*4.3. Reference Materials and Working Solutions*

All reference materials (RMs) of EAs were in desiccated form. Reference solutions were prepared by reconstitution, according to the manufacturer's instructions, obtaining a final concentration of 100 μg mL−<sup>1</sup> for the R epimers of the EAs (ine-epimers) and 25 μg mL−<sup>1</sup> for S epimers of EAs (inine-epimers) respectively. The obtained RMs solutions were stored in amber vials at −20 ◦C.

The working solutions (WS) were prepared by dilution of RMs just before use. For EAs-ine epimers an intermediate working mixed solution at 5 μg mL−<sup>1</sup> was prepared. The intermediate solution of -ine epimers was then combined with single RMs of EAs-inine epimers to obtain a final concentration of 0.5 μg mL−<sup>1</sup> for each molecule.

#### *4.4. Sample Preparation*

Twenty-five grams of sample were weighed in a 250-mL plastic vessel and 100 mL of extraction solution of acetonitrile:ammonium carbonate (200 mg L−<sup>1</sup> (84:16 *v*/*v*)) were added. The samples were mechanically shaken for 30 min. After 15 min of centrifugation at 2780 RCF, 5 mL of extract was collected and loaded into the solid-phase extraction column (MycoSep 150 Ergot). One mL of purified extract was evaporated to dryness at 60 ◦C under a gentle stream of nitrogen. Finally, the sample was reconstituted with 400 μL of ammonium carbonate solution (200 mg/L)/ACN; (50:50 *v*/*v*) and filtered using a 0.2-μm PTFE syringe filter prior to injection into the LC-MS/MS system.

#### *4.5. LC-MS/MS Analysis*

The LC-MS/MS instrumental set up consisted of a Nexera X2 UPLC system (LC-30AD binary pump, CTO-20AC column oven and SIL-30AC autosampler, Shimadzu, Kyoto, Japan, 2015) interfaced to an API 3200 Qtrap mass spectrometer (AB Sciex, Foster City, CA, USA, 2009) equipped with an electrospray (ESI) ion source.

The analysis of EAs was performed in positive ionization mode (ESI+), after separation on an Acquity UPLC BEH C-18 (150 × 2.1 mm, i.d. 1.7 μm) connected to a VanGuard (2.1 × 5 mm) both from Waters (Milford, MA, USA). The column oven was set at 40 ◦C. The flow rate of the mobile phase was 500 μL/min, while the injection volume was 5 μL. Eluent A was a 200-mg/L ammonium carbonate solution and eluent B was acetonitrile. For EAs elution, the starting composition of the eluent was 95% (A) and 5% (B). Then, the following gradient was used: the proportion of eluent B was linearly increased from 5% to 40% over 1 min, then to 50% over the next 3.5 min, then increased to 70%. Finally, it was raised to 99% over 1.5 min and kept constant at 5% for 3 min.

The target mycotoxins were detected in Selected Reaction Monitoring (SRM) mode. The monitored transitions and retention times of single EAs are provided in Table 5. Compliance with SANTE mycotoxin identification criteria for retention time (Rt), chromatographic separation and Ion Ratio (IR) for identification in mass spectrometry was verified (SANTE/12089/2016). Quantification was carried out by external calibration in solvent.


**Table 5.** Retention times and monitored transitions for individual EAs.

Abbreviations: ergometrine (EM), ergometrinine (EMI), ergosine (ES), ergosinine (ESI), ergotamine (ET), ergotaminine (ETI), ergocornine (EC), ergocorninine (ECI), α isomers of ergocryptine (α EKR), α isomers of ergocryptinine (α EKRI), ergocristine (ECR) and ergocristinine (ECRI).

#### *4.6. Method Validation Procedure*

For the method's validation the following parameters were evaluated: LODs, LOQs, instrumental linearity ranges, recovery rates (%), RSDr and RSDWLR, both using relative standard deviation. All parameters' definitions and acceptability criteria are reported in UNI CEN/TR 16059:2010.

4.6.1. Limit of Detection and Limit of Quantification

LOD value were determined according to the "Estimation of LOD via blank samples" method as reported in the "Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food" [26]. Specifically, 10 aliquots of a blank matrix were spiked at 1 μg/kg for all EAs. These spiking levels were fixed as low as possible, considering a S/N ratio ≥3 at the expected LOD. The resulting spiked blank samples were analyzed by LC–MS/MS then an LOD and an LOQ were calculated according to Equations (1) and (2) respectively:

$$\text{LOD} = 3.9 \ast \frac{\text{S}\_{\text{y}\text{b}}}{\text{b}} \tag{1}$$

LOD: limit of detection

Sy,b: standard deviation of the spiked blank signal b: slope of calibration curve

$$\text{LOQ} = \text{3.3} \ast \text{LOD} \tag{2}$$

LOQ: limit of quantification

#### 4.6.2. Linearity Range

Each calibrant solution was prepared by diluting working solutions with acetonitrile/ammonium carbonate solution at 200 mg/L (50:50 *v*/*v*). Calibrant solutions were in the range 0.4–40 ng/mL and were analyzed on three different days over two weeks. Then calibration curve equations were obtained by plotting averaged peak areas vs. concentration of the natural toxin using ordinary least squares (OLS) method, including a (0, 0) point.

The linearity was checked as follows. For each calibration point, y-residuals were obtained by the following Equation (3)

$$y-\text{residuals} = y\_i - \,\beta\_i \tag{3}$$

where *yi* are experimental values used for the regression equation calculation and *y*ˆ*<sup>i</sup>* values are the points on the calculated regression line corresponding to individual x-values.

When, for all points, the residual along y axis were ≤±20%, the calibration curve was considered linear [17].

4.6.3. Recovery, Repeatability and Within-Laboratory Reproducibility

Recoveries, RSDr and RSDWLR for each molecule were evaluated according to UNI CEN/TR 16059:2010.

EAs validation was performed in wheat at three mass fraction levels, specifically 2.5, 5 and 10 μg kg−<sup>1</sup> (corresponding to LOQ, 2xLOQ and 4xLOQ respectively) on two different days by two independent operators under repeatability conditions (eight replicates each). To obtain the WLR data, the two groups were combined and recovery% and RSDWLR were calculated as reported in Table 2.

**Supplementary Materials:** The following are available in online at https://www.mdpi.com/article/ 10.3390/toxins13120871/s1, Table S1: Occurrence data for Ergot Alkaloids: individual data for each toxin.

**Author Contributions:** Conceptualization, V.M.T.L. and I.P.; methodology, I.P. and V.M.T.L.; validation, E.V., S.S., A.C. and B.C.; formal analysis, I.P. and V.M.T.L.; resources, I.P.; data curation, B.C., E.V., I.P.; writing—original draft preparation, B.C., E.V.; writing—review and editing, V.M.T.L. and I.P.; supervision, I.P.; funding acquisition, I.P.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministero della salute (Italian Ministry of Health) (IZSUM 08/16 RC).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in Supplementary Material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

