini ® NX-C18 (3 μm, 150 × 2 mm ID) and a guard column C18 (4 × 2 mm, ID; 3 μm). The mobile phase was a time programmed gradient using water (0.1% formic acid and 5 mM of ammonium formate) as phase A, and methanol (0.1% formic acid and 5 mM of ammonium formate) as phase B [48]. The following gradient was used: equilibration for 2 min at 90% A, 80–20% A in 3 min, 20% A for 1 min, 20–10% A in 2 min, 10% A for 6 min, 10–0% A in 3 min, 100% B for 1 min, 100–50% B in 3 min, return to initial conditions in 2 min and maintain during 2 min. The flow rate was 0.25 mL/min in all steps. The total run time was 21 min.

Regarding mycotoxin analysis, the QTRAP System was used as the triple quadrupole mass spectrometry detector (MS/MS). The Turbo-V ™ source was used in a positive mode to analyze the 15 mycotoxins with the following settings for source/gas parameters: Vacuum Gauge (10 × 10−<sup>5</sup> Torr) 3.1, curtain gas (CUR) 20, ion spray voltage (IS) 5500, source temperature (TEM) 450 ◦C, and ion source gas 1 (GS1) and ion source gas 2 (GS2) 50. The fragments monitored (retention time, quantification, ion, and confirmation ion) and spectrometric parameters (de-clustering potential, collision energy, and cell exit potential) used were those performed previously (Juan et al. 2019), and are shown in Table 1.

### *4.5. Method Validation*

Validation of the LC-MS/MS method was performed for linearity, repeatability (intraday and inter-day precision), and sensitivity, following the EU Commission Decision 2002/657/EC (EC, 2002). Matrix-matched calibration curves were constructed at concentration levels between the LOQ to 1 μg/mL. The matrix effect (ME) was assessed for each analyte by comparing the slope of the standard calibration curve (standard) with that of the matrix-matched calibration curve (matrix) for the same concentration levels. The limit of detection (LOD) and limit of quantification (LOQ) were estimated for a signal-to-noise ratio (S/N) ≥ 3 and ≥ 10, respectively, from chromatograms of samples spiked at the lowest level validated. LOD and LOQ values were established as a mean of the LOD and LOQ for each matrix and a mix with all studied matrixes, in this way taking into account the possible heterogeneity of the samples. Accuracy of the studied mycotoxin extraction from AMP samples was determined by a mix blank samples fortification procedure. The mix blank was prepared using each AMP sample studied (*Origanum vulgare, Rosmarinus officinalis, Matricaria chamomilla Myrtus communis, Verveine officinale*, *Mentha spicata*, *Lavandula Intermedia,* and *Artemisia absinthium*), which initially tested negative, and was fortified before the extraction procedure with three different mycotoxin levels. The concentrations of studied mycotoxins for reproducibility and repeatability studies in AMP samples were at LOQ, 2 LOQ, and 10 LOQ. Three replicates were prepared for each spiking level. Intra-day precision (repeatability) and inter-day precision (reproducibility) of the method were carried out by spiking the mix blank at the three levels previously indicated. Method precision was estimated by calculating the relative standard deviation (RSDR) using the results obtained during intra-day and inter-day replicate analysis (*n* = 9).

#### *4.6. Analysis of Mycotoxin Metabolites by LC-QTOF-MS*

The QTOF LC/MS analysis was carried out using an Agilent Technologies 1200 Infinity Series LC coupled with an Agilent Technologies 6540 UHD Accurate-Mass Q-TOF-LC/MS (Agilent Technologies, Santa Clara, CA, USA), equipped with an electrospray ionization Agilent Technologies Dual Jet Stream ion source (Dual AJS ESI). Chromatographic separation was achieved on a Gemini® NX-C18 (3 μm, 150 × 2 mm ID) and a guard column C18 (4 × 2 mm, ID; 3μm). The mobile phase consisted of 0.1% formic acid in water milli-Q (solvent A) and methanol (solvent B) with a 25 min gradient. The mobile phase gradient (10–95% B) steps were applied as follows: 0–2 min, 10% B; 2–5 min, 70% B; 5–7 min, 80% B; 7–8 min, 90% B; maintained 4 min at 90% B; 12–16 min, 95% B; 16–18 min, 50% B; 18–22 min, and 10% B. The injection volume was 10 μL.

