Pyrrolizidine alkaloids in herbal tea and honey: Report on the 2017 Proficiency testing scheme

Pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) are plant toxins which can enter the food chain through different paths. Two affected foods are herbal infusions and honey. This proficiency testing scheme was executed to assess the capabilities of laboratories to determine PAs. 29 laboratories from nine EU Member States plus Singapore registered. On 04. and 06.09.2017 test items and documentation were dispatched to all of those laboratories. By the dead line of 24.10.2017 26 laboratories had reported back results and filled in a questionnaire. Test item HO (acacia honey) was fortified with six PAs/PANOs (Echimidine, Integerrimine, Intermedine, Senecionine, Seneciphylline-NO, and Senkirkine) and 23 laboratories reported results for this item. The same number of laboratories reported for test item HT (herbal infusion) which was naturally contaminated with four PAs after extraction under reductive conditions (Integerrimine, Retrorsine, Senecionine, and Senecivernine). Laboratories had to report the sums of PA and its respective PANO. Satisfying outcomes could only be registered for Senecionine in test item HT and for Echimidine, Intermedine, and Senkirkine in test item HO with 74 %, 85 %, 85 %, and 91 %, respectively, of reported results having a z'-score smaller or equal to |2|. Only four laboratories reported for Integerrimine in both test items. Contrary to test item HT, Senecionine analysis in test item HO showed very unsatisfactory results. Of the 22 z'-scores calculated for Senecionine nine (41 %) were larger than 3. Senecivernine measurements in test item HT showed a similarly unsatisfying outcome with 47 % of reported results having z'-scores larger than 3. Only three laboratories out of the 26 were able to test for all 10 measurands and only one reported all 10 values with z'-scores smaller or equal to |2|. Overall only five laboratories obtained satisfactory z'-scores (≤ |2|) for all their reported results. There are two groups of three isomeric PAs/PANOs each which apparently caused, for a number of laboratories, problems with quantification. This is an issue which deserves heightened attention. The questionnaire contained queries regarding accreditation and experience, preparation conditions for the two test items, chromatographic separation conditions, detection conditions, calibration approach, and a comments section. The answers were evaluated and for selected questions their correlation to the z'-score of Senecionine in test item HO or Senecivernine in test item HT was analysed. For none of the tested questions a significant influence could be shown.JRC.F.5-Food and Feed Complianc

Report on the 2012 Proficiency Test on pyrrolizidine alkaloids in honey and hay

The purpose of this proficiency test was to investigate the current measurement capacities of testing laboratories for pyrrolizidine alkaloids in honey and plant materials. The scheme consisted of two parts: Benchmarking performance of laboratories against known estimates of pyrrolizidine alkaloids in the samples and checking for methodological differences while measuring naturally contaminated materials. Twenty-eight laboratories expressed their will to participate and analysed multiple analytes in several test samples of honey and plant material. The analysis of spiked honey showed no statistical differences between determining a common sum parameter for alkaloids and individual determination. A significant difference has been found however for of naturally contaminated materials. Individual alkaloid determination showed significantly lower results, possibly because of the presence of substances contributing to the sum parameter that were not in the scopes of the methods applied as well as lack of standard materials available on the market. Satisfactory performance for all of analytes has been achieved by more than half of participants analysing for both: sum parameter, and alkaloids analysed individually.JRC.D.5-Standards for Food Bioscienc

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in cereal products for infants and young children

Tropane alkaloids (TAs) are plant toxins that occur mainly in Datura, Atropa and Hyoscyamus sp, belonging to the Solanaceae family, besides a variety of other families such as Erythroxylaceae, Brassicaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. The TAs occur in all parts of the plants and botanical impurities have been found in a range of crops due to accidental contamination during harvesting. The intoxication via the food leads to anticholinergic effects (e.g. blurred vision, pupil dilation, dry mouth, vomiting, muscle spasms, tachycardia, etc.), culminating in severe intoxications and death. The EFSA CONTAM Panel established a group Acute Reference Dose (ARfD) of 0.016 μg/kg body weight (b.w.) expressed as the sum of (-)-hyoscyamine and ( ) scopolamine, assuming equivalent potency. Infants and young children are the most exposed age classes as they consume a higher amount of cereal-based products per body weight. EFSA estimated that the dietary exposure of toddlers could be up to seven times the group ARfD. Recently, two European legislation acts were published in this field: Commission Recommendation (EU) 2015/976, recommending the monitoring of tropane alkaloids in certain food categories, and Commission Regulation (EU) 2016/239, enforcing maximum levels of tropane alkaloids in certain cereal-based foods for infants and young children. The EURL-Mycotoxins organised a proficiency test (PT) concerning the determination of atropine and scopolamine in cereal-based baby food, aiming to underpin and assess the measurement capability of Member States' (MS) laboratories. Particular focus was given to levels relevant for enforcement of legislation. Two samples were distributed to the participants: one sample labelled “C” – biscuits for infants containing approx. 1.2 µg/kg of atropine and 0.2 µg/kg of scopolamine, and one sample labelled “E” – cereals for porridge containing approx. 7.4 µg/kg of atropine and 1.0 µg/kg of scopolamine. Forty-eight datasets from 18 EU MS laboratories were received. Overall, 81 % of the z-scores were in the range of -2 to 2 and 90 % were in the range of -3 to 3. For the lowest TA level (sample C) still 75 % of z-scores fell into an acceptable range (|z| ≤ 2), while the mass fraction of scopolamine was far below the target level of 1 µg/kg. In line with this observation, the vast majority of reported LOQs were below 1.0 µg/kg. The methodologies used by the participants can be clustered into three groups: the method supplied by the EURL; the RIKILT SOP A1070 and methods based on QuEChERS. The instrumental determination was by LC-MS/MS, with one exception (GC-MS). The recoveries reported by the participants were close to 95 %. No statistically significant dependence of the z scores on the analytical methodology was observed. These results support the assumption that atropine and scopolamine can be reliably determined at the maximum levels proposed by the EU to ensure the protection of infants and young children's health using state-of-the-art analytical instrumentation.  JRC.F.5-Food and Feed Complianc

