semi-annual newsletter - n° 7 january 2012 labinfo€¦ · 2011 has passed off as a roller coaster...

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LNRN A T I O N A L ER E F E R E N T I ELABORATORIA

L A B O R ATO I R E SN A T I O N A U XD E R E F E R E N C ENRLN A T I O N A L

R E F E R E N C ELABORATORIESNRL

Newsletter for the approved food safety laboratories

SEMI-ANNUAL NEWSLETTER - N° 7 JANUARY 2012

FASFCAC-Kruidtuin - Food Safety Center, Kruidtuinlaan 55, 1000 Brussels

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p. 4 Analysis of glyceroltriheptanoate in animal by-products

p. 7 Vitamins

p. 12 Glyphosate in all its forms

p. 17 Migration risks in traditional metal teapots

p. 19 Food allergies

p. 24 NRL-GMO: GMOSeek research project (2009 – 2011) for GMO detection

p. 28 Moving towards better European collaboration in terms of plant quarantine

p. 30 News from the European Reference Laboratory for Milk and Milk Products (EU-RL MMP), Paris, 2011

p. 36 Workshops & Symposia

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LabInfoNewsletter for the approved food safety laboratories

Editors’ groupDirk Courtheyn, Mieke De Mits, Conny De Schepper, Alain Dubois, Marc Evrard, Alain Laure, Bert Vandenborre, Mieke Van de Wiele, Eva Wevers and Marie-Christine Wilem

Authors of this issueGeert De Poorter, Eva Wevers, Mieke Vanbrabant, Séverine Goscinny, Vincent Hanot, Fabien Bolle, Valéry Dumont, Sylvia Broeders, Elodie Barbau-Piednoir, Guillaume Mbongolo Mbella, Nina Papazova, Antoine Pouppez, Nancy Roosens, Isabel Taverniers, Marc De Loose, Frédéric Debode, Gilbert Berben, Eric Janssen, Thibaut Olivier, Anne Chandelier, Stéphan Steyer, Véronique Ninane and Koen De Reu

TranslationTranslation Service of the AgencyEditors’ group and staff of the Laboratories Administration

Photographs and illustrationsSupplied by the laboratories

LayoutGert Van Kerckhove

Editor’s addressLabInfop.a. D. CourtheynFASFCAC-Kruidtuin – Food Safety Center4de verdieping, bureel K04/120218Kruidtuinlaan 551000 BrusselTel.: [email protected]




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Dear reader,

First of all, I wish you a happy and healthy 2012. “Healthy is better than wealthy”, as the saying goes. 2011 has passed off as a roller coaster : a few highs, but mainly lows. Analysts are pessi-mistic about the future, but we may better not revel in defeatism. Continuous innovation and creativity is our duty; it is the only way to create an added value in all sorts of fields. The FASFC and DG Laboratories do want to participate in these efforts.While writing my new management and operational plan I had a lot of conversations with the users of test results of labs performing analyses in the official control programme and the self checking programme, and with the lab managers of those approved labs as well. The main conclusion of those conversations is that users want to get the results ever quicker and at a cheaper cost. The lab managers are mainly looking for a steady legislation, with clear legal limits, and quickly available methods delivering accurate results. In itself, nothing new under the sun, thus, but converting those wishes into concrete objectives is quite a different matter. I’ld like to inform you on some significant initiatives I am going to take in order to meet those longings. All methods and accessory validation files (except those concerning forbidden substan-ces) used by the FASFC labs will be published in 2012 on the protected Intralab site, so that the external approved labs can easily find a method in this quickly changing world, and moreover they no longer have to establish costly and time-consuming validation files; in most of the cases an implementation validation will be sufficient. The functioning of the National Reference Laboratories will be regulated more in accordance with the market, and with an even better quality control.The NARVAL application for consulting legal limits and action limits will be further develo-ped. In view of the wide range of regulated measurands and the complex matrix depen-dence of their limits in food safety legislation, this application surely will aid third laborato-ries in respecting compulsory notification, while it may serve them also for advising their customers. And last but not least, negotiations are being held with PT-providers to inject results directly in our Foodlims PT module, so that the approved laboratories can go in for trend analysis without having to re-type the results. Administrative simplification and an electro-nic laboratory are becoming more and more a reality. This “electronic” Labinfo contains once again a series of interesting articles. Enjoy the read!

Geert De PoorterDirector General Laboratories

EditorialNewsletter for the approved food safety laboratories

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Analysis of glyceroltriheptanoate in animal by-products

In the nineties it was shown that certain transferable animal diseases (e.g. TSE: transferable spongiform encephalo-pathy) can be spread by animal by-products. Moreover, cannibalism (feeding proteins from cadavers of the same species) could entail an additional risk for spreading diseases. To prevent animal by-products that are not destined for human consumption from re-entering the food chain, Regulation 1774/2002 (European Communities, 2002) was drawn up. According to Regulation 1774/2002 animal by-products are divided into three categories: category 1 (high risk), category 2 (average risk) and category 3 (low risk).

At the request of the DG Health and Consumer Protection (DG SANCO) the Joint Research Centre Institute for Reference Materials and Measurements of the European Commission (IRMM) has evaluated the characteristics of the different substances that are suitable for use as marker for animal by-products of category 1 and category 2. In 2002 the IRMM proposed to use glyceroltriheptanoate (GTH) as a marker. After all, GTH proved to be the ideal candidate. GTH is a fat that consists of 3 C7-fatty acids (n-heptane acid) that have been esterifised with glycerol. Therefore, GTH does not appear in nature. The structural formula is illustrated in figure 1. The physico-chemical characteristics of GTH make sure it mixes well with fats and products containing fat. Moreover, GTH cannot be removed after it has been added. It is a safe and non-toxic product that is already used in the food industry for other applications, such as the marking of butter. Apart from that, GTH is a very stable substance that can resist heat treatment under steam pressure at 133°C, as it is applied in the fat rendering industry. GTH is commercially available as a clear transparent fluid. The price is low and not a lot needs to be added (legal requirement: 250 mg/kg fat). After all, there is a sensitive and simple method to analyse GTH.

Figure 1: Structural formula of glyceroltriheptanoate

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The analysis method was developed by the IRMM and is suitable for detecting GTH in animal meal and animal fats. The method consists of three basic steps: (i) an extraction of the fat fraction with petroleum ether (only for animal meal) (ii) purification of the extracted fat (for animal meal) or of the animal fat (for fat samples) and (iii) determination of the GTH concentration by gas chromatography. Two detectors can be used for detection, the Flame Ionisation Detector (FID) or Mass Spectrometer (MS). FID is only suitable for quantification of GTH in the target materials (category 1 and category 2 animal meal and animal fat). With MS, however, the absence of GTH in category 3 animal by-products can also be shown. The in-house validation of the IRMM, in which several performance characteristics (such as robustness, sensitivity, precision (repeatability and reproducibility) and accuracy) were determined, indicated that this method is suitable for the intended purpose.

After the analysis method for GTH determination had been fine-tuned, an implementation study was carried out in cooperation with different rendering plants in Europe. The objective of this study was to check whether the application of GTH as marker in category 1 and category 2 animal by-products is practically feasible under realistic circumstances. The study was carried out by the IRMM and CCL Research (Cooperative Central Laboratory) in close cooperation with DG SANCO and EFRA (European Fat Processors and Renderers Association). Every rendering plant showed high GTH levels in the end products (animal meal and fat). GTH levels were well over the demonstrability limit. These results indicate that GTH can be completely mixed with fat, and this for both marking animal meal and fat. A stability study also showed that GTH is stable in animal meal at room temperature during at least 58 weeks. Therefore, the results of the implementation study make it possible to conclude that GTH is a suitable and a practically usable marker for category 1 and category 2 materials.

On the basis of this study report, the European Commission approved the use of the GTH marker system on September 11, 2007 (European Commission, 2007). Finally, on December 2007 Regulation 1432/2007 was publis-hed (European Union, 2007), which came into force on July 1, 2008. According to this regulation, category 1 and category 2 animal by-products have to be marked permanently with GTH. Marking such materials insures the identification and traceability of products that have to be destroyed and that by no means can end up in the food chain again. The risk of fraude is also eliminated. Adding GTH needs to be done in such a way that the processed products contain a minimum concentration of at least 250 mg per kg of fat. Moreover, GTH needs to be homo-geneously distributed in the substance and has to be added after the products have undergone a prior sanitising heat treatment at a core temperature of at least 80°C. After all, experiments at laboratory scale have indicated that GTH is not stable in raw animal by-products. GTH is cleaved by gut enzymes. These enzymes, however, turn inactive as of the moment when the animal by-products reach a temperature of 80°C. Therefore, GTH is not added in slaughterhouses, but in the processing companies. Figure 2 contains an example of a fat rendering process. The moment of GTH addition is indicated with the little pipette.

