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Through its suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack". Instead, they exploit all the constituents of the haystack. This new form of analytical methods is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing needs to be propagated for the benefit of ones associated in academic research, commercial development, and official control. This paper addresses the frequently asked questions around notations and terminologies surrounding NTMs. The widespread development and adoption of these methods also necessitates the need to develop approaches to NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap to approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions thereof.

Through its suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack". Instead, they exploit all the constituents of the haystack. This new form of analytical methods is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing needs to be propagated for the benefit of ones associated in academic research, commercial development, and official control. This paper addresses the frequently asked questions around notations and terminologies surrounding NTMs. The widespread development and adoption of these methods also necessitates the need to develop approaches to NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap to approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions thereof.

Zusammenfassung Das Next-Generation Sequencing (NGS) besitzt großes Potential im Bereich der Lebensmittelsicherheit und der Authentizitätsprüfung von Lebensmitteln. Die Gesamtgenomsequenzierung mikrobieller Genome kombiniert mit bioinformatischen Auswerteprogrammen ersetzt zunehmend die klassischen Typisierungsmethoden und gilt aufgrund ihres außerordentlichen Auflösungsvermögen mittlerweile als Methode der Wahl im Rahmen von Ausbruchsuntersuchungen. Zur Authentizitätskontrolle z. B. von Fleisch- und Fischproben finden NGS-Methoden als Metabarcoding immer häufiger Anwendung, um Täuschung und Irreführung bis hin zu Lebensmittelbetrug aufzudecken. Einige Untersuchungsbehörden verfügen bereits über die NGS-Technologie und setzen diese auch erfolgreich ein, weitere Einrichtungen werden folgen. Um den mit der Lebensmittelüberwachung betrauten Behörden validierte, leistungsfähige und standardisierte NGS-Methoden zur Verfügung zu stellen, ist eine Aufnahme dieser Methoden in die „Amtliche Sammlung von Verfahren zur Probenahme und Untersuchung von Lebensmitteln“ (ASU) durch die Gründung zweier neuer § 64 LFGB Arbeitsgruppen mit unterschiedlichen thematischen Schwerpunkten vorgesehen. Die Arbeitsgruppe „NGS – Bakteriencharakterisierung“ bearbeitet NGS-Verfahren für die Sequenzierung bakterieller Erreger im Rahmen von Ausbruchsuntersuchungen. Die Arbeitsgruppe „NGS – Speziesidentifizierung“ beschäftigt sich mit NGS-Methoden zur Tierartendifferenzierung in Lebensmitteln. Am 6. März 2019 fand das erste Treffen der Arbeitsgruppe „NGS – Speziesidentifizierung“ und am folgenden Tag, dem 7. März 2019 das der Arbeitsgruppe „NGS – Bakteriencharakterisierung“ auf Einladung des Bundesamts für Verbraucherschutz und Lebensmittelsicherheit (BVL) in Berlin statt. Auf den Sitzungen wurden durch die Mitglieder der Gruppen NGS-Methoden zur Bakteriencharakterisierung bzw. zur Tierartendifferenzierung in Lebensmitteln vorgestellt. Anschließend diskutierten die Mitglieder die ersten thematischen Schwerpunkte der Methodenentwicklung, Validierungskonzepte, Qualitätskontrollmaßnahmen und den Einsatz dieser Methoden in der Lebensmittelüberwachung. Es wurde beschlossen, durch laborübergreifende Vorringversuche die Vergleichbarkeit der verschiedenen NGS-Technologien zu ermitteln sowie die entsprechenden Auswerteparameter, Qualitätskriterien und Validierungsparameter für eine laborübergreifende Validierungsstudie zu erarbeiten.

In the light of recent food fraud cases, the issue of food authenticity is receiving increasing attention. New analytical methods and evaluation approaches are currently being proposed. In this framework, the evaluation of mass spectral profiles constitutes a promising avenue, e.g. for the determination of food origin. Relevant evaluation approaches include principal component analysis, artificial neural networks, random forests, support vector machines, etc. The aim is to derive decision rules for the assignation of unknown samples to different classes. These decision rules are derived on the basis of samples whose origin is known—the training set. Typically, a reliable evaluation requires that the number of samples should be considerably larger than the number of features. However, in the framework of multiparameter mass spectrometry methods, the required ratio between sample and parameter numbers is inverted, with, e.g. 100 samples versus 10 000 features. In this paper, two approaches for the establishment of reliable decision rules in spite of low sample numbers are discussed.

