Determination of four aminoglycoside antibiotics and spectinomycin in feed at cross-contamination level: Development and in-house validation of a LC-MS/MS method for enforcing EU Regulation.

Combating antimicrobial resistance is a top priority worldwide involving a concerted action by several high-level institutions and organisations in the health sector. To ensure that a meaningful progress is achieved, several campaigns and political initiatives have been launched targeting the health professionals, the industry, the farmers, and the general public. The Regulation (EU) 2019/4 on medicated feed contains provisions for the limitation and control of the contamination of non-target compound feed with 24 antimicrobials. The purpose of this work was to develop a reliable and effective method for the determination of four aminoglycoside antibiotics (apramycin, paromomycin, tobramycin and neomycin) and spectinomycin in feed at cross-contamination level, where an absolute lack of suitable methods was identified. Four candidate methods described in the literature failed to provide adequate recoveries of all analytes. Therefore, an in-depth investigation was carried out to identify the bottleneck variable. The optimised method was then in-house validated and showed performance features appropriate for the intended purpose. The selected compounds could be analysed by LC-MS/MS in five animal feeds with LOQs between 2.6 and 9.2 µg kg-1 for the AGs and between 28 and 86 µg kg-1 for spectinomycin. Using isotopically labelled internal standards, the recovery rates varied from 63 % to 103 % and the intermediate precision (RSDip) varied from 1.1 % to 14 %. This work represents a step forward in the reliable determination of antibiotics in compound feed as the developed method has shown to be precise and sensitive. It is expected that this method gains wide acceptance and can supplement the legislation with effective control tools for antibiotic residues.

Determination of urea in pet feed: assessing the suitability of different analytical techniques using proficiency test data.

The determination of urea in pet feed at contaminant levels using the spectrophotometric method described in Commission Regulation (EC) No 152/2009 has been reported by several EU laboratories to lack the required selectivity. Whilst urea is not authorised as an additive in pet feed, the control of urea in pet feed is of economic importance, because the addition of urea may unlawfully increase the apparent protein content. To investigate the capabilities of different analytical techniques, a proficiency test was organised where the participants (EU official control laboratories, laboratories from the academia and private laboratories) were free to use their method of choice for analysing three dog feed test materials, two samples of which were spiked with urea. Twenty-one laboratories submitted results using the following techniques: spectrophotometry (Implementing Regulation (EC) No 152/2009), LC-MS/MS, HPLC-UV, enzymatic-colorimetry, gravimetry and an 'in-house photometric' method. Only two laboratories that used LC-MS/MS were able to quantify urea accurately in the test material containing a mass fraction of 18.9 mg kg-1 whereas satisfactory results at the level of 258.9 mg kg-1 were obtained by one participant that used an 'in-house photometric method' and one that used the enzymatic method, in addition to the five participants using LC-MS/MS. The technique that provided the highest success rate across the three test materials was LC-MS/MS, whereas spectrophotometry, the enzymatic-based and HPLC-UV methods led to overestimated results in addition to a dispersion of results not suitable for compliance analysis. To address the determination of urea in pet feed at low levels, a better performing method than the one described in the legislation is required.

Polar pesticides in food of animal origin: interlaboratory validation to evaluate method fitness-for-purpose of official control.

The present work reports on the design, execution and evaluation of results of an interlaboratory validation study aimed at verifying the fitness-for-purpose of a LC-MS/MS method for the detection of polar pesticides in food of animal origin in official control and monitoring programmes. To this scope, five participant laboratories, with relevant expertise, were recruited. After passing a pre-trial test, the participants were asked to analyse test samples of bovine fat, chicken eggs and cow's milk, contaminated with 11 polar pesticides (group A: Aminomethyl phosphonic acid (AMPA), cyanuric acid, ethephon, glyphosate, fosetyl aluminium, 2-hydroxyethyphosphonic acid (HEPA), maleic hydrazide, N-acetyl-glyphosate, group B: N-acetyl glufosinate (NAG), 3-methylphosphinicopropionic acid (MPP) and glufosinate ammonium) at two different levels (0.05 and 0.25 mg/kg-1 and 0.01 and 0.05 mg/kg-1 for group A and B respectively. The method was based on acidified methanol/water extraction followed by dSPE clean up with C18 sorbent. For LC-MS/MS analysis isotopically labelled standards were used for all targeted analytes. With a couple of exceptions, average recoveries ranged from 85% to 110%, with repeatability (RSDr) ranging from 3% to 25%, and reproducibility (RSDR) from 4% to 26%. The assessment by different laboratories provided also insights on key factors impacting method performance characteristics and its implementation by new users.

