Analysis of Pyrrolizidine Alkaloids in botanicals, herbs, spices and herbal medicinal products
1,2-unsaturated pyrrolizidine alkaloids (PAs) and related N-oxides are secondary plant metabolites that are highly toxic for both human beings and animals. Adherence to the most recently specified limit values for herbal medicinal products and certain foodstuffs constitutes a major challenge throughout the entire supply chain for these products and requires highly specialised analytical techniques.
Although pyrrolizidine alkaloids (PAs) have been identified in around 350 plant species so far, chemotaxonomic studies suggest that they are expected to occur in approximately 6000 plant species. PAs are particularly prevalent in the Boraginaceae, Asteraceae (Senecionae and Eupatoriae tribes) and Fabaceae (Crotalaria genus) families. Concentrations in the single-digit percentage range are found in certain plant organs.
PAs primarily serve the plants as a means of warding off herbivores. PA-producing plants exhibit a characteristic PA spectrum, which is typical for the family, genus or individual species. This being the case, any PAs found in a sample offer a means of identifying the noxious weed concerned.
Structure and classification
Pyrrolizidine alkaloids are monoesters or diesters of 1-hydroxymethyl pyrrolizidine (necine base) and aliphatic monocarboxylic or dicarboxylic acids (necic acids) with a carbon chain length of 5 to 10 carbon atoms. The structural types are differentiated according to the nature and positioning of the esterification: monoester, open chain diester and cyclic diester. Four basic types are defined according to the degree of saturation and the stereochemistry of the necine base (figure). Around 600 different PAs have been identified in the plant kingdom to date.
1,2-unsaturated pyrrolizidine alkaloids are classified as genotoxic carcinogens. The IARC (International Association for Research on Cancer) evaluates PA as being "possibly carcinogenic to humans (group 2b)". It is therefore inappropriate to derive a tolerable daily intake (TDI) on the basis of the relevant toxicological models. Using the BMDL10, as the basis, a margin of exposure (MOE) of 10,000 or more is therefore recommended as the safety margin (EFSA 2011, 2017). This applies to long-term exposure as adopted for the classification of foodstuffs.
Apart from their long-term genotoxic and carcinogenic effects, PAs in higher doses have potentially serious chronic toxic effects (hepatic veno-occlusive disease, HVOD), which manifest themselves after a few weeks to a few months. In the references, a dose limit for these effects is defined between 0.1 µg/kg b.w. and 1.0 µg/kg b.w., whereas the BfR, adopting a conservative approach, defines a health based guidance value (HBGV) of 0.1 µg/kg b.w. (BFR 2013a/b, BFR 2020).
Acute toxic effects including liver failure may occur at very high doses in the mg/kg b.w. range. The BfR defines the boundary between acute and chronic exposure at 14 days (BfR 2013b).
The risk of PA contamination is just as high for herbal medicinal products as it is for plant-based food and feed. As they cannot be avoided completely with current state-of-the-art technology, maximum levels cannot be calculated on the basis of toxicological factors alone. Triggered by the results of a BfR research project on the presence of PAs in herbal infusions and teas published in 2013, industrial associations and official control laboratories gathered data relating to the occurrence of PAs in these and other foods on a massive scale and made the data available to the European Food Safety Authority (EFSA) and the EMA Committee on Herbal Medicinal Products (HMPC). At the same time, collectors, growers and producers of medicinal drugs and herbs invested a great deal of time and effort in researching root causes and on finding and implementing measures to reduce PA contamination. The knowledge acquired in the course of this process, which took several years, provided the foundation for the definition of maximum levels (limit values) for certain foodstuffs and categories of food by the European Commission in 2020 and for herbal medicinal products by the HMPC in 2021.
Limit values for pyrrolizidine alkaloids in food as laid down in Commission Regulation (EU) 2020/2040
|Limit values for pyrrolizidine alkaloids in food as laid down in Commission Regulation (EU) 2020/2040|
|Foodstuffs||Maximum levels (μg/kg)|
|8.4.1||Herbal infusions (dried product) with the exception of the herbal infusions referred to in 8.4.2. and 8.4.4||400|
|8.4.2||Herbal infusions of rooibos, anise (Pimpinella anisum), lemon balm, chamomile, thyme, peppermint, lemon verbena (dried product) and mixtures exclusively composed of these dried herbs with the exception of the herbal infusions referred to in 8.4.4||75|
|8.4.3||Tea (Camellia sinensis) and flavoured tea (Camellia sinensis) (dried product) with the exception of the tea and flavoured tea referred to in 8.4.4||100|
|8.4.4||Tea (Camellia sinensis), flavoured tea (Camellia sinensis) and herbal infusions for infants and young children (dried product)||75|
|8.4.5||Tea (Camellia sinensis), flavoured tea (Camellia sinensis) and herbal infusions for infants and young children (liquid)||1,0|
|8.4.6||Food supplements containing herbal ingredients including extracts with the exception of the food supplements referred to in 8.4.7||400|
|8.4.7||Pollen based food supplements||500|
|8.4.8||Borage leaves (fresh, frozen) placed on the market for the final consumer||750|
|8.4.9||Dried herbs with the exception of the dried herbs referred to in 8.4.10||400|
|8.4.10||Borage, lovage, marjoram and oregano (dried) and mixtures exclusively composed of these dried herbs||1000|
The maximum permissible daily intake of PAs in herbal medicinal products amounts to 1µg (adults). A maximum PA level must be calculated from this maximum daily intake for every medicinal product or active ingredient respectively and monitored by means of a validated procedure that satisfies the requirements imposed by the European Pharmacopeia.