Non-targeted methods (NTMs) are being increasingly developed and adopted to detect food fraud and identify the authenticity of the food. Method validation is a critical step before bringing the NTMs to the routine to be assured of method performance and trust that its outcomes are reliable. However, the paucity of well structured and harmonized validation strategies has been one of the hurdles, withholding the exploitation of NTMs. This report aims to describe a validation framework for methods that involve binary classification, which are prevalent in non-targeted workflows. We foresee this work to contribute to filling the current gap in the provisions for NTM method validation; perhaps also push the dialogue further to collectively resolve existing challenges.

Detection of food fraud and geographical traceability of ingredients is a continually sought goal for government institutions, producers, and consumers. Herein we explore the use of non-target high-resolution mass spectrometry approaches and demonstrate its utility through a particularly challenging case study – to distinguish wheat and spelt cultivars. By employing a data-independent acquisition (DIA) approach for sample measurement, the spectra are of considerable size and complexity. We utilize artificial intelligence (AI) algorithms (artificial neural networks) to evaluate the extensive proteomic footprint of several wheat and spelt cultivars. The AI model thus obtained is used to classify newer varieties of spelt, processed flour, and bread samples. Additionally, we discuss the validation of such a method coupling DIA and AI approaches. The novel framework for method validation enables calculation of precision parameters for facile comparison of the discriminatory power of the method and in the development of a reliable decision rule.

Massenspektrometrische Verfahren verfugen uber ein groses Potenzial fur die Bestimmung von Proteinen in Lebensmitteln. Die Kombination von Flussigchromatografie und massenspektrometrischen Fragmentierungstechniken kann dazu genutzt werden, eine Vielzahl an Proteinen simultan in einem Lebensmittel zu bestimmen, sei es zur Authentizitatsprufung oder zum Nachweis von Allergenen. Jedoch benotigt man fur einen routinemasigen und qualitatsgesicherten Einsatz in der Lebensmitteluberwachung standardisierte Methoden. Aus diesem Grund wurde die § 64 LFGB Arbeitsgruppe „Massenspektrometrische Proteinanalytik“ gegrundet. Die Ziele der Arbeitsgruppe sind die Aufnahme von validierten Flussigchromatografie-Massenspektrometrie-Methoden in die „Amtliche Sammlung von Verfahren zur Probenahme und zur Untersuchung“ und die Entwicklung von Leitfaden fur die Validierung dieser Methoden. Am 13. Marz 2018 fand auf Einladung des Bundesamts fur Verbraucherschutz und Lebensmittelsicherheit in Berlin das erste Treffen der Arbeitsgruppe statt. Auf der Sitzung wurden Methoden zur Proteinbestimmung in Lebensmitteln mittels LC-MS vorgestellt. Anschliesend diskutierten die Mitglieder der Arbeitsgruppe uber das Anwendungspotenzial der Methoden und uber mogliche Validierungskonzepte. Fur eine Methode zur Authentizitatsprufung von Fischproben und fur eine Methode zum Nachweis von Schalenfruchten und Erdnuss wurde beschlossen, eine Uberprufung der Methoden durch laborubergreifende Vorringversuche durchzufuhren, um daran anschliesend Methodenvalidierungsstudien mit mindestens acht Laboren zu realisieren und auszuwerten.

Moderne Analysetechniken besitzen ein groses Anwendungspotenzial im Bereich der Lebensmittel- und Futtermittelsicherheit und der Authentizitatsprufung von Lebensmitteln. Fur die routinemasige Anwendung dieser Verfahren fehlen bisher jedoch entsprechende Standardisierungskonzepte. Um die mit der Lebensmitteluberwachung betrauten Behorden validierte, leistungsfahige und standardisierte Methoden zur Verfugung zu stellen, ist eine Aufnahme dieser Methoden in die „Amtliche Sammlung von Verfahren zur Probenahme und zur Untersuchung (ASU)“ anzustreben. Auf Einladung des BVL berichteten am 14. November 2016 im Rahmen eines § 64 Kick-off Meetings Experten aus den Bereichen der Uberwachung, der Wissenschaft und der beteiligten Wirtschaft uber die bereits existierenden Anwendungsgebiete neuer Analyseverfahren und ihre bisherigen Erfahrungen hinsichtlich der Anwendung dieser Methoden. Die Teilnehmer der Veranstaltung befurworteten die Grundung neuer und spezialisierter § 64-Arbeitsgruppen mit den Schwerpunkten „Moderne molekularbiologische Methoden“ und „Massenspektrometrische Verfahren“. Ziel der neu zu grundenden Arbeitsgruppen ist es zunachst, allgemeine Leitfaden zur Validierung zu erstellen oder bestehende Ansatze auf Anwendbarkeit fur die Methoden zu prufen oder weiterzuentwickeln. Mogliche thematische bzw. anwendungsbezogene Schnittstellen sollen erkannt, und bei der Erstellung entsprechender Leitfaden mit einbezogen werden.