Analysis of feed additives by DART mass spectrometry: method optimisation and applications for product traceability in the European Union focusing on coccidiostats and carotenoids.

In this study, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was used to investigate the accurate characterisation of feed additive formulations containing coccidiostats or carotenoids. The study demonstrates the efficacy of DART-HRMS in identifying the active substances in these formulations and distinguishing between feed additives with the same active substance. The protocol for this method involves two simple steps that are extracting samples with organic solvents and measuring the extracts with DART-HRMS. The study also employs various statistical tools, including a factorial design approach, to optimise the DART-HRMS settings, and multivariate statistics, to establish classification models for feed additive formulations using nominal mass spectra. Our study demonstrates the potential of DART-HRMS in ensuring the correct identification of feed additives containing various coccidiostats or carotenoids and proposes this tool as an additional means for compliance checks with EU regulations.

Advances in selenium supplementation: From selenium-enriched yeast to potential selenium-enriched insects, and selenium nanoparticles.

Selenium (Se) is an essential micronutrient that plays an important role in animal and human development and physiological homoeostasis. This review surveys the role of Se in the environment, plants and animal bodies, and discusses data on Se biofortification with different sources of supplementation, from inorganic to organic forms, with special focus on Se-enriched yeast (Se-yeast). Although Se-yeast remains one of the main sources of organic Se, other emerging and innovative sources are reviewed, such as Se-enriched insects and Se-nanoparticles and their potential use in animal nutrition. Se-enriched insects are discussed as an option for supplying Se in organic form to livestock diets. Se-nanoparticles are also discussed, as they represent a more biocompatible and less toxic source of inorganic Se for animal organisms, compared to selenite and selenate. We also provide up to date information on the legal framework in the EU, USA, and Canada of Se that is contained in feed additives. From the scientific evidence available in the literature, it can be concluded that among the inorganic forms, sodium selenite is still one of the main options, whereas Se-yeast remains the primary organic form. However, other potential sources such as Se-enriched insects and Se-nanoparticles are being investigated as they could potentially combine a high bioavailability and reduced Se emissions in the environment.

DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances.

Feed additives require pre-market authorisation prior to their use in the EU. For the group of coccidiostats, the EU regulations authorising these products include specifications for these substances and the major components of the feed additive formulations. Feed business operators can use only feed additives that meet these criteria. The traceability of these products is supported by the allocation of specific identification numbers that need to be printed on the feed additive label along with other information. In the present study, Direct Analysis in Real Time mass spectrometry (DART-MS) was applied to investigate the correct characterisation of feed additives that contain coccidiostats as active substances. The results of the study demonstrated that this technique allows an unequivocal identification of the active substances in the feed additive formulations when combining DART with high-resolution mass spectrometry. In addition, two feed additives containing the same coccidiostat, but different excipients could be correctly classified according to their composition. The method protocol is very simple and comprises two steps, namely the extraction of the feed additives with an organic solvent and the subsequent measurement with DART-MS. For the evaluation of the MS spectra, chemometrics was applied offering an effective method for classification. Chemometric models were established with nominal masses obtained from the analysis of the samples, thus showing that these feed additives could be correctly classified even using low-resolution mass spectrometry. Moreover, we demonstrated that molecule-specific stable isotope patterns obtained with low-resolution mass spectrometry could be used as an alternative tool for the confirmation of the active substance.

