Although a maximum level of 500 μg/kg was established in European Union for EAs, on 1 July 2024, there will be a reduction of the maximum levels of EAs for some categories of foods to provide a high level of human health protection. To safeguard human and animal health, the CONTAM panel of the EFSA has established a group acute reference dose of 1 μg/kg body weight and a group tolerable daily intake (TDI) for total ergot alkaloids of 0.6 μg/kg of body weight/day
[8].
The limits established by the European Commission are more restrictive when compared to other countries around the world. In 2004, the FAO published “Worldwide regulations for mycotoxins in food and feed in 2003”, where legal mycotoxin limits can be accessed in several countries around the world
[21]. Australia has established 500 mg/kg for the maximum limit for ergot alkaloids, which is extremely higher than the actual limits in Europe
[21].
6. Determination of Ergot Alkaloids
6.1. Sampling
Sampling is a crucial step in ergot alkaloids determination, as their heterogeneous distribution influences the precision of the determination. Concerning cereal samples, matrices can contain tiny fragments of sclerotia or bulks of EAs, making sampling a step of higher importance
[22].
6.2. Sample Pre-Treatment
Extraction is a step of great importance, as it is responsible for the separation of the analyte from the matrix and sometimes can be followed by a clean-up procedure to eliminate possible interference with the analysis. This pre-treatment of samples is required not only to remove interferences but to pre-concentrate the analytes
[9].
Some pre-treatment techniques such as Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS)
[9][23][24] procedures and solid–liquid extractions
[10][25][26] have been applied over the years to ergot alkaloids. Independently of the extraction technique, the choice of the extraction solvent and optimisation of the procedure conditions are critical to obtain satisfactory results
[14].
6.3. Analytical Methods
Many methods have been reported for ergot alkaloids determination, such as liquid chromatography (LC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), gas chromatography (GC), and thin-layer chromatography (TLC)
[9][10]. Gas chromatography is usually coupled with electron capture detection (ECD), and liquid chromatography can be coupled with different detectors, such as ultraviolet light (UV), fluorescence detector (FLD), evaporative light scattering detector (ELSD), and mass spectrometry (MS)
[7][10][26].
Chromatographic methods are based on the separation of components depending on their affinity to a mobile or stationary phase. These different affinities make different movements in the column, leading to a possible separation of the compounds
[27]. This method makes possible the determination of the major EAs individually and summary of them in order to obtain the total ergot alkaloid content; however, this requires a lot of standards, making this process costly. A more cost-effective approach is to transform the EAs into a common structure before the analyses, which can be achieved by a hydrolysis process where EAs and their epimers are cleaved to an uniform lysergic acid hydrolyze
[28].
Since EAs are non-volatile and can decompose in the injector once they are susceptible to heat, gas chromatographic (GC) techniques have become less applied to these compounds. On the other hand, liquid chromatographic methods are commonly used for polar, non-volatile, or thermally labelled mycotoxins such as EAs
[13][15].
Liquid chromatographic methods such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography (UHPLC) have been applied for EAs determination. With its technological advances, UHPLC has shown to be rapid and efficient for compounds separation, which can be justified for the use of columns packed with submicron particles, making this technique more applied to mycotoxin determination
[12][26]. In respect to detectors, UV is used for EAs quantification; however, UV light conducts the epimerization process, interfering with quantification. Thus, FLD detectors began to be applied not only to offer more specificity and sensitivity but because some EAs are naturally fluorescent. However, mass spectrometry (MS) detectors have become widely used for EAs quantification
[14].
Although chromatographic methods are important for official and reference laboratories to control EAs concentration, it seems to be necessary to develop a fast and cost-effective test system for application in the production locations to make a primary screening for EAs possible. In this sense, the enzyme-linked immunosorbent assay (ELISA) has been applied as solid basis for rapid and sensitive screening of ergot alkaloids. This method is based on the interaction between the mycotoxin and antibodies marked with a conjugate toxin enzyme, as binding of the mycotoxin to the conjugate produces color depending on the amount of binding.