The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC–MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.

According to article 32 and 33 of Regulation (EC) No 882/2004, the European Union and National Reference Laboratory for pharmacologically active substances (EURL/NRL) at the Federal Office of Consumer Protection and Food Safety (BVL) is responsible, among other tasks, to support official control laboratories and as competent authorities on a national and EU level. The EURL has used in-house reference materials with residues of β-agonists, coccidiostats, nitroimidazoles, anthelmintics and NSAIDs from animal studies for proficiency testings (PT) for more than 13 years. The presentation provides a detailed picture of how to evaluate PTs for the determination of veterinary drugs. Proficiency testings are evaluated according to the prescriptions of ISO/IEC 17043, ISO 13528 and DIN 38402-45, using the statistical software package “ProLab Plus 2014” (QuoData GmbH in Dresden, Germany). The presentation illustrates the statistical parameters for: (a) Single analyte concentration (robust estimation of assigned value (HAMPEL), reproducibility standard deviation (Q-method) and scoring of laboratory results, (b) Graphical methods for combining performance scores (z-scores overview, combination scores and Mandels-h statistics), (c) Graphical methods for monitoring performance over years (control charts for z-scores and combination scores). In view of false negative results, it was regarded as not sufficient to include only the z-scores and combination scores for the assessment of the laboratories proficiency. Therefore a modified combination score was established that accounts for the overall evaluation of the examination results as well as for the false negative (n. a.—not analysed and n. f.—not found) examination results.

The article Etablierung

According to Art. 32 and 33 of Regulation (EC) No. 882/2004, the European Union and National Reference Laboratory for Pharmacologically Active Substances (EURL/NRL) at the Federal Office of Consumer Protection and Food Safety (BVL) is responsible, among other tasks, for supporting the official control laboratories as well as the competent authorities on a national and EU level. It is thus an important instrument in the overall complex of crisis management. Immediately after the publication of the first rapid alerts in the framework of the European Rapid Alert System for Food and Feed (RASFF), the EURL provided advice to the responsible Commission service with respect to the wording of the Commission Recommendation of 19 February 2013 on a coordinated control plan with a view to establish the prevalence of fraudulent practices in the marketing of certain foods, by detailing the available analytical means and capacities. In connection with the consumer fraud regarding the undeclared presence of horse meat in beef products (convenience food), a possible risk emanating from the non-authorised use of phenylbutazone (PBZ) was taken into consideration. For this reason the Commission Recommendation of 19 February 2013 suggested that at least one sample for every 50 tonnes of horse meat should be tested, each Member State (MS) being required to carry out a minimum of five tests. Moreover there was need for an analysis for PBZ in processed products containing horse meat as undeclared ingredient. The EURL/NRL of the BVL supported the analytical activities of the German control laboratories as well as of the European National Reference Laboratories (NRLs) through numerous measures.

The paper describes in detail how to evaluate interlaboratory studies for the measurement of authorised and non-permitted veterinary drugs. Examples of different kinds of evaluation will be presented including the results of two interlaboratory studies for anthelmintics in bovine milk and muscle. As compared to an interlaboratory study 2008, a clear development regarding the proficiency of the laboratories of the European Residue Control System could be noted in 2009. As a result of this development, the percentage of false-negative and false-positive results had decreased considerably. The described improvements in the analysis of the included anthelmintics were also reflected in the overall assessment. While in 2008 ten (38.5 %) out of 26 NRLs fulfilled the proficiency criteria of the interlaboratory study, this number had increased to fourteen (53.8 %) by 2009. In some cases, the correct quantification needed to be improved.