Validation of a multi-analyte HPLC method for the determination of carotenoids used as feed additives in fish and poultry feed: results of an interlaboratory study.

The performance characteristics of a multi-analyte method for the determination of all 10 carotenoids authorised as feed additives within the EU were assessed via an interlaboratory study. The analytical method is based on reversed phase high performance liquid chromatography (RP-HPLC) coupled to an optical detector set at 410 nm. The analysis is particularly challenging due to the presence of various stereoisomers of each carotenoid, and the use of these compounds via natural or synthetic formulations, requiring a special sample preparation. EU regulations specifying the conditions of use set legal limits for these substances in compound feedingstuffs ranging from 6 mg kg-1 to 138 mg kg-1, depending on the individual carotenoid and the target animal for which the feed is supplemented with this carotenoid. The purpose of the multi-analyte method validated in this paper is to facilitate the monitoring of carotenoids at relevant levels when used as feed additives in compound feedingstuffs and pre-mixtures. The interlaboratory study delivered precision data for 43 different analyte/mass fraction/matrix combinations, covering a mass fraction range of the target analytes from 2.6 mg kg-1 to 3861 mg kg-1. The relative standard deviations for repeatability (RSDr) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSDR) varied from 6.8 to 39 % with a mean value of 21 %. Given the broad scope of the method covering 10 carotenoids added to compound feedingstuffs and pre-mixtures via different formulations, this multi-analyte method is considered fit for the intended purpose.

A simple design for the validation of a FT-NIR screening method: Application to the detection of durum wheat pasta adulteration.

The demand for the development of fast, easy-to-use and low-cost analytical methods for food adulteration analysis has being increasing in the last years. Although infrared spectroscopic techniques offer these advantages, the validation of screening methods requiring the application of multivariate data treatment is less frequently described in literature thus limiting their use as routine tools in control laboratories for food fraud monitoring. In this paper, an EU-validation procedure for screening methods was successfully applied to a multivariate FT-NIR spectroscopic method for the screening of durum wheat pasta samples adulterated with common wheat at the screening target concentration of 3%. Good results in terms of the cut-off value (2.32% mass fraction of soft wheat) and false suspect rates (0.1% for blanks; 13% at 1% mass fraction) demonstrated that the present validation approach would be a proof-of-strategy to be used for multivariate infrared methods applied for screening purposes.

Application of the CEN/ISO Standard for Phytase Activity Measurements to a New Phytase Product: Determination of a Robust Conversion Factor by an Interlaboratory Study for Screening Feed Samples.

Background: Phytase-based preparations are important feed additives currently authorised in the European Union (EU). The European Standard (EN) and International Organization for Standardization (ISO) standard 30024 describes a harmonized method for the determination of phytase activity and is fit-for-purpose for official control of a group of phytase products. However, it is not suitable for the determination of the phytase activity of a new feed additive encoded as 4a16 in the EU Register of Feed Additives, to which a slightly different phytase activity definition has been attributed. Objective: To establish a robust conversion factor to support official control laboratories that apply the EN ISO method when monitoring feed products containing 4a16. Methods: The phytase activity of test materials was determined by the participants using the EN ISO and/or the "applicant" methods. Results: Robust relative SDs for repeatability and for reproducibility of the methods applied for the determination of the phytase activity in the materials containing the 4a16 feed additive ranged from 2.6 to 22% (EN ISO method) and from 2.4 to 39% (applicant method). Conclusions: The data obtained confirmed the performance characteristics published for other phytase-based feeds in the related standard methods. These results allowed us to estimate a factor of 2.68 to convert phytase activities measured with the EN ISO method into the enzyme activity measured with the applicant method. Highlights: The obtained conversion factor will allow EU official laboratories to screen feed samples supplemented with the 4a16 phytase by applying EN ISO Standard 30024.