7. Rapid Alert System for Food and Feed (RASFF) Notifications
In the European Union, a safety tool named the Rapid Alert System for Food and Feed (RASFF) was established in order to facilitate the rapid notification and response in case of risk to human health related to food and feed
[29]. This is an important tool that shares rapid information about direct or indirect risk to humans between the member states, the commission, and the authority
[30].
When a member state identifies a risk and reports it to the RASFF, the first notification is received by the European Commission, which verifies the notification and immediately transmits it to the other members, allowing them to take the necessary actions
[31].
To date, only nine RASFF notifications for ergot alkaloids contaminations have been generated, all of them in very recent years (between September 2021 and March 2023). Looking at the results, it can conclude that from all the cereal and cereal-based products, there is a higher incidence of notifications for rye-flour products. A notification from a product from Ireland was the only one whose notification was not related to cereal or cereal-based products but to dietetic foods, food supplements, and fortified foods. Additionally, all the samples were originally from EU countries, with France having with most notifications. The highest values were found in Belgian and German rye flours, and in addition to this, six of the nine notifications were classified as serious risk; however, two of the notifications are still undecided.
8. Decontamination of Mycotoxins
Since mycotoxins contamination leads to economic losses and health concerns, the search for effective decontamination and detoxification has been of great interest
[32]. Decontamination and detoxification methods for mycotoxins should be effective, simple, and inexpensive; use existing technology; and not alter the nutritional value
[33]. The search for an efficient and effective process for the decontamination of mycotoxins from food and feed still remains a practical and scientific global challenge
[34]. When talking about controlling the levels of EAs in cereals, it need to take into account two main stages. The first stage includes pre-harvest practices, which focus on prevention of mycotoxin production or contamination and are mainly based on good agricultural practices (GAP), good manufacturing practices (GMP), and favorable storage practices
[32][35][36]. Pre-harvest strategies are the best way to prevent mycotoxin production in the field, but once mycotoxin contamination occurs, these strategies might not eliminate them, so post-harvest strategies must be applied
[32]. Therefore, post-harvest strategies are the second stage and are based on processing, chemical, physical, and biological techniques, and application of these strategies aims to decontaminate contaminated products
[32][34]. At both stages, hazard analysis and critical control points (HACCP) plays an important role, which involves strategies for mycotoxin prevention, control, and GMPs for all stages of product management; storage strategies; and sorting, segregation, and cleaning procedures
[32].
A compilation of the pre- and post-harvest strategies applied to mycotoxins decontamination is shown at
Figure 3.
Figure 3. Pre- and Post- Harvest Mycotoxin Decontamination Techniques; GAPs, good agricultural practices; GMPs, good manufacturing practices.
Specifically concerning ergot alkaloids decontamination, pre-harvest strategies remain the most important stage, as they are based on GMPs, GAPs, and favorable storage practices. Relative to post-harvest strategies, only a few have been applied to ergot alkaloid decontamination, namely sorting and cleaning as a physical strategy; frying, baking, and peeling as processing techniques; and ammonization as a chemical strategy
[33][35][37][38][39][40].
9. Conclusions
Cereals and seeds have a high risk of contamination by mycotoxins, namely by ergot alkaloids. Due to climate change and the increase in cereal and cereal-based product consumption, it is one of today’s worldwide food safety concerns. For that reason, monitoring, prevention, and control are imperative to minimizing their occurrence.
Good agricultural and manufacturing practices and controlled storage and transport conditions can prevent ergot alkaloid contamination. These preventive strategies together with control analysis of critical points are fundamental. However, when products are already contaminated, physical, chemical, and biological processes are needed for mycotoxins decontamination. Although decontamination processes can be used, many of them can only reduce the toxicity of the ergot alkaloids by promoting the epimerization process. Therefore, the quantification of both epimers must be taken into account.
Many methods have been developed for the determination and quantification of ergot alkaloids in the search for an efficient, sensitive, and cost-effective method for the quantification of both epimers. QuEChERS has been the preferred method for extraction and purification steps, along with chromatographic methods for quantification, like HPLC and UHPLC. The preference for the tandem mass spectrometry (MS/MS) detector is well known over the years due to its unequivocal advantages.