## 1 Introduction  
In recent years, the awareness about natural toxins of plant origin in food and feed, such as pyrrolizidine alkaloids (PAs), has risen as one of the main issues of food safety. PAs are probably the most widely extended natural plant toxins, as more than 600 different chemical structures of these alkaloids have been identified from over more than 6000 plant species, being the main PA-producing plants belonging to the families Asteraceae, Fabaceae, Doraginaceae, Orchidaceae and Apocynaceae [1]. In this sense, these alkaloids can be introduced into the food chain from different vegetables and botanical sources. In some cases, these PA-producing plants are directly consumed by animals (through forage) and humans (e.g., borage, salads, food supplements, teas and herbal infusions made of PAs producing plants, such as chamomile or rooibos, etc.). However, currently, the major sources of PA consumption in animal and humans seem to be feed and plant-derived products contaminated with PA-producing plants, as many of these plants grow in fields as weeds, leading to the contamination of food crops [2, 3]. Accordingly, several different contamination paths have been reported, such as cross-contamination during harvesting processes, natural horizontal transfer through soil, as well as food fraud and adulteration [2, 3, 4]. As a consequence, many food alerts have notified in the last years high levels of these alkaloids in a wide variety of food products: spices and aromatic herbs (57% of the food alerts notified), teas and herbal teas (15% of the food alerts notified), food supplements (13%of the food alerts notified), herbs (8% of the food alerts notified), pollen (7% of the food alerts notified) and honey (1% of the food alerts notified) [2; 5]. Nonetheless, these alkaloids have also been detected in other plant-derived products, such as cereals, flours and salads, as well as in some animal-derived products such as milk and dairy products, eggs, meat and meat products [2; 6; 7; 8; 9]. However, contamination of PAs in products of animal origin is less frequent, and the concentration levels found of these alkaloids are often low in this type of foodstuff. The intake of PAs represents a potential health risk, as they are known to produce both acute and chronic effects. In this sense, the ingestion of these alkaloids is mainly associated with liver damage (hepatic veno-oclusive disease (HVOD), liver cirrhosis and liver failure), but they can also produce genotoxic and carcinogenic effects at long-term exposure. Some of them (monocrotaline, riddelline, and lasiocarpine) have been classified as potential carcinogens to humans (Group 2B) by the International Agency for Research on Cancer (IARC) [10; 11; 12; 13]. Therefore, due to the wide spread of these alkaloids in a large variety of food products and the potential health risk that their frequent intake may entail for consumers, the analytical control of these alkaloids in food and feed is of utmost importance and constitutes a matter of interest. For this reason, in December 2020, the European Commission published a regulation amending Regulation (EC) No. 1881/2006 to monitor the occurrence of PAs in some food products [14]. This regulation sets maximum total concentration levels of PAs (ranging from 1.0 to 1000 ug/kg) in different foodstuffs, including: tea and herbal infusions, herbal food supplements, pollen-based food supplements, pollen, pollen products, dried herbs and cumin seeds [14]. Moreover, according to this legislation, every analytical methodology used to monitor these contaminants in food or feed must include the analysis of 21 PAs (including their \(N\)-oxide forms, PANOs). At the same time, 14 additional PAs can be considered if the chromatographic method employed enables the individual separation and identification of them without coalition problems, as they are isomers of one or more of the previous mentioned 21 PAs that are known to co-elute with some of them [14]. The coelution of these isomers is one of the main issues in the analysis of PAs.