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Figure 2: Schematic presentation of an example of a fat rendering process. The moment where GTH is added, is indicated with a little pipette.

Finally, the analysis method was validated on the basis of an interlaboratory study, in which the FLVVT (Federal Laboratory for the Safety of the Food Chain Tervuren) also took part. This study’s objective was to determine the performance characteristics of the GTH determination method. The study was carried out by 20 official laborato-ries from 13 member states, so these laboratories were able to immediately check the correct use of GTH in the fat rendering industry after the study was finished. Acceptable values were found for method precision and correct-ness. On the basis of that, it was concluded that the analysis method is suitable for the intended purpose.

More information on GTH, the official analysis method and the reports of the implementation study and the inter-laboratory study, can be found on the IRMM website.(http://www.irmm.jrc.be/activities/marker_for_animal_by_products/Pages/index.aspx)

Eva Wevers, [email protected]

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Vitamins

Definition

Vitamins are essential organic nutrients which, in contrast to other nutrients (carbohydrates, fats and proteins) do not provide any energy or growth boosters, but are necessary for smooth metabolism in the body. Vitamins ap-pear in a large number of natural foods. They are micronutrients, i.e. nutrients of which only a small quantity (a few mg or µg) has to be taken up in order to fulfil its metabolic function. However, the uptake of this small amount is essential, as they are not synthesised by the organism itself or only to an insufficient extent. There is a Recommen-ded Daily Allowance (RDA) for every vitamin. The actual quantity required per individual is defined by a number of different factors such as age, gender, environment, state of health and stress. An unbalanced diet or certain disorders can lead to a vitamin deficiency and possible deficiency disorders. The intake of food supplements or enriching of foods and industrial animal foods with vitamins can avoid a deficiency in humans and animals.

Classification and function

The chemical and physiological properties of vitamins are highly diverse. At present, there are 13 known vitamins, whereby each vitamin represents a group of structurally similar organic compounds with varying biological acti-vity, so-called vitamers. Provitamins are vitamin precursors, i.e. naturally occurring components that are transfor-med into ‘true’ vitamins by normal bodily metabolism.Vitamins are divided into two groups based on their solubility properties. The fat-soluble vitamins contain vita-mins A, D, E and K and around 50 carotenoids. The group of water-soluble vitamins is made up of vitamin C (ascor-bic acid) and all B vitamins, namely thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B8 of H), folic acid (B11 or B9) and cyanocobalamin (B12). The solubility properties also define the distri-bution of vitamins in separate food groups and have direct effects for the applied analytical methods. Fat-soluble vitamins mostly occur in high fat foods and can be stored in fatty tissue and the liver. The water-soluble vitamins B and C appear in all kinds of foods. The body is not able to store these vitamins very well (except for vitamin B12) and an excess leaves the body via urine and sweat. Vitamins are regulators of synthesis and degradation processes and form the foundations of co-enzymes, hormo-nes and other substances. They play a role in growth, the repair and proper functioning of the body and above all have a catalytic function. The majority of water-soluble vitamins are transformed in vivo into co-enzymes that play a crucial role in combination with metabolic enzymes in the catabolism of nutrients to produce energy for the organism. A few vitamins are produced in the body (by intestinal flora bacteria and in the skin under the influence of sunlight). Plants have the ability to synthesise vitamins themselves, with the exception of B12, and they conse-quently serve as the major source of these essential food components. The majority of vitamins are highly unstable compounds and are quickly broken down because of their sensitivity to heating, oxidation, radiation, moisture, water activity, pH, corrosive enzymes and the catalytic effects of metals. To increase stability, derivatives are mostly used for the addition of vitamins to animal and human foods.

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Vitamin analysis

In addition to bioassays for the establishment of the true nutritional value of a vitamin in a product, in vitro analysis techniques such as microbiological assays (MBAs) or physicochemical analysis methods such as HPLC are mainly used for the analysis of enriched animal and human foods. Physicochemical assays allow the quantification of the main constituents which are responsible for biological activity and provide a high level of precision.Currently, (ultra) high performance liquid chromatography (U)HPLC is the preferred method for the detection of vitamin content. HPLC can be used during sample preparation to clean up extracts or to separate and quantify components. MBAs were developed at the start of the 1940s and often still constitute the official methods for the definition of various B vitamins, but in this area, HPLC and LC-MS are also on the rise. The general analytical proce-dure for microbiological and physicochemical assays can be divided into a number of important steps: taking of samples, extraction, possible clean-up, measurement and calculation of the result.

Figure 1: General diagram of the analysis procedure

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Extraction of vitamins

The analyses begin with an extraction of the vitamins from the matrix to make their measurement possible. The chosen method for extraction depends on the required result, the nature of the matrix, the present natural or syn-thetic form of the vitamins, interfering constituents, resistance of the vitamin to heat and extreme pH values and the selectivity and specificity of the analytical method used. For successful determination, it is essential that the vitamins are quantitatively extracted from the matrix in a form that can be accurately measured by the analytical technique used. An efficient extraction procedure homogenises and concentrates the sample, isolates the analyte from its bond with protein, eliminates known interfering substances to the extent possible and destroys endo-genous enzyme activity. Extraction methods for vitamins from food matrices include acid or alkaline hydrolysis (saponification), enzymatic hydrolysis, direct solvent extraction and solid phase extraction.

Liquid chromatography, suitable or not?

HPLC can be used for the determination of added vitamins in enriched human and animal foods. The definition of naturally occurring vitamins via HPLC is more complex due to the different vitamins and as a result there is a need for more complex extraction procedures to take the bound forms into account. HPLC is not sufficiently sensitive for an accurate measurement of the extremely low vitamin B12 and vitamin B 8 content in food. HPLC is also a popular technique for the determination of vitamin C, despite the low absorption of dehydroascorbic acid and its lack of electrochemical activity. However, HPLC does not guarantee that all biologically active forms of a vitamin will be taken into account that could be measured during a microbiological assay and, in this respect, the inherent specificity of HPLC can lead to an underestimation of the total vitamin activity. The use of reversed phase HPLC columns that are suitable for both 100% organic and 100% aqueous mobile pha-ses allows for a separation of strong polar analytes in aqueous mobile phases and hydrophobic analytes in organic mobile phases. As a result, they are suitable for the separation of both fat-soluble and water-soluble vitamins and enable the simultaneous determination of certain vitamin combinations. Recent methods are above all directed towards the fast and efficient simultaneous determination of as many vitamins as possible in as many matrices as possible. The finding of an appropriate extraction method, the elimination of matrix effects and the optimisation of HPLC conditions however, represent a substantial challenge for the simultaneous determination of vitamins. The most widely used detection methods are UV and fluorescence detection, of which FL detection shows a higher sensitivity. Photodiode array detection on the other hand, offers the possibility to identify analytes via UV absorption at various wavelengths and can test peak purity at the same time. A more recent development is the use of LC-MS as a sensitive and selective means for the determination of one or more vitamins in food.

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The future of vitamins

Over the coming years, increasing emphasis will be placed on the accurate definition of types of vitamins and vitamin concentrations in foodstuffs (food supplements, infant foods, baby foods, etc.) in European legislation. The introduction of stricter regulations makes the development of well validated, internationally accepted analytical methods with high accuracy and precision even more necessary. However, it is not clear whether the current standardised methods can be used for the definition of certain vitamins in certain matrices. Unfortunately, the development of new methods for the analysis of vitamins is a very slow process, above all for water-soluble vita-mins. A harmonisation and/or extension of the current standardised methods and matrices of the various official organisations would already be a first step to ensure compliance with the new regulations and to maintain an overview of the available methods.

