**1. Introduction**

The EAs are mycotoxins produced by several species of fungi in the genus *Claviceps*. In Europe *Claviceps purpurea* is the most widespread and it commonly affects cereals such as rye, wheat, triticale, barley, millets and oats [1]. During fungi infection, healthy kernels are replaced by dark mycelial masses known as sclerotia (also known as ergots, or ergot bodies) that contain high concentrations of various EAs [2].

The toxicity of EAs is well known and has been characterized [3,4]. Though some are cytotoxic and antimicrobial, most are primarily neurotropic. Today, ergotism has practically been eliminated as a human disease, but it remains an important veterinary problem, particularly in cattle, horses, sheep, pigs and poultry [5].

Based on the twelve EAs predominantly present in the sclerotia of *C. purpurea*, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) concluded that chemical analysis should focus mainly on ergometrine (EM), ergometrinine (EMI), ergosine (ES), ergosinine (ESI), ergotamine (ET), ergotaminine (ETI), ergocornine (EC), ergocorninine

**Citation:** Lattanzio, V.M.T.; Verdini, E.; Sdogati, S.; Caporali, A.; Ciasca, B.; Pecorelli, I. Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities. *Toxins* **2021**, *13*, 871. https://doi.org/10.3390/ toxins13120871

Received: 28 October 2021 Accepted: 29 November 2021 Published: 6 December 2021

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(ECI), mixture of α- and β-isomers of ergocryptine (EKR) and ergocryptinine (EKRI), ergocristine (ECR) and ergocristinine (ECRI) (Figure 1). The -inine epimers are described to be biologically inactive, however, an interconversion occurs under alkaline or acidic conditions and, thus, the CONTAM Panel based its risk assessment on both forms (-ine and -inine) [3].

**Figure 1.** Structure of ergot alkaloids.

Although, today, advanced cleaning procedures prior to milling are rather effective, EAs are still found in food and feed commodities, sometimes at relatively high levels [6,7]. The occurrence data on EAs in food and feed submitted to EFSA indicates that ET, ECR, ES and EC mostly contribute to the overall content of EAs. Furthermore, the highest concentrations of EAs were reported for rye (grains, milling) products and by-products [3,4]. The CONTAM Panel recommended, that efforts should continue to collect more data on occurrence of the above EAs in relevant food and feed commodities. Special attention should be paid to processed food and to specific foods consumed by vegetarians or raw grain consumers. Moreover, the CONTAM Panel underlined the need for commercially available reference standards, such as for isotope-labelled internal standards and certified reference materials (CRM) for the analysis of EAs.

As a follow up to the conclusions and the information contained in the EFSA opinion, a Commission Recommendation on the monitoring of the presence of EAs in feed and

food has been in force since 2012 to stimulate analytical data collection regarding the occurrence of EAs identified in the EFSA opinion in relevant food and feed commodities. Furthermore, the Commission Recommendation encouraged the collection of specific information on the relationship between the presence of ergot sclerotia and the level of individual EAs in food and feed in order to set appropriate limits [8]. Finally, in response to EFSA Recommendation, regarding "harmonised performance criteria for the analysis of EAs in feed and food" [3], the Committee agreed that the method of analysis used for the monitoring of ergot alkaloids should have a limit of quantification (LOQ) of 20 μg/kg as a minimum acceptable criterion, but preferably, this value should be 10 μg/kg or lower [9].

Recently, on 24 August 2021, the European Commission published Regulation (EU) 2021/1399 [10], amending Regulation (EC) 1881/2006 [11]. The new Regulation sets the maximum permitted limits for the sum of the above mentioned 12 EAs as lower-bound concentrations (i.e., calculated on the assumption that all values of the different individual ergot alkaloids below the limit of quantification are equal to zero) in cereal-based food products (Table 1). The limits for these alkaloids relate to barley, wheat, spelt, oats, rye and processed cereal-based foods for infants and children and will apply from 1 January 2022. In the Regulation, a higher maximum permitted level is set for milling products containing bran (identified on the base of ash content) taking into account the absorption by cereals of dust containing high levels of EAs.

**Table 1.** Maximum permitted level for ergot alkaloids in food by the European Commission (Commission Regulation (EU) 2021/1399). <sup>a</sup> Effective from 1 July 2024.


The maximum levels of EAs set in Regulation (EU) 2021/1399 imply that the analytical methods, for enforcement purposes, should have a LOQ lower than the value previously established by the EU document [9]. Specifically, if calculated according to the formula reported in the UNI CEN/TR 16059:2010 [12] the LOQ for monitoring of milling products, bran milling products/grain for human consumption for cereal other than rye and processed cereal-based baby foods shall be equal to 4.0, 6.0 and 0.8 μg/kg per each ergot compound, respectively.

Different methods have been reported in the literature for the analysis of ergot alkaloids, mainly liquid chromatographic methods coupled to fluorometric or tandem massspectrometric detectors (FLD or MS/MS). A critical review can be found in Chung 2021 [13], discussing the advantages and disadvantages of available methods for determination of EAs in cereals and feed, covering the period from 2008 to 2020. The review points out that, although both LC-FLD and LC-MS/MS can be used for the analysis of the 12 EUrecommended EAs, the latter has a greater sensitivity, but it is affected by a matrix effect especially, for EM and EMI. Another analytical challenge, stressed in the review, is the co-elution of alpha and beta isomers of EKR (α-EKR, β-EKR) for most of the reported methods, due to the use of C18 analytical columns. A proficiency test, conducted in 2017, revealed that an acceptable resolution was obtained with phenyl-hexyl as a stationary phase [14]. Finally, the review underlines that only very few reported methods can fulfil

the regulated LOQs for individual epimers in processed cereal-based food for infants and young children owing to its lower limit. Recently a modified QuEChERS-based method coupled to LC-MS/MS as a detection technique was successfully validated for the detection and quantification of EAs in dry cereal-based baby foods with individual LOQs of 0.5 μg/kg [15], however the method did not provide the separation of the α and β isomers of EKR and EKRI.

A standardized method, for the determination of EAs in cereals and cereal products by dispersive solid phase extraction (dSPE) clean-up and LC-MS/MS, has been recently issued by the European Committee for Standardization for official control purposes [16]. The method has been validated in the range of 13.2 μg/kg to 168 μg/kg for the sum of the twelve EAs, in rye flour, rye bread and cereal products (breakfast cereals, infant breakfast cereals and crispbread) that contained rye as an ingredient, as well as seeded wholemeal flour and a barley and rye flour mixture. Method performances were satisfactory in the range 24.1 μg/kg to 168 μg/kg for sum of EAs, whereas for concentrations below 24.1 μg/kg the method resulted to be only suitable for screening purposes.

Due to analytical challenges in the EAs determination, the occurrence of data available in the literature are scarce and provide a limited picture of EAs distribution worldwide.

The present manuscript reports on monitoring data of EAs in cereal and cereal-derived products collected in Italy over the period 2017–2020, as requested by the national implementation of the monitoring recommendations [8]. To these purposes a LC-MS/MS method for the determination of EAs in cereal and derived products has been optimized and in house validated to verify its fitness for purpose. Validation data will be reported and discussed, also taking into account the recently issued Regulation requirements.

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