Skip to main content
SpringerLink
Log in
Book cover

Natural Products pp 1049–1068Cite as

Methods for Analysis of Pyrrolizidine Alkaloids

  • Reference work entry
  • First Online:

Abstract

Pyrrolizidine alkaloids poison animals grazing on toxic wild plants and those fed contaminated feed, causing economic losses. They poison humans through deliberate consumption of certain foods and herbal medicines and through consumption of food contaminated by wild plants, such as via transport of the toxins by bees into honey. Analytical methods are required for different purposes – to detect the presence of pyrrolizidine alkaloids, to quantify the total level of the toxins, or to measure the quantity of individual compounds. The task is made more challenging by the variety of PAs, their widespread nature and their different forms. Analytical methods are based on color reactions, enzyme linked immunosorbent assays (ELISAs), spectroscopy, and the full range of chromatographic techniques. A lack of reference standards and validated methods has hampered the application of available methods to food surveys, but has not prevented the development of a number of very useful methods as are described in this chapter.

This is a preview of subscription content, log in via an institution.

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   2,999.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   549.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Abbreviations

ASE:

Accelerated solvent extraction

EFSA:

European Food Safety Authority

ELISA:

Enzyme linked immunosorbent assay

GC:

Gas chromatography

GC-MS:

Gas chromatography with mass spectrometric detection

HPLC:

High performance liquid chromatography

LC (High performance):

Liquid chromatography

LC-MS:

High performance liquid chromatography with mass spectrometric detection

LC-MS/MS:

Liquid chromatography with tandem mass spectrometric detection

LC-UV:

Liquid chromatography with ultra-violet detection

LOD:

Limit of detection

LOQ:

Limit of quantification

MRM:

Multiple reaction monitoring

NMR:

Nuclear magnetic resonance spectroscopy

PA:

Pyrrolizidine alkaloid

PANO:

Pyrrolizidine alkaloid N-oxide

SCX:

Strong cation exchange

SCX-SPE:

Strong cation exchange with solid phase extraction

SIM:

Selected ion monitoring

SPE:

Solid phase extraction

SRM:

Selected reaction monitoring

TLC:

Thin layer chromatography

TOF:

Time of flight mass spectrometry

UV:

Ultra-violet

References

  1. Roeder E (1999) Analysis of pyrrolizidine alkaloids. Curr Org Chem 3:557–576

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. EFSA (2011) EFSA panel on contaminants in the food chain (CONTAM). Scientific opinion on pyrrolizidine alkaloids in food and feed. EFSA J 11:2406

    Google Scholar 

  4. Boppré M, Colegate SM, Edgar JA, Fischer OW (2008) Hepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollen. J Agric Food Chem 56:5662–5672

    Article  Google Scholar 

  5. Joosten L, Mulder PPJ, Vrieling K, van Veen JA, Klinkhamer PGL (2010) The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods. Phytochem Anal 21:197–204

    CAS  Google Scholar 

  6. Lebada R, Schreier A, Scherz S, Resch C, Krenn L, Kopp B (2000) Quantitative analysis of the pyrrolizidine alkaloids senkirkine and senecionine in Tussilago farfara L. by capillary electrophoresis. Phytochem Anal 11:366–369

    Article  CAS  Google Scholar 

  7. Mroczek T, Glowniak K, Wlaszczyk A (2002) Simultaneous determination of N-oxides and free bases of pyrrolizidine alkaloids by cation-exchange solid-phase extraction and ionpair high-performance liquid chromatography. J Chromatogr A 949:249–262

    Article  CAS  Google Scholar 

  8. Hartmann T, Toppel G (1987) Senecionine N-oxide, the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Senecio vulgaris. Phytochemistry 26:1639–1643

    Article  CAS  Google Scholar 

  9. Oberlies NH, Kim NC, Brine DR, Collins BJ, Handy RW, Sparacino CM, Wani MC, Wall ME (2004) Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids. Pub Health Nutr 7:19–24

    Article  Google Scholar 

  10. Colegate SM, Edgar JA, Knill AM, Lee ST (2005) Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum. Phytochem Anal 16:108–119

    Article  CAS  Google Scholar 

  11. Beales K, Colegate SM, Edgar JA (2003) Experiences with the quantitative trace analysis of pyrrolizidine alkaloids using GC-MS and LCMS. In: Acamovic T, Stewart CS, Pennycott TW (eds) Poisonous plants and related toxins. CAB International, Wallingford

    Google Scholar 

  12. Langer T, Franz C (1997) Determination of pyrrolizidine alkaloids in commercial samples of borage seed oil products by GC-MS. Scientia Pharmaceut 65:321–328

