Use of a new LC-MS method for the determination of pyrrolizidine alkaloids in feeds

Open access


Introduction: Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

Material and Methods: PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis.

Results: The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%.

Conclusions: A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

1. Bodi D., Ronczka S., Gottschalk C., Behr N., Skibba A., Wagner M., Lahrssen-Wiederholt M., Preiss-Weigert A., These A.: Determination of pyrrolizidine alkaloids in tea, herbal drugs, and honey. Food Addit Contam Part A 2014, 31, 1886–1895.

2. Bolechová M., Čáslavský J., Pospíchalová M., Kosubová P.: UPLC–MS/MS method for determination of selected pyrrolizidine alkaloids in feed. Food Chem 2015, 170, 265–270.

3. Boppré M.: The ecological context of pyrrolizidine alkaloids in food, feed, and forage: an overview. Food Addit Contam Part A 2011, 28, 260–281.

4. Boppré M., Colegate S.M., Edgar J.A.: Pyrrolizidine alkaloids of Echium vulgare honey found in pure pollen. J Agric Food Chem 2005, 53, 594–600.

5. Chen L., Mulder P.P.J., Louisse J., Peijnenburg A., Wesseling S., Rietjens I.M.C.M.: Risk assessment for pyrrolizidine alkaloids detection in (herbal) teas and plant supplements. Regul Toxicol Pharm 2017, 86, 292–302.

6. Cramer L., Schiebel H.M., Ernst L., Beuerle T.: Pyrrolizidine alkaloids in the food chain: development, validation, and application of a new HPLC-ESI-MS/MS sum parameter method. J Agric Food Chem 2013, 61, 11382–11391.

7. Crews C., Berthiller F., Krska R.: Update on analytical methods for toxic pyrrolizidine alkaloids. Anal Bioanal Chem 2010, 396, 327–338.

8. de Nijs M., Mulder P.P.J., Klijnstra M.D., Driehuis F., Hoogenboom R.L.A.P.: Fate of pyrrolizidine alkaloids during processing of milk of cows treated with ragwort. Food Addit Contam Part A 2017, 34, 2212–2219.

9. Danezis G.P., Anagnostopoulos C.J., Liapis K., Koupparis M.A.: Multi-residue analysis of pesticides, plant hormones, veterinary drugs, and mycotoxins using HILIC chromatography MS/MS in various food matrices. Anal Chimica Acta 2016, 942, 121–138.

10. Dreger M., Stanislawska M., Krajewska-Patan A., Mielcarek S., Mikolajczak P.L., Buchwald W.: Pyrrolizidine alkaloids - chemistry, biosynthesis, pathway, toxicity, safety, and perspectives of medicinal usage. Herba Pol 2009, 55, 127–147.

11. Dübecke A., Beckh G., Lüllmann C.: Pyrrolizidine alkaloids in honey and bee pollen. Food Addit Contam Part A 2011, 28, 348–358.

12. Edgar J.A., Colegate S.M., Boppre M., Molyneux R.J.: Pyrrolizidine alkaloids in food: a spectrum of potential health consequences. Food Addit Contam Part A 2011, 28, 308–324.

13. Edgar J.A., Molyneux R.J., Colegate S.M.: Pyrrolizidine alkaloids: potential role in the aetiology of cancers, pulmonary hypertension, congenital anomalies, and liver disease. Chem Res Toxicol 2015, 28, 4–20.

14. Edgar J.A., Roeder E., Molyneaux R.J.: Honey from plants containing pyrrolizidine alkaloids: a potential threat to health. J Agric Food Chem 2002, 50, 2719–2730.

15. European Food Safety Authority, EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific opinion on pyrrolizidine alkaloids in food and feed. EFSA J 2011, 9:2406, 1–134.

16. Fu P.P., Xia Q., Lin G., Chou M.W.: Pyrrolizidine alkaloids-genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev 2004, 36, 1–55.

17. Griffin C.T., Danaher M., Elliott C.T., Kennedy D.G., Furey A.: Detection of pyrrolizidine alkaloids in commercial honey using liquid chromatography-ion trap mass spectrometry. Food Chem 2013, 136, 1577–1583.

18. Gottschalk C., Ronczka S., Preiß-Weigert A., Ostertag J., Klaffke H., Schafft H., Lahrssen-Wiederholt M.: Pyrrolizidine alkaloids in natural and experimental grass silages and implications for feed safety. Anim Feed Sci Tech 2015, 207, 253–261.

19. Hoogenboom L.A., Mulder P.P., Zeilmaker M.J., van den Top H.J., Remmelink G.J., Brandon E.F., Klijnstra M., Meijer G.A., Schothorst R., Van Egmond H.P.: Carry-over of pyrrolizidine alkaloids from feed to milk in dairy cows. Food Addit Contam Part A 2011, 28, 359–372.

20. Huybrechts B., Callebaut A.: Pyrrolizidine alkaloids in food and feed on the Belgian market. Food Addit Contam Part A 2015, 32, 1939–1951.

21. Kowalczyk E., Kwiatek K.: Determination of pyrrolizidine alkaloids in selected feed materials with gas chromatography-mass spectrometry. Food Addit Contam Part A 2017, 34, 853–863.

22. Kowalczyk E., Sieradzki Z., Kwiatek K.: Determination of pyrrolizidine alkaloids in honey with sensitive gas chromatography-mass spectrometry method. Food Anal Methods 2018, 11, 1345–1355.

23. Martinello M., Cristofoli C., Gallina A., Mutinelli F.: Easy and rapid method for the quantitative determination of pyrrolizidine alkaloids in honey by ultra performance liquid chromatography-mass spectrometry: An evaluation in commercial honey. Food Control 2014, 37, 146–152.

24. Merz K.H., Schrenk D.: Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines. Toxicol Lett 2016, 263, 44–57.

25. Mulder P.P.J., Beumer B., Oosterink E., de Jong J.: Dutch survey pyrrolizidine alkaloids in animal forage, RIKILT Report No 2009.018, Wegeningen 2009.

26. Mulder P.P.J., López Sánchez P., These A., Preiss-Weigert A., Castellari M.: Occurrence of pyrrolizidine alkaloids in food. 2015, EFSA:EN-859, 1–116.

27. Mulder P.P.J., de Witte S.L., Stoopen G.M., van der Meulen J., van Wikselaar P.G., Gruys E., Groot M.J., Hoogenboom R.L: Transfer of pyrrolizidine alkaloids from various herbs to eggs and meat in laying hens. Food Addit Contam Part A 2016, 33, 1826–1839.

28. SANTE/11945/2015 Guidance document on analytical quality control and method validation procedures for pesticides residues analysis in food and feed. Supersedes SANCO/12571/2013, Implemented by 01/01/2016, 1–42.

29. Schulz M., Meins J., Diemert S., Zagermann-Muncke P., Goebelm R., Schrenkm D., Schubert-Zsilaveczm M., Abdel-Tawabm M.: Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas. Phytomedicine 2015, 22, 648–656.

30. Stegelmeier B.L., Edgar J.A., Colegate S.M., Gardner D.R., Schoch T.K., Coulombe R.A., Molyneux R.J.: Pyrrolizidine alkaloid plants, metabolism and toxicity. J Nat Toxins 1999, 8, 95–116.

Journal of Veterinary Research

formerly Bulletin of the Veterinary Institute in Pulawy

Journal Information

IMPACT FACTOR J Vet Res 2017: 0.811

CiteScore 2017: 0.68

SCImago Journal Rank (SJR) 2017: 0.29
Source Normalized Impact per Paper (SNIP) 2017: 0.484


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 304 304 46
PDF Downloads 200 200 17