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Development of a sensitive analytical method for determining 44 pyrrolizidine alkaloids in teas and herbal teas via LC-ESI-MS/MS

  • Florian KaltnerEmail author
  • Benedikt Stiglbauer
  • Michael Rychlik
  • Manfred Gareis
  • Christoph Gottschalk
Research Paper

Abstract

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.

Keywords

Pyrrolizidine alkaloids Herbal tea Method development Liquid chromatography tandem-mass spectrometry 

Abbreviations

AcIm

Acetylintermedine

AcImN

Acetylintermedine-N-oxide

AcLy

Acetylycopsamine

AcLyN

Acetylycopsamine-N-oxide

BfR

German Federal Institute for Risk Assessment

BMDL10

Benchmark dose lower confidence limit 10%

Ec

Erucifoline

EcN

Erucifoline-N-oxide

Em

Echimidine

EmN

Echimidine-N-oxide

Eu

Europine

EuN

Europine-N-oxide

EFSA

European Food Safety Authority

ESI+

Positive electrospray ionisation

HILIC

Hydrophilic interaction liquid chromatography

HPLC

High-performance liquid chromatography

Ht

Heliotrine

HtN

Heliotrine-N-oxide

Ic

Indicine

IcN

Indicine-N-oxide

Ig

Integerrimine

IgN

Integerrimine-N-oxide

Im

Intermedine

ImN

Intermedine-N-oxide

Jb

Jacobine

Jb

Jacobine-N-oxide

Jl

Jacoline

JlN

Jacoline-N-oxide

Lc

Lasiocarpine

LcN

Lasiocarpine-N-oxide

LC-MS/MS

Liquid chromatography tandem mass spectrometry

LOD

Limit of detection

LOQ

Limit of quantification

Ly

Lycopsamine

LyN

Lycopsamine-N-oxide

Mc

Monocrotaline

McN

Monocrotaline-N-oxide

Mk

Merenskine

MkN

Merenskine-N-oxide

MOE

Margin of exposure

MRM

Multiple reaction monitoring

Mx

Merepoxine

MxN

Merepoxine-N-oxide

MS

Mass spectrometry

MS3

Multiple tandem mass spectrometry

PA

Pyrrolizidine alkaloid

PANO

Pyrrolizidine alkaloid-N-oxide

RP

Reversed phase

Rs

Retrorsine

RsN

Retrorsine-N-oxide

RSD

Relative standard deviation

S/N

Signal-to-noise ratio

Sc

Senecionine

ScN

Senecionine-N-oxide

SCX

Strong cation exchange

Sl

Sceleratine

SlN

Sceleratine-N-oxide

Sp

Seneciphylline

SPE

Solid phase extraction

SpN

Seneciphylline-N-oxide

Sk

Senkirkine

Sv

Senecivernine

SvN

Senecivernine-N-oxide

Td

Trichodesmine

Notes

Acknowledgements

The authors gratefully acknowledge the skilful technical assistance of Carmen Piller, Michaela Freitag and Helmut Ziemann during the course of the study.

Funding information

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.

Ethical approval

This article does not contain any studies involving human participants or animals performed by any of the authors.