Mieke Vanbrabant, [email protected]

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Glyphosate in all its formsS. Goscinny and V. HanotScientific Institute for Public HealthPesticides Unit

A breakthrough molecule

Since its first appearance on the market in 1974, glyphosate has become the most widely used phytosanitary compound worldwide (one million tons sold in 2010). Its growing popularity is closely linked to its agronomic efficiency, its low cost and, above all, its favourable toxicological profile (see CV). The main breakdown product of glyphosate is aminomethylphosphonic acid (AMPA), which has the same chemical and toxicological properties as the parent compound. In foodstuffs, AMPA was only detected in 2% of cases, which is why the maximum residue limit only concerns glyphosate. As they share the same analytical difficulties, the same methods for the deter-mination of glyphosate are also implemented for AMPA. This not only provides access to more data for the risk assessment but also provides for better preparedness, in the event of any change to the residue definition in the future, without having to go through an additional development and validation phase.

Glyphosate

Curriculum Vitae

Chemical Properties

C3H8NO5P = N-(phosphonomethyl)glycine

Molecular mass = 169.07

pKa: 0.78-2.29-5.96-10.98

weak organic acid

water solubility: 12 g/L at 25°C

Mechanism of action

Inhibits the enzyme 5-enolpyruvoyl-shikimate-3-phosphate synthase (EPSPS) involved in the shikimic acid metabolic pathway. This pathway is absent in animals, which explains the high oral LD50values:

• Female goat 3 530 mg/kg • Female rat 2 686 mg/kg

Agronomic Use

Broad spectrum systemic herbicide, which provides great flexibility of use: on all crop types and for the maintenance of urban spaces (green spaces, gardens, roads, railways). Widely used with genetically modified crops

Toxicology Breakdown

Moderately persistent in soil and with a half-life of 20 to 100 days, it undergoes microbial breakdown, which finally metabolises it into carbon dioxide and simple inorganic compounds. However, its widespread use calls into question the issue of its “relatively low toxicity” for the environment.

Exposure

Only slightly soluble in fats, thereby reducing the risks of bio accumulation. It is incompletely absorbed (15 to 40%) after oral administration.

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The “Prima Donna” of analytical chemistry

Compared to other herbicides, glyphosate is the most difficult to analyse. The source of these difficulties lies in its physicochemical properties, which complicate each stage of the analysis:

Sample preparation

Glyphosate is very polar, water soluble and insoluble in organic solvents, thus making the possibilities for its ex-traction limited. As a result, the use of water is essential for this stage of the analysis. However, within the extract, other water soluble matrix compounds can also be found (sugars, amino acids, salts, etc.) that may interfere with the determination of glyphosate. A column purification stage is problematic due to the amphoteric nature of glyphosate. A liquid-liquid extraction purification method is less problematic in terms of yield and is, as a result, the most common method referred to in literature. However, the latter requires more handling and extended analysis time.

Separation and detection

Not only are these compounds very polar and ionic, but in addition, they do not possess chromophore groups to allow fluorescence or ultraviolet absorption detection. At this stage of the analysis, a difficult choice has to be made: whether or not to derivatise the glyphosate.The derivatisation technique allows for the analysis of compounds which cannot be analysed directly by gas chromatography (GC) or liquid chromatography (LC). Through a chemical reaction, an agent (chromophore) is grafted onto one or a number of sites of the target molecule. Changes in properties occur with the derivatised compound in favour of its analysis. In the case of glyphosate, derivatisation adds a chromophore, reduces its pola-rity and, depending on the chosen derivatising reagent, even allows for its GC analysis. However, this technique is not highly regarded by analysts as it requires the optimisation of a number of parameters (temperature, reaction time, concentration and purity of the reagents, laboratory handling time). If omitting this heavy task is deemed preferable, there are other viable options using liquid chromatography, such as hydrophilic interaction chroma-tography (HILIC), ion exchange chromatography (IC) or ion-pair chromatography suitable for very polar and ionic molecules. However, the robustness of these separation techniques is heavily dependent on enhanced purificati-on or requires a high dilution factor of the extract and therefore a highly sensitive detection system. Derivatisation and direct analysis both have their advantages and disadvantages. The choice of technique should be made in a rational manner to ensure a cost-effective method. In both cases, mass spectrometric detection is possible and recommended in order to avoid the problems of interference and sensitivity that can occur with other detectors (UV, electrochemical etc.)

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Background of the method

The analysis of glyphosate in cereal products has been mandatory since the beginning of 2011 for all laboratories involved in the European control plan. This obligation means that a robust analysis method must be developed and validated, which must be capable of being easily incorporated into the laboratory’s routine analyses. Within this framework, an analysis method for glyphosate and its metabolite AMPA was developed at the SIPH in partner-ship with the Austrian Agency for Health and Food Safety (AAHFS). Direct analysis methods (non derivatised) may be deemed attractive since they do not require management of the derivatisation stage. However, they impose chromatographic changes (mobile phases, columns, operation time) which may not be compatible with the routine analyses. We have favoured a protocol that allows the instrumental conditions of the multi-residue ana-lyses to be maintained (conventional C18 column and mobile phase mixtures of methanol water buffered with ammonium acetate at pH ± 6). We have therefore opted for a method that incorporates a derivatisation stage. The chosen reagent is 9-fluorenylmethyl-chloroformate (FMOC) which allows, thanks to its hydrophobic grouping, the polarity of the target molecule to be reduced and thus its retention on a reverse phase C18 column to be incre-ased (see chromatogram) as well as the molecular mass, which will facilitate MS-MS detection. FMOC binds to the amine functions by releasing a HCl molecule. The reaction must take place in an alkaline medium (a borate buffer is used-Na pH=9) in order to neutralise the acid formed and to help push the reaction towards the formation of glyphosate-FMOC. Unfortunately, the FMOC is not specific to glyphosate but reacts with all the amine functions and is highly reactive with water. The reagent concentration is of key importance in order to ensure a robust me-thod, which is why the chosen concentration (1 mg/ml) is significantly high, thereby ensuring:- that the surrogate and target compounds are not in competition with each other and reacting with the FMOC at the same time;- the reaction with the water and amines from the extract is not harmful to the target compounds.

The excess of the reagent is removed by washing with dichloromethane and the product of the reaction is di-rectly filtered before being injected.

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Fig.1: UPLC-ESI+-MS/MS chromatogram for glyphosate-FMOC and AMPA-FMOC in a calibration solution at 100 µg/kg

Fig.2: UPLC-ESI+-MS/MS chromatogram for glyphosate-FMOC and AMPA-FMOC in a spiked sample of oats at 10 x LOQ (200 µg/kg)

Glyphosate-FMOC

Glyphosate-FMOC

AMPA-FMOC

AMPA-FMOC

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A “tailored” method

The method was validated in accordance with the guidelines set out in the SANCO/10684/2009 document. As a result of this:• there is no matrix effect, the calibration curve is therefore prepared by derivatising the compounds outside of the matrix and covers a range of concentrations from 20 µg/kg to 500 µg/kg; • the derivatisation conditions are well adapted since they achieve yields at the limit of quantification (20 µg/kg) of 101.1 % (RSD of 6.9 %) and 112.9 % (RSD of 11.9 %) for glyphosate and AMPA respectively.

The determination of glyphosate at trace level is unquestionably complicated. However, if the correct analytical method is chosen, rather effective results can be achieved. In our case, the choice of isotope dilution quantifi-cation has allowed us to achieve a good yield without having to worry about potential losses during the rapid stage (1 min) of simultaneous extraction-purification. Derivatisation has been a success since it allows us to easily incorporate the analysis of glyphosate into routine analyses without making any changes instrumentally, which therefore represents a considerable time saving benefit.

[email protected]@wiv-isp.be

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Migration risks in traditional metal teapots

In Brussels, the use of a traditional teapot has led to cases of lead poisoning involving an entire family. Various teapot samplings were carried out by the ISP (Scientific Institute of Public Health) in partnership with the Brussels Intermunicipal Laboratory for Chemistry and Bacteriology and the FASFC (Federal Agency for the Safety of the Food Chain), which proceeded with the withdrawal of these pots from the market.

The concentration of toxic metals (Lead and Nickel) migrating into the tea is a cause of particular concern and can pose a serious poisoning risk. When looking at the measured values, it is clear that the use of these teapots can lead to cases of acute lead poisoning.Indeed, the exceedance of the lead and nickel toxicological reference values is very disturbing. Although the assumptions made are not intended to be exhaustive, they demonstrate the possibility of exceeding the lead Ac-ceptable Daily Intake (ADI) by up to 90 times. However, factors such as a longer seeping time (migration up to 9 times higher after 1 hour in comparison with 15 min) may aggravate the problem further.The use of lemon can be an equally aggravating factor since the level of migration can be up to 10 times higher than with a plain tea.The sustained intake by vulnerable groups such as children (as in the case referred to above) is also worth noting since the latter are even more sensitive than adults to lead poisoning (5 times higher assimilation).

