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 2011 Proficiency Test of the European Union Reference Laboratory for Mycotoxins, for the Network of National Reference Laboratories: Determination of aflatoxin B1 in baby food, maize powder, animal feed and test solution

This report presents the results of a proficiency test of the EU-RL for Mycotoxins which focused on the determination of aflatoxin B1 in food and feed samples. Sixty nine participants from 28 countries registered for the exercise. Sixty-one sets of results were reported for the solution, 58 for the baby food, 67 for the maize powder and 62 for the animal feed. One laboratory did not report any results. In total about 90% of the attributed z scores were below an absolute value of two, which indicated that most of the participants performed satisfactory or better.JRC.D.5-Food Safety and Qualit

REPORT OF THE FOLLOW-UP COLLABORATIVE STUDY Determination of the sum of Fumonisin B1 and B2 in Compound Animal Feed and Maize by Immunoaffinity Column Clean-up and High Performance Liquid Chromatography with Fluorometric Detection

The accurate determination of mycotoxins in food and feed matrices for which EU legislative limits apply require robust and reliable analytical techniques. The robustness and reliability are best shown through validation by a collaborative study. Previous collaborative studies dealing with other mycotoxins have shown that it is possible to achieve performance characteristics which are fit-for-purpose provided suitable methodology is available. As with any interlaboratory comparison homogeneity between the test units is of utmost importance. Due to the complexity of food and feed matrices particular care has to be taken during test material preparation to achieve this. Methods for the determination of Fumonisin B1 (FB1) and Fumonisin B2 (FB2) have been subject to a collaborative study in the past and the methodology used involved immunoaffinity clean-up to purify the sample extracts. Detection was afforded by derivatisation of the Fumonisins to yield fluorescent derivatives before a chromatographic separation. The reagent used was o-phtaldialdehyde and mercaptoethanol. However, pre-column derivatisation does have disadvantages related to more demanding chromatography and the instability of the derivatives. Strict time control of all processes is required to obtain adequate repeatability which necessitates the use of programmable auto liquid samplers (ALS). This may be circumvented by using post column derivatisation instead. Here the native Fumonisins are separated and reagents are added constantly to the effluent of the chromatographic column. An additional pump, a mixing Tee, and additional tubing are needed for post column derivatisation replacing the need for a sophisticated ALS. During method development it could be shown that both methods can perform equally well with respect to the requirements by EU legislation for method performance and working range. A collaborative study to validate a method for the "Determination of Fumonisin B1 and B2 in Baby Food, Breakfast Cereals and Animal Feed by Immunoaffinity Column Clean-up with High Performance Liquid Chromatography and Fluorometric Detection" failed partially because of problems with the immunoaffinity columns (IAC) used for the study. After modifications to the method protocol regarding a check of proper performance of the IAC and the sample extract clean-up we describe below the results of a repeat of the study for compound animal feed and maize.JRC.D.8-Food safety and qualit

Report on the 2008 Proficiency Test of the Community Reference Laboratory for Mycotoxins, for the Network of National Reference Laboratories, regarding the Determination of Deoxynivalenol in a Cereal Product and a Test Solution

A proficiency test was conducted by the Community Reference Laboratory for Mycotoxins with 33 European National Reference Laboratories (NRLs) for Mycotoxins and 2 Laboratory from candidate countries, thus a total of 35 participants. Test materials were a deoxynivalenol (DON) solution in acetonitrile and three cereal test materials. Laboratories determined the DON content by either enzyme linked immuno sorbent assay (ELISA), gas chromatography (GC) or reverse-phase high-performance liquid-chromatography (RP-HPLC). One NRL did not report any results. Applying the Horwitz equation as a basis for the target standard deviation (19% in the case of this proficiency test), 27 out of the remaining 34 laboratories reported values within the z-score limit of 2 after recovery correction of the result for the DON-positive sample. Twenty-five laboratories reported results within a z-score limit of 1. Thus, 79 % of the participating laboratories performed satisfactorily in the proficiency test. No z scores were calculated for the blank material.JRC.D.8-Food safety and qualit