    CAS  Google Scholar 

  13. Wretensjö I, Karlberg B (2003) Pyrrolizidine alkaloid content in crude and processed borage oil from different processing stages. J Am Oil Chem Soc 80:963–970

    Article  Google Scholar 

  14. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and ‘dispersive solidphase extraction’ for the determination of pesticide residues in products. J AOAC Int 86:412–431

    CAS  Google Scholar 

  15. Kempf M, Wittig M, Reinhard A, von der Ohe K, Blacquière T, Raezke K-P, Michel R, Schreier P, Beuerle T (2011) Pyrrolizidine alkaloids in honey: comparison of analytical methods. Food Addit Contam A 28:332–347

    Article  CAS  Google Scholar 

  16. Hartmann T, Theuring C, Witte L, Pasteels JM (2001) Sequestration, metabolism and partial synthesis of tertiary pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi. Insect Biochem Mol Biol 31:1041–1056

    Article  CAS  Google Scholar 

  17. Kempf M, Heil S, Hasslauer I, Schmidt L, von der Ohe K, Theuring C, Reinhard A, Schreier P, Beuerle T (2010) Pyrrolizidine alkaloids in pollen and pollen products. Mol Nutr Food Chem 54:292–300

    Article  CAS  Google Scholar 

  18. Cooper RA, Bowers RJ, Beckham CJ, Huxtable RJ (1996) Preparative separation of pyrrolizidine alkaloids by high-speed counter-current chromatography. J Chromatogr A 732:43–50

    Article  CAS  Google Scholar 

  19. Kim NC, Oberlies NH, Brine DR, Handy RW, Wani MC, Wall ME (2001) Isolation of symlandine from the roots of common comfrey (Symphytum officinale) using countercurrent chromatography. J Nat Prod 64:251–253

    Article  CAS  Google Scholar 

  20. Hoogenboom LAP, Mulder PPJ, Zeilmaker MJ, van den Top HJ, Remmelink GJ, Brandon EFA, Klijnstra M, Meijer GAL, Schothorst R, Van Egmond HP (2011) Carry-over of pyrrolizidine alkaloids from feed to milk in dairy cows. Food Addit Contam A 28:359–372

    Article  CAS  Google Scholar 

  21. Hosch G, Wiedenfeld H, Dingermann T, Roder E (1996) A new high-performance liquid chromatography method for the simultaneous quantitative analysis of pyrrolizidine alkaloids and their N-oxides in plant materials. Phytochem Anal 7:284–288

    Article  Google Scholar 

  22. Cao Y, Colegate SM, Edgar JA (2008) Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination. Phytochem Anal 19:526–533

    Article  CAS  Google Scholar 

  23. Beales KA, Betteridge K, Colegate SM, Edgar JA (2004) Solid phase extraction and LC–MS analysis of pyrrolizidine alkaloids in honeys. J Agric Food Chem 52:6664–6672

    Article  CAS  Google Scholar 

  24. Betteridge K, Cao Y, Colegate SM (2005) Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: application to Echium vulgare honeys. J Agric Food Chem 53:1894–1902

    Article  CAS  Google Scholar 

  25. Kempf M, Beuerle T, Bühringer M, Denner M, Trost D, von der Ohe K, Bhavanam VBR, Schreier P (2008) Pyrrolizidine alkaloids in honey: risk analysis by gas chromatography–mass spectrometry. Mol Nutr Food Res 52:1193–1200

    Article  CAS  Google Scholar 

  26. Zhou Y, Li N, Choi FF-K, Qiao C-F, Song J-Z, Li S-L, Liu X, Cai Z-W, Fu PP, Lin G, Xu H-X (2010) A new approach for simultaneous screening and quantification of toxic pyrrolizidine alkaloids in some potential pyrrolizidine alkaloid-containing plants by using ultra performance liquid chromatography–tandem quadrupole mass spectrometry. Anal Chim Acta 681:33–40

    Article  CAS  Google Scholar 

  27. Dübecke A, Beckh G, Lüllmann C (2011) Pyrrolizidine alkaloids in honey and pollen. Food Addit Contam A 28:348–358

    Article  Google Scholar 

  28. Mattocks AR (1967) Detection of pyrrolizidine alkaloids on thin-layer chromatograms. J Chromatogr A 27:505–508

    Article  CAS  Google Scholar 

  29. Dann AT (1960) Detection of N-oxides of the pyrrolizidine alkaloids. Nature 186:1051

    Article  CAS  Google Scholar 

  30. Mattocks AR, Jukes R (1987) New improved field test for toxic pyrrolizidine alkaloids. J Nat Prod 50:161–166

    Article  CAS  Google Scholar 

  31. Witte L, Rubiolo P, Bicchi C, Hartmann T (1993) Comparative analysis of pyrrolizidine alkaloids from natural sources by gas chromatography–mass spectrometry. Phytochemistry 32:187–196