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Table 1:The following table illustrates, for the teapots analysed, the identified lead concentration, the ingestion based on the assumption of consuming 4 cups per day and the Acceptable Daily Intake (ADI) percentage that such con-sumption generates.

Lead (assumption of 4 cups/day [800 ml]) and seeping time of 15 minutes in the teapot

n° Type of tea prepared in the teapot mg/l mg/D % ADI

Teapot n°1 Plain tea 1,74 1,4 650











Teapot n°1 Lemon tea 23,97 19,2 8961











In a joint opinion (Sci Com 06-2011 and SHC N°8726) approved by the Scientific Committee of the FASFC on the 24th of June 2011 and approved by the Board of the Superior Health Council (SHC) on the 6th of July 2011, the following was concluded: “Although only one in-depth study, including biomonitoring, has been able to unequi-vocally reflect the risk associated with the use of traditional brass teapots, we recommend advising against the use of such teapots and the prohibition of their sale. Moreover, in addition to this type of teapot, other contact materials (ceramic pots or plates, e.g. tagines), whose use is more culturally defined, have also been proven to be a significant risk factor in terms of exposure to metallic elements (and to lead in particular).

Consequently, a targeted communication strategy underlining the risks of such contact materials is strongly recommended. Moreover, the FASFC control programme should take into account the migration of metallic ele-ments (lead, as well as other metals including nickel) from similar artisanal utensils (e.g. brass, enamelled cast iron) and ceramic materials that come into contact with foodstuffs.”

Fabien Bolle, [email protected]

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Food allergies

A public health problem

Food allergy is a significant public health problem that has increased in recent years. It affects 2 to 3% of the worldwide population and its prevalence has reached 6 to 8% in children. Furthermore, an increase in the fre-quency and severity of allergic reactions has been observed over the last decade (Superior Health Council, publi-cation N°8513). According to the World Health Organisation, food allergies are the 4th largest health problem.

Food allergies should be distinguished from food intolerances such as lactose intolerance or celiac disease. Aller-gies are abnormal IgE-dependent immunologic reactions to a particular foodstuff. Intolerances are either non-im-munological reactions dependent on individual susceptibility such as lactose (with individuals that have a lactase deficiency), or immunological reactions that are not IgE-dependant such as celiac disease or gluten intolerance.

The symptoms vary (urticaria, diarrhoea, constipation, etc.) and can be severe ( respiratory or cardio-vascular problems and anaphylactic shock). In serious cases, an allergic reaction can result in death. Minute amounts of allergens, in units of mg of protein, may cause reactions in sensitive patients. Only complete avoidance of the allergenic food can prevent an allergic reaction in such patients. The full and accurate labelling of all foodstuffs is therefore essential for these patients. The European directive 2007/68/EC has made the labelling of 14 allergenic ingredients for pre-packaged products mandatory:• cereals containing gluten (namely wheat, rye, barley, oats, spelt, kamut or their hybridised varieties)• seafood• eggs• fish• peanuts• soja• milk• nuts (namely almonds (Amygdalus communis L.), hazelnuts (Corylus avellana), walnuts (Juglans regia), cashew nuts (Anacardium occidentale), pecan nuts [Carya illinoiesis (Wangenh.) K. Koch], Brazil nuts (Bertholletia excelsa), pistachio nuts (Pistacia vera), Macadamia nuts and Queensland nuts (Macadamia ternifolia)• celery• mustard• sesame• molluscs• white lupine• sulphitesand products derived from these foodstuffs (with some exceptions).This legislation relates to allergens intentionally added to products. However, allergens can be added non inten-tionally as a result of cross-contamination between production lines, for example through raw materials or via staff. These hidden allergens constitute a risk for the allergic consumer. In order to evaluate the presence of these allergens, it is essential to have reliable and approved analytical tools at hand.

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The methods of analysis

There are two main techniques currently used for detecting allergens: PCR (Polymerase Chain Reaction) and immunochemical methods such as ELISA (Enzyme-Linked ImmunoSorbent Assay) or LFD (Lateral Flow Device). Table 1 shows a comparison of both techniques. The PCR is based on the detection of allergen-specific DNA in the product tested. The advantage of this technique is its high specificity. However, highly refined products which still contain DNA but are protein free will be detected positive without posing a risk to allergic consumers. In addition, certain foods such as milk, eggs and food extracts contain a very low level of DNA in relation to the amount of protein. As a result they will not be detected by the PCR.

The immunochemical methods are based on the interaction between the antibodies and their antigens, or in this particular case, the food allergen proteins. ELISA is currently the most frequently used method for allergen detection. This method is simple, quick, sensitive (in units of mg/kg) and specific. Various approaches have been chosen in the development of ELISA methods for allergens. The antibodies are either directed against specific proteins with or without an allergenic potential, or against soluble protein extracts. Unlike DNA detection, protein detection can be interpreted as a potential risk for allergic consumers. However, certain proteins are sensitive to processing during the manufacturing stage (cooking, sterilisation, fermentation) and can be partially degraded or modified. As a result, they cannot be detected by the antibody test although their allergenic potential may be un-affected or even increased. Neo-allergens may also be produced during food processing as described in the case of the groundnut roasting process. Another problem of immunochemical methods is the capacity of the antibody to recognise protein motifs similar to the target protein. This is known as cross-reactivity, which may lead to false positive results. This may be the case for example with the nut family that has homologous proteins.LFDs are very quick tests (5 - 10 min). If an allergen is present in a sample, a coloured line appears on the test strip. Currently, these tests pose problems in the control of allergens in foodstuffs following numerous false positive results and due to a lack of reproducibility.

Mass spectrometric analysis (MS) is increasingly described in literature as a means of addressing the disadvantages of the two other methods: specificity, the targeting of allergenic proteins, the possibility of analysing several aller-gens at the same time, etc. Following extraction, the proteins are directed by an enzyme (mainly trypsine), thereby forming peptides. Next, these peptides are separated by chromatography and identified by the mass spectrome-ter. The extraction efficiency and matrix interference are two parameters which limit the sensitivity of this techni-que. Moreover, this technique requires costly materials and highly qualified staff. As a result, it is not used routinely.

The lack of data

Currently, there is very little validation data available to compare the results obtained with these methods. This is no doubt due to the lack of internationally harmonised validation protocols and recognised reference materials. In fact, the standards are developed by research teams and are specific to the test used. A harmonised validation protocol has been published for the ELISA allergen detection methods (Abbott et al., 2010). It recommends the use of reference standards and supplemented reference materials. However, fully representative reference material is scarce. The protein profile of allergenic foods varies depending on the type of food, climatic conditions, crop location, production processes, etc. In addition, depending on the matrix, the allergen can be found in a raw state (milk powders in a soja milk powder) or in a processed state (milk powder in bread dough before cooking). Several international projects (MoniQA, Europrevall) aim to develop such model materials that are representative of the cross-contamination that can occur (Dumont et al., 2010). The results of interlaboratory validations based on the harmonised protocol should soon be published in order to ensure the comparability of the ELISA methods used.

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Findings

Significant advances have been made over recent years in the area of food allergies, both on a clinical basis and on an assessment basis, using analytical tools and cross-contamination risk management in the agri-food industry.Much more extensive research is still required in terms of, among other things, determining the analytical method detection limits based on the clinical sensitivity thresholds and interpreting and correlating the results of the dif-ferent methods and techniques. The FASFC has therefore designated the CER Group and ILVO-T&V as the National Reference Laboratory whose main task will be to answer such questions.

