Pyrrolizidine alkaloids and tropane alkaloids in fennel seeds

According to Commission Regulation (EU) 2020/2040 of the European Commission amending regulation (EC) No 1881/2006 maximum levels of pyrrolizidine alkaloids apply to certain foodstoff from 1 July 2022 on. According to Commission Regulation (EU) 2021/1408 new maximum levels of tropane alkaloids apply from 1 September 2022 on.

The maximum level refers to 35 pyrrolizidine alkaloids as mentioned in the respective regulation. A selection of the 35 pyrrolizidine alkaloids are within the scope of this ring test. Futhermore, the tropane alkaloids hyoscyamine and scopolamine are covered by this test. 

Up to now, there is no standardised method available for the quanification of PAs in foodstuff.

As a consequence, questions are raised like

  • how to deal with co-eluting PAs,
  • which analytical standards should be used for quantification (especially for co-eluting PAs),
  • is there a need for (labelled) internal standards,
  • what is the state-of-the-art in separating the co-eluting PAs,
  • how should the co-eluting PAs be reported,
  • and many more...

Thus, the aim of the ring test is to summarise and work out the state-of-the-art in analysing PAs in foods like fennel seeds. 


  • milled fennel seeds are provided as test material. The test material contains spiked and incurred levels of pyrrolizidine alkaloids as well as spiked levels of tropane alkaloids. The blank material might contain incurred levels of pyrrolizidine alkaloids.
  • a questionnaire is provided, where the participants are asked for details related to the applied analytical methods.
  • the labs are asked to provide a chromatogramm of the co-eluting PAs (standard substance and probably also related to the test material) in order to show how good/bad the separation of the co-eluting PAs works in the respective lab.
  • the correct identification of the spiked PAs is part of the evaluation of the ring test.
  • the results are evaluated with respect to the comparability (z-score) and the trueness (70 to 120 % of the spiked level) for all spiked PAs and the TAs.
  • The results are evaluated with respect to the comparability (z-score) for all incurred PAs. 

For reasons of fairness, only laboratories, which submit chromatograms resp. answers related to the questionnaire receive the respective part of the evaluation of the ring test.

Reporting limits of 2 µg/kg are required for all PAs as well as reporting limits of 2.5 µg/kg are required for the TAs.


echimidine (Em), echimidine-N-oxide (EmN), europine (Eu), europine-N-oxide (EuN), heliotrine (Hn), heliotrine-N-oxide (HnN), intermedine (Im), intermedine-N-oxide (ImN), lasiocarpine (Lc), lasiocarpine-N-oxide (LcN), lycopsamine (La), lycopsamine-N-oxide (LaN), retrorsine (Re), retrorsine-N-oxide (ReN), senecionine (Sc), senecionine-N-oxide (ScN), seneciphylline (Sp), seneciphylline-N-oxide (SpN), senecivernine (Sv), senecivernine-N-oxide (SvN), senkirkine (Sk), echinatine, echinatine-N-oxide, heliosupine, heliosupine-N-oxide, indicine, indicine-N-oxide, integerrimine, integerrimine-N-oxide, rinderine, rinderine-N-oxide, spartioidine, spartioidine-N-oxide, usaramine, usaramine-N-oxide, hyoscyamine, scopolamine

Expected time schedule

Please register before:
10 January 2022
Dispatch of samples:
22 March 2022
Deadline for reporting results:
11 April 2022

Costs of P2202-MRT

Ring test and material Amount Price per unit
Pyrrolizidine alkaloids and tropane alkaloids in fennel seeds 850 €
Test material
Fennel seeds, milled, with spiked levels of TAs and and spiked and incurred levels of PAs.
50 g included
Blank material
Fennel seeds, milled, unspiked, probably with incurred levels of PAs.
50 g included

Note: PROOF-ACS reserves the right to cancel tests in case of less than ten participants.