One PT targeted determination of the most prominent EAs in Claviceps purpurea: ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine and their related –inine epimers.
Matrices where EAs are monitored are cereals (wheat, barley, buckwheat, rye, triticale and oat) but also cereal products (cereal flour, bakery products) and compound feed.
Levels in the rye test material varied from 116 (ergometrine/inine) to 752 μg/kg (ergocristine/inine).
Majority of labs gave correct results
Thirty-seven labs, among them 26 National Reference Laboratories for mycotoxins in food and feed from 21 member states plus Iceland and Norway and 11 official control labs participated.
The rating of laboratories' performance was by z-scores. A total of 91% of results were satisfactory while 3.7% were unsatisfactory.
All results for ergocornine/inine were satisfactory, whereas 76 and 86% for a-ergocryptine/inine and ergometrine/inine were satisfactory.
“About 20% more laboratories had a satisfactory performance in the determination of the –ine than in the –inine epimers.
“This can be explained by the fact that the amount of –inine epimers in the test material was lower than the amount of –ine epimers by a factor of 2.7, on average, rendering the analysis more challenging.
“Despite the overall good performance of the laboratories analysing EAs in the test item, this PT highlighted the need to seek a harmonised approach for quantifying a- and β-ergocryptine/inine, as currently only the a-isomers of this EA type are available as pure reference materials.”
Reported challenges
Separation of a- and β-ergocryptinine, both in the sample, has been shown to be challenging under conventional reverse-phase chromatographic conditions.
Hindrances related to matrix effects; matrix interferences for ergometrine/ine and ergosine/ine; retention time fluctuation; EA' levels exceeding the normal calibration range and unusual recoveries and sensitivity of the instrument not being as good as it was.
Participants were asked to submit validation data, such as the method's limits of detection (LODs), limits of quantification (LOQs) and method recovery estimates (%).
Most labs (29) used LC-MS/MS-based methods while six used HPLC-FLD and two LC-HRMS (Orbitrap). One lab used both HPLC-FLD and LC-MS/MS depending on the analyte.
Most used clean-up materials were MycoSep 150 > SPE Alumina > Bondesil PSA.
The majority of participants (81%) said they had prior experience on determination of ergot alkaloids, spanning from one to more than 10 years (typically three).
Typically, labs analyse between 20 and 50 samples per year however, there are some that do not analyse them routinely.
Determining DON in wheat
The second PT was on the determination of deoxynivalenol (DON) in wheat.
Test materials were four naturally contaminated wheat materials.
Assigned values, established by an exact-matching double isotope dilution mass spectrometric technique (EMD-IDMS), were 551 μg/kg DON for material A; 1556 μg/kg for material B; 4405 μg/kg for material C and 1160 μg/kg for material D.
Fifty-nine participants from 32 countries (among them 41 NRLs and 18 OCLs) registered.
Twenty-seven labs analysed samples with LC-MS, 25 labs used HPLC-UV, four used ELISA, one HPLC with Diode-Array Detection (HPLC-DAD), one GC-MS and one lateral flow strips (screening test).
The PT found 93% of the attributed z-scores were below an absolute value of two for test items A and C, 95% for test item B and 92% for test item D.
One NRL reported a result with an unsatisfactory z-score.
A total of 85 to 100% assessed compliance/non-compliance of test materials A, C and D correctly.
Only 36% of labs assessed correctly the non-compliance of Material B and 56% classified it as compliant providing a proper justification.
This was mainly due to the larger uncertainty estimations of labs.
“The remaining laboratories (8-12%) wrongly interpreted their analytical results. This clearly indicates that compliance assessment remains to be improved.”