Table 1: Comparison of the two main allergen analysis techniques. (Kerbach et al., 2009)

Immunochemical PCR

Detection Proteins DNA

Specificity Possible cross reactivity Very specific

Sensitivity In units of 1 mg/kg In units of 10 mg/kg

Handling Simple and quick More time consuming

Signal modification Biological variabilityClimatic variabilityExtraction efficiencyModification (glycosylation, phosp-horylation, etc.) following proces-sing during the production stageMatrix effect

Stable genotypeExtraction efficiencyStable DNA at high temperatures but may fragment at low pH levels.Matrix effect - PCR inhibitors in the matrix

References:

Kerbach S., Alldrick A.J., Crevel R.W.R., Dömötör L., DunnGalvin A., Mills E.N.C., Pfaff S., Poms R.E., Popping B., Tömösközi S. (2009) Managing food allergens in the food supply chain – viewed from different stakeholders per-spectives. Qual. Assurance & Safety of Crops & Foods 1(1), 50-60Abbott M., Hayward S., Ross W., Godefroy S.B., Ulberth F., Van Hengel A.J., Roberts J., Akiyama H., Popping B., Yeung J.M., Wehling P., Taylor S.L., Poms R.E., Delahaut P. (2010) Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. J AOAC Int 93(2), 442-450Dumont, V., Kerbach, S., Poms, R., Johnson, P., Mills, C., Popping, B., Tömösközi, S. and Delahaut, P. (2010), Develop-ment of milk and egg incurred reference materials for the validation of food allergen detection methods. Quality Assurance and Safety of Crops & Foods, 2(4), 208–215.

Valéry Dumont, CER Group, Health Department, [email protected]

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NRL-GMO: GMOSeek research project(2009 – 2011) for GMO detection

IPH: Sylvia Broeders, Elodie Barbau-Piednoir, Guillaume Mbongolo Mbella, Nina Papazova, Antoine Pouppez, Nancy Roosens ILVO: Isabel Taverniers, Marc De LooseCRA-W: Frédéric Debode, Gilbert Berben, Eric Janssen

Introduction

The GMOseek project (SAFEFOODERA: “Food Safety – forming a European platform for protecting consumers against health risks”) was financed by the European Commission under the ERA-NET platform for protecting consumers against health risks and was running from 1/06/2009 till 31/05/2011. It consisted of six European part-ners from four different member states: Centre Wallon de Recherches Agronomiques (CRA-W, BE), Instituut voor Landbouw- en Visserijonderzoek (ILVO, BE), Wetenschappelijk Instituut Volksgezondheid (WIV-ISP, BE), Bayerische Landesamt für Gesundheit und Lebensmittelsicherheit (LGL, DE), Joint Research Centre – Institute for Health and Consumer Protection (JRC-IHCP, IT) and Nacionalni Inštitut za Biologijo (NIB, SI). The three Belgian laboratories which took part in the project compose further the National Reference Laboratory for Genetically Modified Orga-nisms (NRL-GMO). The project consisted of different work packages (WP) and each laboratory participated in at least 2 work packages.The project dealt with the detection of Genetically Modified Organisms (GMO). Given the extensive legislation concerning GMO’s, the fast increasing amount of authorized GMO’s being placed on the market and the growing chance of the presence of unauthorized GMO’s (UGM), it is necessary to develop new detection techniques and to refine the existing ones. The principal results of the project were:• Construction of a database gathering all information on existing GM events• Establishment of a bio-informatics system that allows selecting a maximal number of EU-authorized GM events based on the screening of an optimal set of genetic elements• Development and in-house validation of SYBRGreen qPCR screening methods • Development and in-house validation of TaqMan qPCR screening methods• Development and in-house validation of a pentaplex qPCR screening method• Transfer of the new qPCR screening methods to a partner laboratory• Establishment of a document concerning validation guidelines for qualitative qPCR methodsHereafter, a small outline of the work done by the three Belgian NRL-GMO laboratories is given.

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Wetenschappelijk Instituut Volksgezondheid (WIV-ISP)

The GMOlab of the unit Platform Biotechnology and molecular Biology of the WIV-ISP was involved in the GMO-seek project in work packages WP2 and WP4. In WP2, the work of the lab consisted in the extraction and characterization of genomic DNA (gDNA). Hereto Cer-tified Reference Materials (CRM) were purchased and gDNA was isolated using extraction methods that are being applied on European level. The lab could show that the DNA extracted from the different materials was sufficient to cover the needs of the partner laboratories to develop their methods, that it was of good quality and that no inhibition was present. The extracted gDNA material was distributed to the partners according to their needs. Ad-ditionally, a plasmid was constructed for each method developed and validated by the GMOlab.In WP4, the GMOlab developed and validated three new screening methods targeting the genetic elements pNOS, pFMV and Cry3Bb which are present in different GM events. These methods allow covering more GM events and to increase the discriminating power of the previously validated and patented ‘Combinatory SY-BRGreen qPCR screening’ (CoSYPS) system. The system allows determining which GM events could possibly be present in a sample through a combination of genetic elements and is used for routine analysis of food and feed samples in the GMOlab under ISO 17025 accreditation. For each new genetic marker, the GMO lab could show that the parameters, that need to be evaluated during in-house validation, were within the acceptance criteria. All information was gathered in a validation dossier. In a last step, the three methods were transferred to the partner laboratory JRC-IHCP together with the necessary materials and the protocol to follow. Comparison of the results from both laboratories showed the reproducibility of the three methods which is important seen the harmoniza-tion of detection methods the EU is aiming for. Finally, it could be concluded that the three new screening me-thods were fit for the detection of GMO in food and feed samples and could be integrated in the CoSYPS system allowing a more cost and time efficient GMO detection.

Instituut voor Landbouw- en Visserijonderzoek (ILVO)

ILVO contributed in three of the GMOseek project work packages: WP1 on Bioinformatics, WP3 on DNA-based hybridization methods, and WP4 on Reference material and Guidelines for validations. Within WP1, an in-house “GMOmatrix” database was set up containing relevant information of all GM events existing worldwide. Regular check-up and maintenance of this database formed one of the tasks where ILVO was actively involved in. Each six months, all GM events (records) in the database were checked for completeness and correctness, errors were indicated and novel data on GM events or novel GM events were added into the data-base. This database further formed the basis for the development of novel bioinformatics tools, useful in the selec-tion and design of GMO screening methodologies as a first step in official laboratories’ GMO testing procedures. The task within WP3 entitled ‘NAIMA platforms for multiplex screening on microarray’, aimed at optimising and evaluating the NASBA-Integrated Multiplex Amplification (NAIMA) screening technology developed by NIB (Ljubljana, Slovenia). After optimisation and in-house validation, done at NIB, ILVO acted as transfer lab for further testing the hexaplex method. ILVO performed amplification reactions (NASBA) and hybridization of the reactions to pre-designed microarray plates, using materials and following the exact protocols from NIB. However the arrays were sent back to NIB for scanning and data analysis. Based on the low robustness and difficult transfer and imple-mentation of the technology in general, the NAIMA multiplex platform was not considered as relevant for further inter-laboratory testing.

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Centre Wallon de Recherches Agronomiques (CRA-W) The Unit Traceability and authentication of CRA-W contributed to the success of the project by its implication in WP1, WP2 and WP4 (development of plasmid reference material).WP1 of the project consisted of the construction of a GMO matrix by collecting all information about the genes, promoters and terminators used in GM constructs. CRA-W supplied the matrix with the information in his posses-sion and participated to the theoretical and practical checking of the information. CRA-W also developed simple Excel-based tools to facilitate the research of information in the matrix or for decision support. This tool will be freely accessible and will be very useful for control or research laboratories.In WP2, the aim was to develop new screening methods. Therefore, CRA-W developed new screening methods based on real-time PCR using TaqMan probes. Two methods targeting the Ubiquitin promoter of maize (pUbi) and the terminator E9 of pea (tE9) were developed and in-house validated taking into account the acceptance criteria such as specificity, sensitivity, limit of detection, efficiency of amplification, rates of false positive and false negative results. The pUbi and tE9 methods were successfully transferred to the LGL laboratory for evaluation. On the other hand, CRA-W evaluated the simplex (p35S-nptII junction) and multiplex methods (duplex PAT/bar, triplex p35S/tNOS/CTP2-CP4-EPSPS, pentaplex PAT/bar/ p35S/tNOS/CTP2-CP4-EPSPS) methods designed by LGL.Two additional methods were developed at CRA-W and several acceptance criteria were evaluated. In order to avoid false positive results with the tE9 target due to the presence of pea (the tE9 terminator is coming from pea) in a food product, a specific target for pea detection was developed. A last screening target for the Cry1Ab genes was also created and the variations of sequence of this target were studied in the different GM events for which reference material was available.