References

  1. 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
  2. 2.
    Smith LW, Culvenor CCJ. Plant sources of hepatotoxic pyrrolizidine alkaloids. J Nat Prod. 1981;44:129–52.CrossRefGoogle Scholar
  3. 3.
    Mattocks AR. Chemistry and toxicology of pyrrolizidine alkaloids: Academic; 1986.Google Scholar
  4. 4.
    Boppré M. The ecological context of pyrrolizidine alkaloids in food, feed and forage: an overview. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011;28:260–81.CrossRefGoogle Scholar
  5. 5.
    Fu PP, Xia Q, Lin G, Chou MW. Pyrrolizidine alkaloids—genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev. 2004;36:1–55.CrossRefGoogle Scholar
  6. 6.
    Roder E. Pyrrolizidine alkaloid containing medicinal plants. Dtsch Apoth Ztg. 1992;132:2427–35.Google Scholar
  7. 7.
    Merz KH, 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.CrossRefGoogle Scholar
  8. 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. 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
  10. 10.
    Anjos BL, Nobre VMT, Dantas AFM, Medeiros RMT, Oliveira Neto TS, Molyneux RJ, et al. Poisoning of sheep by seeds of Crotalaria retusa: acquired resistance by continuous administration of low doses. Toxicon. 2010;55:28–32.CrossRefGoogle Scholar
  11. 11.
    Kakar F, Akbarian Z, Leslie T, Mustafa ML, Watson J, Van Egmond HP, et al. An outbreak of hepatic veno-occlusive disease in western Afghanistan associated with exposure to wheat flour contaminated with pyrrolizidine alkaloids. J Toxicol. 2010;2010:313280.CrossRefGoogle Scholar
  12. 12.
    Ruan J, Yang M, Fu P, Ye Y, Lin G. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis. Chem Res Toxicol. 2014;27:1030–9.CrossRefGoogle Scholar
  13. 13.
    Edgar JA, Colegate SM, Boppré M, Molyneux RJ. Pyrrolizidine alkaloids in food: a spectrum of potential health consequences. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011;28:308–24.CrossRefGoogle Scholar
  14. 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. 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. 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. 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
  18. 18.
    Cao Y, Colegate SM, Edgar JA. Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination. Phytochem Anal. 2008;19:526–33.CrossRefGoogle Scholar
  19. 19.
    Valese AC, Molognoni L, de Sá Ploêncio LA, de Lima FG, Gonzaga LV, Górniak SL, et al. A fast and simple LC-ESI-MS/MS method for detecting pyrrolizidine alkaloids in honey with full validation and measurement uncertainty. Food Control. 2016;67:183–91.CrossRefGoogle Scholar
  20. 20.
    Cramer L, Schiebel HM, 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;61:11382–91.CrossRefGoogle Scholar
  21. 21.
    Mädge I, Cramer L, Rahaus I, Jerz G, Winterhalter P, Beuerle T. Pyrrolizidine alkaloids in herbal teas for infants, pregnant or lactating women. Food Chem. 2015;187:491–8.CrossRefGoogle Scholar
  22. 22.
    Dübecke A, Beckh G, Lüllmann C. Pyrrolizidine alkaloids in honey and bee pollen. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011;28:348–58.CrossRefGoogle Scholar
  23. 23.
    Bodi D, Ronczka S, Gottschalk C, Behr N, Skibba A, Wagner M, et al. Determination of pyrrolizidine alkaloids in tea, herbal drugs and honey. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2014;31:1886–95.CrossRefGoogle Scholar
  24. 24.
    Chung SWC, Lam CH. Development of an analytical method for analyzing pyrrolizidine alkaloids in different groups of food by UPLC-MS/MS. J Agric Food Chem. 2018;66:3009–18.CrossRefGoogle Scholar
  25. 25.
    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. Anal Bioanal Chem. 2013;405:9375–83.CrossRefGoogle Scholar
  26. 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. 27.
    Roder E, Wiedenfeld H, Kersten R. The pyrrolizidine alkaloids of Senecio aquaticus huds. Sci Pharm. 1990;58:1–8.Google Scholar
  28. 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. 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
  30. 30.
    Hessel S, Gottschalk C, Schumann D, These A, Preiss-Weigert A, Lampen A. Structure-activity relationship in the passage of different pyrrolizidine alkaloids through the gastrointestinal barrier: ABCB1 excretes heliotrine and echimidine. Mol Nutr Food Res. 2014;58:995–1004.CrossRefGoogle Scholar
  31. 31.
    Kaltner F, Rychlik M, Gareis M, Gottschalk C. Influence of storage on the stability of toxic pyrrolizidine alkaloids and their n-oxides in peppermint tea, hay, and honey. J Agric Food Chem. 2018;66:5221–8.CrossRefGoogle Scholar
  32. 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
  33. 33.
    Greco G, Grosse S, Letzel T. Serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine. J Sep Sci. 2013;36:1379–88.CrossRefGoogle Scholar
  34. 34.
    Van de Schans MGM, Blokland MH, Zoontjes PW, Mulder PPJ, Nielen MWF. Multiple heart-cutting two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry of pyrrolizidine alkaloids. J Chrom A. 2017;1503:38–48.CrossRefGoogle Scholar
  35. 35.
    Picron JF, Herman M, Van Hoeck E, Goscinny S. Analytical strategies for the determination of pyrrolizidine alkaloids in plant based food and examination of the transfer rate during the infusion process. Food Chem. 2018;266:514–23.CrossRefGoogle Scholar
  36. 36.
    Mulder PPJ, López Sanchez P, Castelari M, Bodi D, Ronczka S, Preiß-Weigert A, et al. Occurrence of pyrrolizidine alkaloids in animal- and plant-derived food: results of a survey across Europe. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2018;35:118–33.CrossRefGoogle Scholar
  37. 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
  38. 38.
    Colegate SM, Edgar JA, Knill AM, Lee ST. Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum. Phytochem Anal. 2005;16:108–19.CrossRefGoogle Scholar
  39. 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
  40. 40.
    Bredenkamp MW, Wiechers A, van Rooyen PH. A new pyrrolizidine alkaloid from Senecio latifolius DC. Tetrahedron Lett. 1985;26:929–32.CrossRefGoogle Scholar
  41. 41.
    Griffin CT, O’Mahony J, Danaher M, Furey A. Liquid chromatography tandem mass spectrometry detection of targeted pyrrolizidine alkaloids in honeys purchased within Ireland. Food Anal Methods. 2015;8:18–31.CrossRefGoogle Scholar
  42. 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
  43. 43.
    Mathon C, Edder P, Bieri S, Christen P. Survey of pyrrolizidine alkaloids in teas and herbal teas on the Swiss market using HPLC-MS/MS. Anal Bioanal Chem. 2014;406:7345–54.CrossRefGoogle Scholar
  44. 44.
    Schulz M, Meins J, Diemert S, Zagermann-Muncke P, Goebel R, Schrenk D, et al. Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas. Phytomedicine. 2015;22:648–56.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Florian Kaltner
    • 1
    • 2
    Email author
  • Benedikt Stiglbauer
    • 2
  • Michael Rychlik
    • 2
  • Manfred Gareis
    • 1
  • Christoph Gottschalk
    • 1
  1. 1.Chair of Food Safety, Faculty of Veterinary MedicineLudwig-Maximilians-University of MunichOberschleissheimGermany
  2. 2.Chair of Analytical Food Chemistry, TUM School of Life Science WeihenstephanTechnical University of MunichFreisingGermany

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