Validation of an Analytical Method to Determine the Content of Fumonisins in Baby Food, Breakfast Cereals and Animal Feed

An inter-laboratory comparison was carried out to evaluate the effectiveness of a method based on immunoaffinity column clean-up followed by derivatisation and high performance liquid chromatography with fluorimetric quantification (HPLC-FL). The method was tested for the determination of Fumonisins B1 and B2 (FB1 & FB2) in baby food, breakfast cereals and animal feed to monitor compliance with limits according to Regulation 1881/2006/EC and Recommendation 576/2006/EC. The test portion of the sample was extracted with methanol:water. The sample extract was filtered, diluted, passed over an immunoaffinity column for clean-up and evaporated. The redissolved and purified eluate was separated and determined by reverse-phase high performance liquid chromatography (HPLC) and fluorescence detection after the fumonisins had been derivatised to their fluorescent isoindols in the presence of o-phthaldehyde and a thiol-coupling reagent with either pre- or post-column derivatisation. Baby food, breakfast cereal and animal feed samples, both blank and naturally contaminated with FB1 and FB2, were sent to 40 laboratories from 19 EU Member States, and a laboratory in Uruguay. For recovery determination extra test portions of the blank samples were to be spiked by the participants at levels of 135 µg/kg for the sum of FB1 and FB2 in baby food, 400 µg/kg in breakfast cereals, and 3700 µg/kg in animal feed. All samples were sent as blinded duplicates. Mean recoveries were calculated as 71 % for baby food, and 87 % for breakfast cereals. Based on results for the spiked and naturally contaminated samples the relative standard deviations for reproducibility (RSDR) in baby food were 31 % at a spiked level of 135 µg/kg, 44 % at a natural contamination level of 267 µg/kg, and 33 % at a natural contamination level of 501 µg/kg. For breakfast cereal these figures were 15 % at a spiked level of 400 µg/kg, and 33 % at a natural contamination level of 1034 µg/kg. The values for RSDr in those materials ranged from 5 to 29 % in baby food and 12 to 14 % in breakfast cereal.JRC.DG.D.6-Food Safety and Qualit

Melamine Proficiency Test 2009 - Assessing the Capabilities of Control Laboratories to Measure Melamine in Skimmed Milk Powder and Starch-containing Foods

A proficiency test to assess the capabilities of laboratories in the EU and beyond to determine melamine in a milk powder and a baking mix, representing starch-containing foods like bread and biscuits, was carried out in January of 2009 by the Joint Research Centre upon request of the European Commission¿s Directorate-General for Health and Consumer Protection (DG SANCO). The need for such an interlaboratory comparison arose from a health scare in China in 2008 about melamine tainted powdered milk. Laboratories of 31 countries, including Australia, China, India, Japan, New Zealand, the United States of America, and 21 of the 27 Member States of the European Union, participated and reported back 114 results for the milk powder and 112 for the baking mix test materials. The reported results were compared to reference values determined by exact-matching double isotope dilution mass spectrometry. The so determined assigned values were 10.0±0.6 mg/kg melamine in the milk powder and 3.18±0.17 mg/kg melamine in the baking mix. A coverage factor k of 2 was applied to calculate the expanded uncertainties. Three-quarters of all reported results for both materials had associated z-scores which were satisfactory (z=|2|). 90% of the results were accompanied by a measurement uncertainty statement and the majority of the measurement uncertainty ranges were reasonable. A number of laboratories were found to underestimate their measurement uncertainties. Isotope dilution mass spectrometry with stable-isotope labelled melamine was shown to be clearly advantageous with regards to the accuracy of the results. However, no significant influence by other method parameters could be identified.JRC.D.8-Food safety and qualit

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the network of National Reference Laboratories: Determination of aflatoxin B1 in defatted peanut powder