All partners

The three NRL-GMO laboratories together with the three other partners were involved in WP4 on the establish-ment of general guidelines for the validation of qualitative multiplex methods for GMO testing. Based on official IUPAC, ISO and Codex Alimentarius guidelines, a document was drafted as project deliverable, on agreed require-ments for method development and in-house validation for a GMO screening method. It was further agreed that these criteria have to be fulfilled before a method goes further to full validation through an official inter-laboratory comparative study.

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The project participants

[email protected]@[email protected]

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Moving towards better European collaboration in terms of plant quarantine

T. Olivier, A. Chandelier and S. Steyer CRA-W: Pest Biology and Biovigilance Unit

Although the European Union has had a common protection regulation against the introduction of harmful or-ganisms in plants and plant products in place since 1976, the harmonious application of the regulation within the various Member States currently remains a challenge. In fact, such harmonisation is faced with several constraints that pose a challenge to the various players working in the field of plant quarantine. The following non-exhaustive examples are given: the lack of a community reference laboratory for plant quarantine, the problems with adap-ting the regulation to emerging pests and diseases, international trade pressure, climate change, the challenges associated with the integration of new Member States and, above all, the lack of a European wide Pest Risk Analy-sis (PRA) for the majority of quarantine pests covered by directive 2000/29/EC. It is worth noting that the application of the directives on plant quarantine remains the national responsibility of each Member State. The National Plant Protection Organisations (NPPOs) must therefore find a balance between the protection of their national agriculture and free trade, advocated by World Trade Organisation (WTO) and the European Customs Union. This balance must comply with the sanitary and phytosanitary measures of the WHO (SPS - Sanitary and Phytosanitary Measures agreement) which requires that the restrictions on imports for quaran-tine purposes must be based on a transparent scientific analysis (PRA). For the import sector studied, this analysis has a twofold objective. On the one hand, the pest risk is evaluated by assessing the likelihood of the entry and establishment of a new pathogen that may be harmful to the agriculture and environment of a given territorial area, thereby establishing scientific evidence. On the other hand, the pest risk is managed, which consists of pro-viding concrete measures which may be taken to avoid the entry and establishment of the pathogen in terms of the economic and environmental repercussions at stake.

It is in this difficult context that different European initiatives have recently emerged in order to harmonise the Union policy built around the unifying theme of the European PRA. The European Commission and the European Food Safety Authority (EFSA), in close collaboration with the various NPPO’s and The European and Mediterranean Plant Protection Organisation (EPPO), were the driving forces behind these initiatives which led to the funding of various research projects.

Given the central role of the PRAs in the harmonisation of the European Union’s plant quarantine policy, the European Commission and the EFSA each decided to finance a research project related to this subject. On the one hand, this concerns the PRATIQUE project that forms part of the 7th Framework Programme for research and technological development (FP7) and, on the other, a Prima Phacie project financed by the EFSA. The PRATIQUE project aims to improve the PRA procedures (the EPPO scheme mainly) in order to improve their reliability and manageability for the end users. This improvement means, among other things, putting in place a structured inventory of the data required for the EU PRAs and improving the integration of the notion of uncertainty in the final result of the PRAs. The Prima Phacie project tests several PRA methodologies on harmful organisms classified within the various biological kingdoms in order to evaluate their robustness. The aim is to select one or a number of methodologies for future application by the EFSA in order to fulfil, in collaboration with expert groups and the

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EPPO, its role as pest risk assessor for the EU.

In order to initiate and populate these PRAs with scientific data, the European Commission has financed the research project Euphresco II (as a follow up to the first project under the same name). In this way, the Commis-sion sought to establish a self-supporting networking structure for the various research institutions and European financiers. It was done in order to combine the expertise and financing of the various Member States, to avoid the problems of duplicates and inconsistencies and to identify emerging issues. This structure, in which the Belgian NRL is involved (CRA-W and ILVO) will therefore perform a lobbying role with the Commission in order to orien-tate its subsidy policy and its choice of PRAs towards the themes identified by the Euphresco network of experts. Lastly, the Q-Bol project, also financed by the Commission through the FP7 and which aims to develop DNA bar-coding for a fast and precise identification of the quarantine organisms and in turn the improved monitoring of the EU territory, supplements the overall efforts.

In conclusion, although the exact role of the various players mentioned above remains to be defined, the forma-tion of two highly interconnected networks is emerging (see figure 1). First of all, the PRA network consists of the Directorate General for Health and Consumers (DG SANCO), in partner-ship with the NPPOs, as pest risk manager for the EU on the one hand, and the EFSA, supported by the EPPO and its group of experts for the assessment of this risk, on the other. The second network, which can be described as the plant quarantine research network, consists of the Euphresco structure in partnership with the EPPO on the one hand, and the Directorate General for Research and Innovation (DG Research) on the other. The latter network will initiate and populate the European PRAs undertaken by the first network.

Via the projects referred to in this article, the structure of the future European quarantine Phytopathology research network is beginning to form.

Figure 1: Diagram of the two PRA and Plant Quarantine Research interconnected networks

[email protected]

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News from the European Reference Laboratory for Milk and Milk Products (EU-RL MMP), Paris, 2011

Véronique Ninane* & Koen De Reu***CRA-W, Département Valorisation des Productions, 24 Chaussée de Namur, B-5030 Gembloux**ILVO-T&V, Instituut voor Landbouw en Visserijonderzoek – Eenheid Technologie en Voeding, Brusselsesteenweg 370, B-9090 Melle

Introduction

Each year, the EU-RL MMP holds a workshop for the national reference laboratories (NRL). The latest session, that took place on 2 and 3 May 2011, was dedicated to the entire field of analyses that is covered by the EU-RL MMP. The state of progress, the needs with respect to analysis and legislation concerning total flora, somatic cells and alkaline phosphatase were discussed by means of presentations and debates.

Performance of the European Reference Laboratories (EU-RLs)

In his introduction to the workshop, Klaus Kostenzer, representing DG SANCO of the European Commission, gave a detailed overview of the recent evaluation of the activities conducted by the different EU-RLs. The quality of the performances of the EU-RL MMP was judged good to very good.

Conducting inter-laboratory tests

The heart of the matter was approached through an item that includes all analysis parameters, i.e. the carrying out of comparative inter-laboratory tests. Soraya Amarouche of the French Laboratoire national de métrologie et d’essai (LNE) described the broad outline of the standards ISO 17043 (2010): General requirements for proficiency testing and ISO 13528 (2005): Statistical methods for use in proficiency testing by interlaboratory comparison. The latter standard is a supplement to ISO Guide 43. It gives a detailed description of the statistical methods that must be used by those who set up profi-ciency tests and makes recommendations with respect to the use of these methods. The standard, among other things, mentions an indicative threshold value for judging the homogeneity of samples ; but Soraya Amarouche emphasized that this value is not always appropriate and may therefore be adjusted. The practical organization of inter-laboratory tests was discussed by the representatives of two NRLs, Karin Knapp-stein of the German NRL and Koen De Reu (ILVO-T&V) of the Belgian NRL, and by Thomas Berger of Agroscope (Switzerland). The aim of inter-laboratory tests was different for each of these speakers: comparison between routine devices and comparison between routine devices and with the reference method or determination of the value of reference material. In addition, Koen De Reu (ILVO-T&V) explained the process used for evaluating the

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performance of the Belgian NRL by means of performance indicators as well as the financial penalty system linked to that process. It appeared from these accounts that the Belgian system is up to date when compared to the European developments in the field of analytical guidance. The representatives of all Member States were impres-sed by the completeness of the inter-laboratory tests presented and by the fact that part of these tests are done under accreditation: at the ILVO-T&V, some inter-laboratory tests are prepared under accreditation. It is therefore not surprising that many questions were asked to Koen De Reu when he had finished his presentation. Furthermore, the setting up of inter-laboratory tests was also at the centre of the presentations concerning the progress of EU-RL MMP studies and study projects on stability. As it is, the EU-RL MMP develops projects with a view to defining protocols on the treatment and the management of samples that are suitable for distributing tests among geographically distant laboratories. In that respect, the stability of raw goat’s milk samples, to which preservatives had or had not been added, was tested at 12°C over a 10 days period (Rabed Miled). This study showed that adding a mixture of boric acid, glycerol and potassium sorbate makes raw goat’s milk stable with respect to total flora. A project has been planned that deals with the formulation of raw cow milk samples that are naturally contaminated with somatic cells and are stable for that parameter (Alexandra Cauquil). The project was discussed and completed, more particularly on the basis of the information given by Koen De Reu on the method of preparation applied by the Belgian NRL to raw cow milk samples intended for inter-laboratory tests on somatic cell counting. Finally, more details were given on the protocol for checking the homogeneity and the stability of milk samples distributed for the purpose of the inter-laboratory test set up by the EU-RL MMP with a view to the determination of alkaline phosphatase (Caroline Vignaud, ANSES, Paris).