A mass spectrometry analysis was used with the following QTOF-MS conditions: drying gas flow (N2), 8.0 L min−1; nebulizer pressure, 45 psi; gas drying temperature, 370 ◦C; capillary voltage, 3500 V; fragmentor voltage, 130 V; skimmer voltage, 65 V; and octopole RF peak, 750 V. The Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI) interface was used in the positive and negative ionization modes, and ions were acquired in the range of 100–1000 *m*/*z* for MS scans, and 50–1000 *m*/*z* for auto MS/MS scans, at a scan rate of five spectra/s for MS and three spectra/s for MS/MS, respectively. Automatic acquisition mode MS/MS was carried out using the following collision energy values: *m*/*z,* 20 eV; *m*/*z,* 30 eV and 40 eV. Internal mass correction was enabled by using two reference masses at 121.0509 and 922.0098 *<sup>m</sup>*/*<sup>z</sup>*. Instrument control and data acquisition were performed using Agilent MassHunter Workstation software B.08.00. All of the MS and MS/MS data of the validation standards were integrated by MassHunter Qualitative Analysis B.10.0 and MassHunter Quantitative Analysis B.10.0 (Agilent Technologies).

#### *4.7. Risk of Dietary Exposure*

Risk of dietary exposure calculation/evaluation, in the present study, consisted of measuring the presence of mycotoxins in analyzed samples. It also consisted of characterizing the distribution of one or more mycotoxin for estimating population exposure upon the consumption of average or extremely high amounts [49]. The probable daily intake (PDI, ng.kg−1.bw.day−1) of each mycotoxin through AMP consumption was estimated based on the concentration of each mycotoxin detected in the samples, the daily consumption rate of AMPs, and the average body weight of an individual consumer (70 kg). It is important to highlight that this is for genotoxic substances and substances classified as carcinogenic by the International Agency for Research on Cancer (IARC) [41] (such as AFs or OTA). EFSA, through a CONTAM Panel, considered the possibility of applying a margin of exposure (MOE) approach, with a benchmark dose lower confidence limit, for a benchmark response of 10% (BMDL10) [50]. Nonetheless, recent studies from Portugal [26] have conducted this calculation for AFs by using the TDI of 0.2 ng/kg b.w./day already proposed [51]. To characterize the risk for each mycotoxin, the PDIs were compared with the TDIs of mycotoxins established by JECFA (2001) [52] and SCF (2002) [53]. The percentage of tolerable daily intake (%TDI) from the consumption of green tea was calculated as follows: %TDI = PDI/TDI × 100.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2072-6 651/13/2/125/s1, Figure S1: Total ion current (TIC) chromatogram from a positive sample and its extracted ion chromatogram (EIC) of β-ZEL and ZEN-14-Glc (a), scan and product ion spectrum of β-ZEL (b), and scan and product ion spectrum of ZEN-14-Glc (**c**)., Table S1: Recoveries of intraday (*n* = 3) study of spiked AMP samples and matrix.

**Author Contributions:** Data curation, A.E.J.; Formal analysis, C.J.; Funding acquisition, A.Z.; Investigation, A.J.-G. and C.J.; Methodology, C.J.; Project administration, A.Z. and J.M.; Resources, A.E.J. and A.Z.; Supervision, A.Z., A.J.-G., J.M., S.E., and C.J.; Visualization, C.J.; Writing—original draft, A.E.J. and C.J.; Writing—review & editing, A.E.J., A.J.-G., and C.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Spanish Ministry of Science and Innovation PID2019- 108070RB-I00ALI and the Generalitat Valenciana GVPROMETEO2018-126, Generalitat Valenciana GV 2020/020 and PHC Maghreb project (09MAG20). A. Zinedine would like to thank the CNRST of Morocco and the Moroccan Ministry of Higher Education and Scientific Research (MENESRSFC) for their support.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare that there are no conflicts of interest regarding the publication of this paper.