The Joint Research Centre (JRC), a Directorate-General of the European Commission, operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise proficiency tests (PTs) among appointed National Reference Laboratories (NRLs). This report presents the results of the PT on the determination of aflatoxins in defatted peanut powder. The test items for this PT were two contaminated defatted peanut powder samples. The materials were produced by the JRC and dispatched to the participants mid October 2016. Each participant received one bottle per test material containing approximately 55 g each. Fifty-six participants from thirty countries (among them 40 NRLs and 16 official food control laboratories) registered for the exercise and 54 sets (Sample A and B) of results were reported. The assigned values, established by an exact-matching double isotope dilution mass spectrometric technique, were 2.80 µg/kg (± 0.19 µg/kg) aflatoxin B1 in sample A and 3.20 µg/kg (± 0.20 µg/kg) in sample B. Participants' results were rated with z-scores and zeta-scores for aflatoxin B1 in accordance with ISO 13528:2015. The z-score compares the participant's deviation from the reference value with the target standard deviation accepted for the PT, whereas the zeta-score provides an indication of whether the participant's estimate of uncertainty is consistent with the observed deviation from the assigned value. Only z-scores were used for the evaluation whether an individual laboratory underperformed. In total, 96 % of the attributed z scores were below an absolute value of two for sample A and and 92 % for sample B. This indicates that most of the participants performed satisfactorily. The few participants that had z-scores above an absolute value of 2 will have to investigate the reasons for the deviation (root-cause analysis) and report the planned corrective actions to the EURL.JRC.F.5-Food and Feed Complianc

Equivalence testing of histamine methods - Final Report

Histamine fish poisoning is an allergy-like form of food poisoning that continues to be a major problem in seafood safety. The FAO/WHO Codex Alimentarius as well as EU legislation have therefore set maximum limits for histamine in fish and fish products. The analytical methods requested by Codex and by EU are different and concern has been raised that this could lead to disputes in the international trade of seafood. This report describes the outcome of a study, commissioned by DG Health and Consumers and carried out by DG Joint Research Centre, that compared the performance of the method for determining histamine in fish as mandated by Regulation (EC) No 2073/2005 to the method mandated in Codex Alimentarius Standard 165-1989. The EU mandated method is based on HPLC separation of histamine and subsequent detection by a UV detector. It was published in the Journal of AOAC International, but has not been validated by a collaborative study. The Codex method is AOAC 977.13, which is a based on the formation of a fluorescent derivative of histamine and subsequent measurement in a fluorimeter; it has been validated by collaborative trial. The correct implementation of both methods by JRC was assessed by carrying out performance verification studies using various canned and fresh scromboid fish samples (tuna, macrel, and herring) taken from the Belgian market. Repeatability (RSDr) and intermediate precision (RSDip) as well as recovery data were generated. Both methods conformed to specifications. Various approaches were followed to test the equivalency of both methods, which were based on statistical hypothesis testing (t-test), regression analysis and benchmarking against established reference values. All approaches indicated that the two methods are not fully equivalent. The EU mandated method has a tendency to overestimate, while the Codex method has a tendency to underestimate the histamine content in fish. It was recognised that the EU mandated method was very accurate when applied to fresh tuna. A distinct matrix influence was noticed for all other fish species tested, leading to an overestimation of the histamine content. It is therefore recommended to optimise the EU method so that matrix effects can be eliminated, or at least taken into account in an appropriate manner, In addition, a collaborative trial for the HPLC method to establish reproducibility data for the method should be organised. In line with current practice the collaborative study should also require to correct the reported data for recovery. Furthermore, as an ad-hoc measure the replacement of the HPLC method mentioned in Regulation (EC) No 2073/2005 by a ring-tested HPLC method, which are already available, could be considered.JRC.D.5-Standards for Food Bioscienc

2014 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories - Determination of Zearalenone in Maize Oil

This report presents the results of the PT of the EURL for Mycotoxins which focused on the determination of zearalenone in maize oil. Forty-eight participants from thirty countries (among them 32 NRLs, 2 Non-EU Reference Laboratories and 13 official food control laboratories) registered for the exercise and 46 sets (Sample A and B) of results were reported. Only z-scores were used for the evaluation whether an individual laboratory underperformed. In total, 87 % of the attributed z scores were below an absolute value of two, which indicates that most of the participants performed satisfactorily.JRC.D.5-Standards for Food Bioscienc