Total flora

As for the criterion « total flora », the validation of alternative methods and the establishment of their conversion relation are the main problems that need to be solved. The total flora of raw milk can be counted by means of an alternative method if that method has been validated against the reference method and when the rules of the ISO 16140 standard have been complied with. These rules have been established for foodstuffs and are not very appropriate and not very clear for the matrix in ques-tion: raw milk. In order to make these rules more explicit, the EU-RL MMP completed them with specific standards for milk, the FIL/IDF 161 standard that is being reviewed ( ISO 16297) and 128, and prepared a practical synthesis of the references to be consulted when implementing each of the validation points mentioned in standard ISO 16140 (Véronique Deperrois, ANSES, Paris). As for the conversion relation for alternative methods, the total flora of raw milk is determined by means of instru-mental methods and specific conversion relations for each of the countries involved. At the previous meeting was made a suggestion to establish a working group that would have to examine whether these conversion relations can be harmonised at EU level (Ninane & Werbrouck, 2010). The EU-RL MMP initiated the feasibility study and sug-gested three statistical approaches that may be used to establish the incidence of the regional factor (geographic origin) on the conversion relation (Laurent Guillier, ANSES, Paris). The members who committed themselves to participate in the working group, including Véronique Ninane (CRA-W) for the Belgian NRL, will be invited to con-tribute to the further development of the study. Finally, the EU-RL MMP set up a study with a view to identify the factors that affect the conversion relation of the Bactocount (Bentley, USA) for counting the total flora in raw goat’s milk (Rabed Miled).

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Somatic cells

The discussions on somatic cell count focused on the capacity of the reference laboratories to use the micros-copic method that is described in the ISO 13366-1 standard, which is the reference method for that parameter. In order to evaluate the capacity of the European analysis network to use that method, the EU-RL MMP set up an inter-laboratory test at the end of 2010 (Alexandra Cauquil). That test revealed that 85 % of the participating laboratories, among which the Belgian NRL, use the method in an appropriate way. An accurate identification of the difficulties met by other laboratories, carried out with the aid of the EU-RL MMP, should lead to an increase of that percentage. The results of the test also reveal that the overall performance of the European network in terms of repeatability and reproducibility have improved when compared to the previous test (2008). The EU-RL MMP intends to further improve these performances by means of a practical training. In addition, the development of a structure for producing reference material for the somatic cell count should lead to an improvement of both the performance of the reference method and the mutual consistency of routine methods. The International Dairy Federation (IDF) and the International Committee for Animal Recording (ICAR, Italy) have joined their efforts to develop a suitable organisation structure, a so-called reference system (Thomas Berger, Ascroscope, Liebefeld-Posieux, Switzerland). The Belgian NRL is involved in the development of this system: Véronique Ninane (CRA-W) takes part in the activities of this working group of the IDF and the ICAR.

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Alkaline phosphatase

Several analytical aspects have been examined with regard to alkaline phosphatase the activity of which is used to detect pasteurization.One of these aspects is the validation of the fluorometric method for use in goat’s milk that is described in the ISO 11816-1 standard (2006). In 2010, the EU-RL MMP set up an inter-laboratory test for the purpose of determining the repeatability and the reproducibility of the method in goat’s milk. 22 laboratories were involved in that test, including the ILVO-T&V for the Belgian NRL. The test related to 6 alkaline phosphatase activity rates (Anne-Cécile Boitelle, ANSES, Paris). The quality of the results of the inter-laboratory test is such that the test may be used for validation, which was the purpose for which it was set up.

Figure 1: Fluorophos instrument with incubator for the determination of the alkaline phosphatase activity

Figure 2: Positive sample (left) and negative sample (right) analyzing the alkaline phosphatase activity

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Another aspect that was discussed during the session is that of the residual alkaline phosphatase activity in pasteurized goat’s milk, and more in particular, the legally acceptable limit value of alkaline phosphatase activity. In a concrete manner, the EU-RL MMP examined the possibility to use for goat’s milk the limit value of 350 mU/l that was set by the EU for cow’s milk. This study showed that the alkaline phosphatase activity following pasteu-rization of goat’s milk is generally below the limit value set for cow’s milk but that this value is exceeded in goat’s milk coming from two Member States (Marina Nicolas, ANSES, Paris). DG SANCO should now take the initiative for legislation since it has enough information to make a decision.The determination of an activity limit value for cheese made from pasteurized milk was also on the agenda of the workshop. The adoption of a limit value is based upon the inventory of values found in practice and, hence, on the capacity of the laboratories to determine the alkaline phosphatase activity in cheese. In 2009, the EU-RL MMP set up a practical workshop on standard ISO 11816-2 (which was then being reviewed) that includes a description of the reference method for that parameter, with a view to improving the testing capacity of the NRL network. Fol-lowing that workshop, the capacity of the NRLs to use the method was evaluated by means of an inter-laboratory test. Four out of the 19 participating laboratories did not have a sufficient capacity and were re-briefed and then subjected to an evaluation test. After these two briefing and evaluation sessions, 17 laboratories, among which the ILVO-T&V for the Belgian NRL, were found capable of determining the alkaline phosphatase activity in cheese in a correct way. The list of the residual alkaline phosphatase activity values found in practice may now be com-pleted in order to be more representative of the real situation for cheese in Europe (Marina Nicolas, ANSES, Paris).In the meantime, the EU-RL MMP suggested to adopt a working limit value of 10 mU/g which may later be adjus-ted if necessary. That value has already been tested on some cheese types. So, the Swiss Agroscope laboratory tested the limit value on a certain number of Swiss cheeses made of cow’s milk, using the reference method very accurately (Charlotte Egger, Agroscope,Liebefeld-Posiux, Switzerland). All cheeses of raw or thermised milk that were examined, showed an activity rate of more than 10 mU/g whereas most of the cheeses made from pasteurized milk had a residual alkaline phosphatase activity of less than 10 mU/g. One soft cheese, Limburger, showed a residual alkaline phosphatase activity of more than 10 mU/g although it was made from pasteurized milk. In that particular case, the superficial flora contributed to the alkaline phosp-hatase activity that was measured: when the activity was measured after removal of a thicker rind (exceeding the standard thickness for that cheese, i.e.0.5 cm instead of “as thin as possible”), it was indeed less than 10 mU/g. The importance of the place,where the sample was taken, was then shown by means of large wheels (80-100 cm diameter) of cheese made from thermised milk: the wheels are so big that the cheese at the centre of the wheel stays warm long enough to deactivate alkaline phosphatase in the same way as in the case of pasteurisation.Whereas for Swiss cheeses, an adjustment of the standard seems at first sight sufficient to make the proposed limit value acceptable, this is not so for other cheese types. As a matter of tact, the EU-RL MMP showed that the manufacturing process of some cheeses may be at the origin of a change in alkaline phosphatase activity and may lead to a misinterpretation of results (Marina Nicolas, ANSES, Paris). During the manufacturing process of mozzarella type cheese e.g., alkaline phospatase is sometimes deactivated, leading to an activity of less than 10 mU/g, although this cheese is made from raw milk. The participants’ attention was also drawn to the opposite phenomenon: cheese made from pasteurized milk that showed an alkaline phosphatase activity of more than 10 mU/g. In the latter case, an activity rate of more than 10 mU/g was measured at the centre of the cheese and not only on the surface, such as in the case of Limburger cheese; so, removing a thicker rind does not solve the problem. It is very likely that the microflora is at the origin of this abnormally high alkaline phosphatase activity. As for the analysis, the ISO 11816-2 method for determining alkaline phosphatase in cheese still needs to be validated. In that respect, the EU-RL MMP intends to carry out a preliminary study on the characterization of the method in 2011 and to deal with the inter-laboratory aspects of validation in 2012.In order to check pasteurization of milk of other animal species (such as camels), the use of other alkaline phosp-hatase markers, such as lactoperoxidase of γ-GT, is being considered.