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins: Determination of regulated mycotoxins and enniatins and beauvericin in cereals

The number of known mycotoxins, their precursors and metabolites has been steadily increasing over the past years. The European Commission puts special emphasis on the need to monitor the co-occurrence of mycotoxins of various families at levels that allow for a sound risk assessment, taking into account possible additive or synergistic effects. Prior any regulatory action is taken for mycotoxins for which a health concern has been expressed (e.g. enniatins and beauvericin) valid data on their prevalence in food is required. LC-MS-based multi-mycotoxin methods have the potential of streamlining and widening the monitoring work carried out by the official control laboratories. Although many practical advantages have been recognised, such methodologies have not been adopted by all routine laboratories and only a handful of such methods are just on the verge to become standardised. It is of great interest to assess how well laboratories using diverse sample preparation methodologies and determination techniques perform. Therefore, a proficiency test was organised by the European Union Reference Laboratory (EURL) for Mycotoxins for this purpose. The focus was the assessment of the measurement performance of EU Member States laboratories regarding the determination of aflatoxin B1, deoxynivalenol, zearalenone, fumonisins B1 & B2,T-2 & HT-2 toxins, enniatins B, B1, A, A1 and beauvericin in two test materials (corn and oat) using single- or multi-mycotoxin methodologies. Fifty-three laboratories, among them thirty-six National Reference Laboratories for mycotoxins in food and feed from the 28 EU Member States and 17 Official Control Laboratories, participated in the PT. For the regulated mycotoxins, 83.7 % of the results were rated with satisfactory z-scores. The performance of the laboratories was best for AFB1 (94 %), followed by DON (91 %), ZON (89 %), FB1 (87 %), FB2 (78 %), T-2 (75 %) and HT-2 (64 %). Additionally, 11 laboratories submitted results for all enniatins and beauvericin. LC-MS/MS is gaining much preference as it allowed for the determination of all the proposed analytes (12) in the test materials. Nevertheless, the esults provided by multi-mycotoxin methodologies did not differ statistically from those produced by singleanalyte procedures. Many participants uphold the will to implement a methodology to analyse enniatins and beauvericin in the near future, while other laboratories' methods require improvements in the extraction efficiency and sensitivity.JRC.F.5 - Food and Feed Complianc

Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimized method using liquid chromatography – tandem mass spectrometry

Alternaria toxins and citrinin are mycotoxins produced by fungi growing on agricultural commodities. Maximum levels for these toxins in foods are currently under consideration by the European Commission as a risk management measure. In this study, a new quantitative method is described for the determination of five Alternaria toxins and citrinin in tomato and tomato juice samples based on liquid chromatography with tandem mass spectrometry detection. Samples were extracted with pure methanol, followed by a derivatization step with 2,4 dinitrophenylhydrazine to improve the determination of tenuazonic acid and to decrease the wide polarity difference between the compounds of interest. Samples were purified on hydrophilic modified styrene polymer solid-phase extraction cartridges. High performance liquid chromatographic columns packed with different core-shell materials have been tested for the separation of toxins, and a C-18 phase with 2.7 µm particle size was applied in the final method to achieve sufficient separation of all relevant analytes. A key element of this approach was to prove successful transferability of the method to three different triple quadrupole mass spectrometers. A full single laboratory method validation has been performed on two liquid chromatography – tandem mass spectrometric systems and performance characteristics met the predefined requirements. Moreover, the method has been used in an international proficiency test, and the satisfactory z-scores obtained (-0.1 – 0.8 in tomato juice samples) demonstrated the reliability of the described approach. The method will be validated in an inter-laboratory collaborative study and if criteria for method precision are met, the method will be proposed to as a new Work Item to the European Committee for Standardization.JRC.F.5-Food and Feed Complianc