The requirements imposed on methods for the quantitative analysis of PAs have now been mandatorily defined in the European Pharmacopeia (Ph.Eur. 10.6) on the basis of the LC-MS/MS procedure qualified by the BfR within the framework of interlaboratory proficiency testing. Since PA limits for herbal medicinal products are defined as maximum daily intake values (in this case 1 µg/day for an adult) rather than maximum levels, suitable specifications must be derived for each individual product according to the daily intake of the active ingredient. Commission Regulation (EU) 2020/2040 of 11 December 2020 lays down limit values and assessment criteria for PAs in foodstuffs. The procedure developed in PhytoLab's laboratory has been fully validated and accredited and it includes all 21(35) of the pyrrolizidine alkaloids required for food analysis and all 28 of the pyrrolizidine alkaloids required for the analysis of medicinal products.
We conduct quantitative analysis for pyrrolizidine alkaloids in medicinal herbs and herbal preparations under GMP conditions in accordance with the requirements laid down in the European Pharmacopeia, chapter 2.8.26.
The performance, evaluation and documentation of pyrrolizidine alkaloid analyses require a particularly high level of technological excellence and analytical and regulatory know-how. PhytoLab has occupied a leading position in the PA analysis of dried raw botanical materials and extracts, and products made from these, such as botanicals, tea, herbal infusions, spices and food supplements, for many years now.
Our experts would be happy to help you. Feel free to contact us.
BfArM 2016: Bekanntmachung zur Prüfung des Gehalts an Pyrrolizidinalkaloiden zur Sicherstellung der Qualität und Unbedenklichkeit von Arzneimitteln, die pflanzliche Stoffe bzw. pflanzliche Zubereitungen oder homöopathische Zubereitungen aus pflanzlichen Ausgangsstoffen als Wirkstoffe enthalten https://www.bfarm.de/SharedDocs/Bekanntmachungen/DE/Arzneimittel/besTherap/bm-besTherap-20160301-pa-pdf.html (Seite besucht am 20.07.2021)
BfR (Bundesinstitut für Risikobewertung) Updated risk assessment on levels of 1,2-unsaturated pyrrolizidine alkaloids (PAs) in foods. BfR Opinion 026/2020 issued 17 June 20201 DOI 10.17590/20200805-100055
Bodi, D; Ronczka, S.; Gottschalk, CH.; Behr, N.; Skibba, A.; Wagner, M.; Lahrssen-Wiederholt, M.; Preiss-Weigert, A.; These, A. (2014): Determination of pyrrolizidine alkaloids in tea, herbal drugs and honey, Food Additives and Contaminants: Part A, DOI: 10.1080/19440049.2014.964337
EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2011. Scientific Opinion on Pyrrolizidine alkaloids in food and feed. EFSA Journal 2011;9(11):2406, 134 pp. https://doi.org/10.2903/j.efsa.2011.2406
EFSA (European Food Safety Authority), 2016. Dietary exposure assessment to pyrrolizidine alkaloids in the European population. EFSA Journal 2016;14(8):4572, 50 pp. doi:10.2903/j.efsa.2016.4572
EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Knutsen HK, Alexander J, Barreg_ard L, Bignami M, Br€uschweiler B, Ceccatelli S, Cottrill B, Dinovi M, Edler L, Grasl-Kraupp B, Hogstrand C, Hoogenboom LR, Nebbia CS, Oswald IP, Petersen A, Rose M, Roudot A-C, Schwerdtle T, Vleminckx C, Vollmer G, Wallace H, Gomez Ruiz JA and Binaglia M, 2017. Statement on the risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA Journal 2017;15(7):4908, 34 pp. https://doi.org/10.2903/j.efsa.2017.4908
EC 2020a: COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs, last amended by Commission Regulation (EU) 2020/1322 of 23 September 2020
EC 2020: COMMISSION REGULATION (EU) 2020/2040 of 11 December 2020 amending Regulation (EC) No 1881/2006 as regards maximum levels of pyrrolizidine alkaloids in certain foodstuffs. Official Journal of the European Union, 14.12.2020
EMA Herbal Medicinal Products Committee (HMPC): Public statement on the use of herbal medicinal products containing toxic, unsaturated pyrrolizidine alkaloids (PAs) EMA/HMPC/893108/2011
European Pharmacopeia, 2.8.26 contaminant pyrrolizidine alkaloids
Kaltner F, Rychlik M, Gareis M, Gottschalk CH: Occurrence and Risk Assessment of Pyrrolizidine Alkaloids in Spices and Culinary Herbs from Various Geographical Origins. Toxins 2020, 12, 155; doi:10.3390/toxins12030155
Mulder, P.P.J.; Lopez, Sanchez P.; Castelari, M.; Bodi, D.; Ronczka, S.; Preiss-Weigert, A.; These, A. Occurrence of pyrrolizidine alkaloids in animal- and plant-derived food: Results of a survey across Europe. Food additives and contaminants - Part A [35 (1), 2018, S. 118-133]
These, A.; Bodi, D.; Ronczka, S.; Lahrssen-Wiederholt, M.; Preiss-Weigert, A.. Structural screening by multiple reaction monitoring as a new approach for tandem mass spectrometry: presented for the determination of pyrrolizidine alkaloids in plants. Analytical and bioanalytical chemistry [405 (29), 2013, S. 9375-9383]
Schrenk, D.; Gao, L.; Lin, G.; Mahony, C.; Mulder, P.P.J.; Peijnenburg, A.; Pfuhler, S.; Rietjens, I.M.C.M.; Rutz, L.; Steinhoff, B.; These, A.. Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment - A review. Food and chemical toxicology [136, 2020, 111107]