Development of a sensitive analytical method for determining 44 pyrrolizidine alkaloids in teas and herbal teas via LC-ESI-MS/MS
Pyrrolizidine alkaloids (PA) and PA-N-oxides (PANO) are a large group of secondary plant metabolites comprising more than 660 compounds. Exhibiting geno- and hepatotoxic properties, they are responsible for multiple cases of food and feed poisoning over the last 100 years. For food and feed safety reasons, relevant PA/PANO should be monitored extensively in the main sources of PA/PANO intake. In this study, a sensitive analytical method was developed for detecting a broad range of 44 commercially available PA/PANO compounds, and in-house validation procedures were performed for several (herbal) teas. Various extraction solvents and procedures, as well as solid phase extraction materials for sample clean-up and analyte concentration, were tested to establish the methods’ efficiency and effectiveness. Chromatographic conditions were optimised to obtain the best possible separation of isomers for the 44 PA/PANO analytes. The final method was proven very sensitive and accurate, with detection limits ranging from 0.1 to 7.0 μg/kg and precisions between 0.7 and 16.1%. For 40 of the analytes, the recovery rates ranged from 60.7 to 128.8%. The applicability and trueness of the method were examined by analysing tea samples from a local supermarket and comparing them to a reference material. At least one PA/PANO analyte was detected in 17 of the 18 samples under investigation, and the sum contents of the samples ranged from 0.1 to 47.9 μg/kg. Knowledge of the PA/PANO composition in a sample can be used to indicate the botanical origin of the impurity and, thus, the geographical region of cultivation.
KeywordsPyrrolizidine alkaloids Herbal tea Method development Liquid chromatography tandem-mass spectrometry
German Federal Institute for Risk Assessment
Benchmark dose lower confidence limit 10%
European Food Safety Authority
Positive electrospray ionisation
Hydrophilic interaction liquid chromatography
High-performance liquid chromatography
Liquid chromatography tandem mass spectrometry
Limit of detection
Limit of quantification
Margin of exposure
Multiple reaction monitoring
Multiple tandem mass spectrometry
Relative standard deviation
Strong cation exchange
Solid phase extraction
The authors gratefully acknowledge the skilful technical assistance of Carmen Piller, Michaela Freitag and Helmut Ziemann during the course of the study.
This IGF Project No. 19010 of the FEI was supported via AiF within the programme for promoting the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs and Energy (BMWi), based on a resolution of the German Parliament. This project was additionally supported by the Adalbert-Raps Foundation (Kulmbach, Germany) and the Brigitte and Wolfram Gedek-Foundation (Ismaning, Germany).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies involving human participants or animals performed by any of the authors.
- 1.Hartmann T, Witte L. Chemistry, biology and chemoecology of the pyrrolizidine alkaloids. In: Pelletier SW, editor. Alkaloids: chemical and biological perspectives: Elsevier; 1995. p. 155–233.Google Scholar
- 3.Mattocks AR. Chemistry and toxicology of pyrrolizidine alkaloids: Academic; 1986.Google Scholar
- 6.Roder E. Pyrrolizidine alkaloid containing medicinal plants. Dtsch Apoth Ztg. 1992;132:2427–35.Google Scholar
- 8.Colegate SM, Stegelmeier BL, Edgar JA. Dietary exposure of livestock and humans to hepatotoxic natural products. Animal Feed Contamination: Effects on Livestock and Food Safety 2012:352–382.Google Scholar
- 9.Chauvin P, Dillon JC, Moren A. An outbreak of Heliotrope food poisoning, Tadjikistan, November 1992-March 1993. Sante. 1994;4:263–8.Google Scholar
- 14.Schneider J, Tsegaye Y, Tensae MW, Selassie SG, Haue T, Bane A, et al. Veno-occlusive liver disease: a case report. Ethiop Med J. 2012;50:47–51.Google Scholar
- 15.EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Knutsen HK, Alexander J, Barregard L, Bignami M, Brüschweiler B, et al. Statement on the risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J. 2017;15:4908–42.Google Scholar
- 16.Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Off J Europ Communities L. 2002;140:10–22.Google Scholar
- 17.BfR. Opinion no. 030/2016: pyrrolizidine alkaloids: levels in foods should continue to be kept as low as possible. Berlin: BfR (Federal Institute for Risk Assessment); 2016.Google Scholar
- 26.Zhu L, Ruan JQ, Li N, Fu PP, Ye Y, Lin G. A novel ultra-performance liquid chromatography hyphenated with quadrupole time of flight mass spectrometry method for rapid estimation of total toxic retronecine-type of pyrrolizidine alkaloids in herbs without requiring corresponding standards. Food Chem. 2016;194:1320–8.CrossRefGoogle Scholar
- 27.Roder E, Wiedenfeld H, Kersten R. The pyrrolizidine alkaloids of Senecio aquaticus huds. Sci Pharm. 1990;58:1–8.Google Scholar
- 28.DIN 32645:2008-11 Chemical analysis decision limit, detection limit and determination limit under repeatability conditions—terms, method, evaluation. Beuth Verlag GmbH: Berlin.Google Scholar
- 29.BfR. PT-PA01 final report: international proficiency testing on the determination of pyrrolizidine alkaloids in herbal teas. Berlin: BfR (Federal Institute for Risk Assessment); 2016.Google Scholar
- 32.Gottschalk C, Huckauf A, Dübecke A, Kaltner F, Zimmermann M, Rahaus I, et al. Uncertainties in the determination of pyrrolizidine alkaloid levels in naturally contaminated honeys and comparison of results obtained by different analytical approaches. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2018;35:1366–83.CrossRefGoogle Scholar
- 37.Joosten L, Mulder PPJ, Vrieling K, Van Veen JA, Klinkhamer PGL. The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods. Phytochem Anal. 2010;21:197–204.Google Scholar
- 39.EC. Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs (text with EEA relevance). Off J Europ Communities L. 2006;70:12–34.Google Scholar
- 42.Mulder PPJ, López Sánchez P, These A, Preiss-Weigert A, Castelari M. Occurrence of pyrrolizidine alkaloids in food. EFSA Supporting Publication; 2015(EN-859): 116 pp.Google Scholar