    Article  Google Scholar 

  32. Stelljes ME, Kelley RB, Molyneux RJ, Seiber JN (1991) GC–MS determination of pyrrolizidine alkaloids in four Senecio species. J Nat Prod 54:759–773

    Article  CAS  Google Scholar 

  33. Bredenkamp MW (1991) The mass spectrometry of pyrrolizidine alkaloids. In: Rizk AM (ed) Naturally occurring pyrrolizidine alkaloids. CRC Press, Boca Raton

    Google Scholar 

  34. Bicchi C, Caniato R, Tabacchi R, Tsoupras G (1989) Capillary gas-chromatography positive and negative-ion chemical ionization mass-spectrometry on pyrrolizidine alkaloids of Senecio inaequidens using ammonia and hydroxyl ions as the reagent species. J Nat Prod 52:32–41

    Article  CAS  Google Scholar 

  35. Huizing HJ, Deboer F, Hendriks H, Balraadjsing W, Bruins AP (1986) Positive and negative-ion chemical ionization mass spectrometry of trimethylsilyl derivatives of pyrrolizidine alkaloids using (NH)H+ and (OH)H− as the reactant ions. Biomed Environ Mass Spectrom 13:293–298

    Article  CAS  Google Scholar 

  36. Edgar JA, Lin HJ, Kumana CR, Ng MM (1992) Pyrrolizidine alkaloid composition of three Chinese medicinal herbs Eupatorium cannabinum, E. japonicum and Crotalaria assamica. Am J Chin Med 20:281–288

    Article  CAS  Google Scholar 

  37. Culvenor CCJ, Edgar JA, Smith LW (1981) Pyrrolizidine alkaloids in honey from Echium plantagineum L. J Agric Food Chem 29:958–960

    Article  CAS  Google Scholar 

  38. Hovermale JT, Craig AM (1998) A routine method for the determination of retronecine. Fresen J Anal Chem 361:201–206

    Article  CAS  Google Scholar 

  39. Böhlen M, Kast C, Dübecke A, Zoller O (2011) Sum-analytical determination of pyrrolizidine alkaloids in Swiss honey by GC–MS. In: 5th International symposium on recent advances in food analysis, 1–4 Nov 2011, Prague. Available http://www.rafa2011.eu/pdf/boa2011.pdf. Accessed 8 Nov 2012

  40. Xiong AZ, Yang L, Zhang F, Yang XJ, Wang CH, Wang ZT (2009) Determination of total retronecine esters-type hepatotoxic pyrrolizidine alkaloids in plant materials by pre-column derivatisation high-performance liquid chromatography. Biomed Chromatogr 23:665–671

    Article  CAS  Google Scholar 

  41. Xiong A, Li Y, Yang L, Gao J, He Y, Wang C, Wang Z (2009) Simultaneous determination of senecionine, adonifoline and their metabolites in rat serum by UPLC-ESIMS and its application in pharmacokinetic studies. J Pharm Biomed Anal 50:1070–1074

    Article  CAS  Google Scholar 

  42. Gray DE, Porter A, O′Neill T, Harris RK, Rottinghaus GE (2004) A rapid cleanup method for the isolation and concentration of pyrrolizidine alkaloids in comfrey root. J AOAC Int 87:1049–1057

    CAS  Google Scholar 

  43. Hartmann T, Theuring C, Beuerle T, Ernst L, Singer MS, Bernays EA (2004) Acquired and partially de novo synthesized pyrrolizidine alkaloids in two polyphagous arctiids and the alkaloid profiles of their larval food-plants. J Chem Ecol 30:229–254

    Article  CAS  Google Scholar 

  44. Pawar RS, Grundel E, Mazzola E, White KD, Krynitsky AJ, Rader J (2010) Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicum. J Sep Sci 33:200–205

    Article  CAS  Google Scholar 

  45. Joosten L, Cheng D, Mulder PPJ, Vrieling K, van Veen JA, Klinkhamer PGL (2011) The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris. Phytochemistry 72:214–222

    Article  CAS  Google Scholar 

  46. Mulder PPJ, Beumer B, Oosterink E, de Jong J (2009) Dutch survey pyrrolizidine alkaloids in animal forage. RIKILT report 2009.018. Available from http://edepot.wur.nl/135952. Accessed 8 Nov 2012