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Reference

Ninane V. & Werbrouck H. (2010). Workshop of the NRLs for Milk and Milk Products, Paris, 2010. Labinfo AFSCA, 5, 25-27.

[email protected]@ilvo.vlaanderen.be

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Workshops & Symposia

Date Subject Place More information (site web)

26.01.2012 Workshop Nieuwe EU-wetge-ving: Wat verandert er op het etiket?

De Biltse Hoek, de Bilt, the Netherlands

http://www.fooddoctors.com/Workshops/Programma.pdf

31.01-1.02.2012 Second International Sympo-sium on Hyphenated Techni-ques for Sample Preparation (HTSP-2)

Bruges, Belgium http://www.ordibo.be/htc/

1-3.02.2012 Twelfth International Symposium on Hyphenated Techniques in Chromatograp-hy and Hyphenated Chromatographic Analyzers (HTC-12)


13-14.02.2012 3rd International Fresenius FEED ConferenceEfficacy – Claims – Mode of Action

Cologne, Germany http://www.akademie-fresenius.com/en-glish/konferenz/output.php?kurs=306

28-29.02.2012 10th International Fresenius Conference “Food Safety and Dietary Risk Assessment”

Mainz, Germany http://www.akademie-fresenius.com/2030This conference will provide you with an up-to-date overview on the subject of assessing the risks of pesticide residues in food.

11-15.03.2012 Pittcon 2012 Orlando, FL, USA Pittcon is the world’s largest annual conference and exposition for laboratory science.

20-23.03.2012 IDF Regional Conference on Domestic Milk Supply and Demand Systems : Sharing Success Beyond Challenges!

Seoul, Korea http://www.2012idfrc.or.kr/

20-21.03.2012 International Fresenius ConferenceThe New Food Information Regulation

Cologne, Germany http://www.akademie-fresenius.com/en-glish/konferenz/output.php?kurs=312For professionals working in food manufac-turing and in the food trade industry as well as in food law and in the regulatory affairs field.

Spring of 2012 4th International Fresenius Conference “Health Claims”

Germany European and international specialists, with backgrounds in the industry, the legal field and the monitoring authorities, will discuss all the latest Health Claims developments. For more information: Telephone +49 231 75896-54 or [email protected].

19/04/2012 AOAC-LL Symposium:“Food Allergens”

Breda, the Netherlands www.aoaclowlands.nl

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Date Subject Place More information (site web)

26.01.2012 Workshop Nieuwe EU-wetge-ving: Wat verandert er op het etiket?

De Biltse Hoek, de Bilt, the Netherlands

http://www.fooddoctors.com/Workshops/Programma.pdf

31.01-1.02.2012 Second International Sympo-sium on Hyphenated Techni-ques for Sample Preparation (HTSP-2)


1-3.02.2012 Twelfth International Symposium on Hyphenated Techniques in Chromatograp-hy and Hyphenated Chromatographic Analyzers (HTC-12)


13-14.02.2012 3rd International Fresenius FEED ConferenceEfficacy – Claims – Mode of Action

Cologne, Germany http://www.akademie-fresenius.com/en-glish/konferenz/output.php?kurs=306

28-29.02.2012 10th International Fresenius Conference “Food Safety and Dietary Risk Assessment”

Mainz, Germany http://www.akademie-fresenius.com/2030This conference will provide you with an up-to-date overview on the subject of assessing the risks of pesticide residues in food.

11-15.03.2012 Pittcon 2012 Orlando, FL, USA Pittcon is the world’s largest annual conference and exposition for laboratory science.

20-23.03.2012 IDF Regional Conference on Domestic Milk Supply and Demand Systems : Sharing Success Beyond Challenges!

Seoul, Korea http://www.2012idfrc.or.kr/

20-21.03.2012 International Fresenius ConferenceThe New Food Information Regulation

Cologne, Germany http://www.akademie-fresenius.com/en-glish/konferenz/output.php?kurs=312For professionals working in food manufac-turing and in the food trade industry as well as in food law and in the regulatory affairs field.

Spring of 2012 4th International Fresenius Conference “Health Claims”

Germany European and international specialists, with backgrounds in the industry, the legal field and the monitoring authorities, will discuss all the latest Health Claims developments. For more information: Telephone +49 231 75896-54 or [email protected].

19/04/2012 AOAC-LL Symposium:“Food Allergens”

Breda, the Netherlands www.aoaclowlands.nl

20-24.05.2012 IDF International Symposium on Cheese Ripening and Technology

Madison, Wisconsin, USA http://www.idfcheeseus2012.com/

21-25.05.2012 QBOL EPPO Conference on DNA Barcoding and diagnostic methods for plant pests

Haarlem, the Netherlands www.eppo.org

22.05.2012 64th International Symposium on Crop Protection

Gent, Belgium http://www.iscp.ugent.be/

June 2012 2nd Global conference on GMO analysis

Como,Italy

http://mbg.jrc.ec.europa.eu/home/

4-8.06.2012 2012 IDF/ISO Analytical Week Tel Aviv, Israel http://www.idf-iso-analytical-week.org

19-21.06.2012 IDF/INRA International Symposium on Spray Dried Dairy Products

St. Malo, France http://www.fil-idf.org/Public/SiteEventType.php?ID=23123http:/www.colloque.inra.fr/sddp2012

25-26.06.2012 14th International Fresenius AGRO ConferenceBehaviour of Pesticides in Air, Soil and Water

Mainz, Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=3&kurs=295

25-28.06.2012 9th European Pesticide Residue Workshop(EPRW 2012)

Vienna, Austria http://www.ages.at/ages/eprw2012/The European Pesticide Residue Workshop (EPRW) is the premier European meeting for the presentation and discussion of the latest concepts and developments in the field of pesticide residues in food and drink.

Summer of 2012 5th International Fresenius Conference“Food Allergens”

Mainz, Germany http://www.akademie-fresenius.com/english/konferenz/output.php?thema=3&kurs=294

Summer of 2012 2nd International Fresenius Conference “Residues of Food Contact Materials in Food”

Germany This conference is primarily aimed at food and packaging producers concerned with the safety and quality of food and pac-kaging materials. For more information: Telephone +49 231 75896-82 or [email protected].

1-4.07.2012 Second International Woody Ornamental Symposium

Gent, Belgium http://www.ilvo.vlaanderen.be/woodyorna-mentals2012

12-17.08.2012 58th ICoMSTInternational Congress of Meat Science and Technology

Montréal, Canada http://www.biocircle-project.eu/me-dia/6894/icomst_2012_brochure_belgique.pdf

26-31.08.2012 32nd International Symposium on Halogenated Persistent Organic Pollutants (POP’s) - DIOXIN 2012

Cairns, Australia http://www.dioxin2012.org/

11-13.09.2012 Fourth international conference on Feed Safety – Methods and Challenges

Beijing, P.R. China http://www.feedsafety.org/

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20-21.09.2012 17th Conference on Food Microbiology

Brussels, Belgium www.bsfm.be

30.09-3.10.2012 126th AOAC Annual Meeting & Exposition

Las Vegas, USA http://www.aoac.org

3-9.11.2012 IDF World Dairy Summit Cape Town, South Africa http://www.fil-idf.org/Public/SiteEventType.php?ID=23123

5-9.11.2012 7th Conference of The World Mycotoxin Forum® and XIIIth IUPAC International Symposium on Mycotoxins and Phycotoxins

Rotterdam, the Netherlands http://www.wmfmeetsiupac.org/html/wel-come.aspThis unique combined event, WMF meets IUPAC, will build on the success of the previ-ous conferences which were held separately all over the world.The aim of WMF meets IUPAC is to increase awareness of human and animal health risks due to natural toxicant contamination in agricultural commodities and seafood, and of potential risk management options, technologies and strategies for minimized contamination. The event will focus in particular on mycotoxins, phycotoxins and plant toxins.

7-10.05.2013 EuroFoodChem XVII Istanbul, Turkey http://www.arber.com.tr/eurofoodchemxvii.org/index.php/homewith state-of-the-art knowledge and appli-cations in food chemistry and complemen-tary disciplines

25-29.08.2013 24th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)

Perth Exhibition Centre, Perth, Western Australia

http://www.waavp.org/

October 2013 IDF World Dairy Summit Yokohama, Japan http://www.fil-idf.org/Public/SiteEventType.php?ID=23123

semi-annual newsletter - n° 7 january 2012 labinfo€¦ · 2011 has passed off as a roller coaster...

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