  47. Craig JC, Purushothaman KK (1970) An improved preparation of tertiary amine N-oxides. J Org Chem 35:1721–1722

    Article  Google Scholar 

  48. Logie CG, Grue MR, Liddell JR (1994) Proton NMR spectroscopy of pyrrolizidine alkaloids. Phytochemistry 37:43–109

    Article  CAS  Google Scholar 

  49. Roeder E (1990) Carbon-13 NMR-spectroscopy of pyrrolizidine alkaloids. Phytochemistry 29:11–29

    Article  CAS  Google Scholar 

  50. Segall HJ, Dallas JL (1983) H-1-NMR spectroscopy of pyrrolizidine alkaloids. Phytochemistry 22:1271–1273

    Article  CAS  Google Scholar 

  51. Fletcher MT, Hayes PY, Somerville MJ, De Voss JJ (2011) Crotalaria medicaginea associated with horse deaths in northern Australia: new pyrrolizidine alkaloids. J Agric Food Chem 59:11888–11892

    Article  CAS  Google Scholar 

  52. Nuringtyas TR, Choi YH, Verpoorte R, Klinkhamer PG, Leiss KA (2012) Differential tissue distribution of metabolites in Jacobaea vulgaris, Jacobaea aquatica and their crosses. Phytochemistry 78:89–97

    Article  CAS  Google Scholar 

  53. Lee ST, Schoch TK, Stegelmeier BL, Gardner DR, Than KA, Molyneux RJ (2001) Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide. J Agric Food Chem 49:4144–4151

    Article  CAS  Google Scholar 

  54. Roeder E, Pflueger T (1995) Analysis of pyrrolizidine alkaloids: a competitive enzyme-linked immunoassay (ELISA) for the quantitative determination of some toxic pyrrolizidine alkaloids. Nat Tox 3:305–309

    Article  CAS  Google Scholar 

  55. Bober MA, Milco LA, Miller RB, Mount M, Wicks B, Kurth MJ (1989) A competitive enzyme linked immunosorbent assay (ELISA) to detect retronecine and monocrotaline in vitro. Toxicon 27:1059–1064

    Article  CAS  Google Scholar 

  56. Bober MA, Kurt MJ, Milco LA, Roseman DM, Miller RB, Segal HJ (1991) A pyrrolizidine alkaloid enzyme-linked immunosorbent assay detection strategy. ACS Symp Ser 451:176–183

    Article  Google Scholar 

  57. Zundorf I, Wiedenfeld H, Roeder E, Dingermann T (1998) Generation and characterization of monoclonal antibodies against the pyrrolizidine alkaloid retrorsine. Planta Med 64:259–263

    Article  CAS  Google Scholar 

  58. Roseman DM, Wu X, Kurth MJ (1996) Enzyme-linked immunosorbent assay detection of pyrrolizidine alkaloids: immunogens based on quaternary pyrrolizidinium salts. Bioconjug Chem 7:187–195

    Article  CAS  Google Scholar 

  59. Nivarlet N, Andrianne D, Campbell K, Huet A-C, Swinkels A, Crews C, Mulder P, Delahaut Ph, Elliott C, van Egmond H, Granier B (2011) Multiplex lateral flow immunoassays for the detection of pyrrolizidine, tropane and ergot alkaloids. Poster in: ASSET2011: food integrity and traceability conference, Belfast, 21–24 Mar 2011. Available http://www.conffidence.eu/img/enewsletter/enews_ALKALOIDS_DIPSTICK_WP4A_2_.pdf. Accessed 8 Nov 2012

  60. Yu LJ, Li SFY (2005) Dynamic pH junction-sweeping capillary electrophoresis for online preconcentration of toxic pyrrolizidine alkaloids in Chinese herbal medicine. Electrophoresis 26:4360–4367

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Food and Environment Research Agency (FERA), Sand Hutton, York, UK

    Colin Crews

Authors
  1. Colin Crews
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Colin Crews .

Editor information

Editors and Affiliations

  1. Botany Department, M.L. Sukhadia University, Durga Nursery Road, Udaipur, 313001, Rajasthan, India

    Kishan Gopal Ramawat

  2. Institute of Vine and Wine Sciences, Biological-Active Plant Substances Study, University of Bordeaux, 210 Chemin de Leysotte CS 50008, Villenave d'Ornon, 33882, France

    Jean-Michel Mérillon

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer-Verlag Berlin Heidelberg

About this entry

Cite this entry

Crews, C. (2013). Methods for Analysis of Pyrrolizidine Alkaloids. In: Ramawat, K., Mérillon, JM. (eds) Natural Products. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-22144-6_186

Download citation

Publish with us

Policies and ethics

Search

Navigation