Rapid screening of ochratoxin A in wheat by infrared spectroscopy.

The use of infrared spectroscopy for the screening of 229 unprocessed durum wheat samples naturally contaminated with OTA has been investigated. Samples were analysed by both Fourier Transform near- and mid-infrared spectroscopy (FT-NIR, FT-MIR). Partial-Least Squares-Discriminant Analysis (PLS-DA) and Principal Component-Linear Discriminant Analysis (PC-LDA) classification models were used to differentiate highly contaminated durum wheat samples from low contaminated ones and the performances of the resulting models were compared. The overall discrimination rates were higher than 94% for both FT-NIR and FT-MIR range by using a cut-off limit set at 2 µg/kg OTA, independently from the classification model used thus confirming the reliability of the two statistical approaches used. False compliant rates of 6% were obtained for both spectral ranges and both classification models. These findings indicate that FT-NIR, as well as FT-MIR analysis, might be a promising, inexpensive and easy-to-use screening tool to rapidly discriminate unprocessed wheat samples for OTA content.

Evaluation of Mycotoxin Screening Tests in a Verification Study Involving First Time Users.

(AFB1) in maize and wheat using LFD and LC-HRMS, respectively. The results of analyses were used to calculate intermediate precision (RSDip, covering the inter-analyst variability in preparing the analytical samples and the precision under repeatability conditions) cut-off values and false suspect rates. RSDip ranged from 6.5% to 30% for DON, and from 16% to 33% for AFB1. The highest obtained variances were associated with the AFB1 analyses due to working with much lower mass fractions. The rate of false suspect results were lower than 0.1% for all tested methods. All methods showed a fit-for-purpose method performance profile, which allowed a clear distinction of samples containing the analytes at the screening target concentration (STC) from negative control samples. Moreover, the first time users obtained method performances similar to those obtained for validation studies previously performed on the screening methods included in the training course.

Fourier transform near-infrared and mid-infrared spectroscopy as efficient tools for rapid screening of deoxynivalenol contamination in wheat bran.

BACKGROUND: Deoxynivalenol (DON) is the most common Fusarium mycotoxin occurring in wheat and wheat-derived products, with several adverse and toxic effects in animals and humans. Although bran fractions produced by milling wheat have numerous health benefits, cereal bran is the part of the grain with the highest concentration of DON, thus representing a risk for consumers. Increased efforts have been made to develop analytical methods suitable for rapid DON screening. RESULTS: The applicability of Fourier transform near-infrared (FTNIR), or mid-infrared (FTMIR) spectroscopy, and their combination for rapid analysis of DON in wheat bran, was investigated for the classification of samples into compliant and non-compliant groups regarding the EU legal limit of 750 µg kg-1 . Partial least squares-discriminant analysis (PLS-DA) and principal component-linear discriminant analysis (PC-LDA) were employed as classification techniques using a cutoff value of 400 µg kg-1 DON to distinguish the two classes. Depending on the classification model, overall discrimination rates were from 87% to 91% for FTNIR and from 86% to 87% for the FTMIR spectral range. The FTNIR spectroscopy gave the highest overall classification rate of wheat bran samples, with no false compliant samples and 18% false noncompliant samples when the PC-LDA classification model was applied. The combination of the two spectral ranges did not provide a substantial improvement in classification results in comparison with FTNIR. CONCLUSIONS: Fourier transform near-infrared spectroscopy in combination with classification models was an efficient tool to screen many DON-contaminated wheat bran samples and assess their compliance with EU regulations. © 2018 Society of Chemical Industry.

Revision of a European Union Official Method to Enforce Legal Provisions: The Case of Diclazuril.

Background: Diclazuril is a coccidiostat currently authorized as feed additive in the European Union (EU), with a legal limit set at 1 mg/kg. For official control, an official EU method based on reversed-phase HPLC coupled with UV detection at 280 nm needs to be applied. Recently, the EU Reference Laboratory for feed additives was informed that the recovery rate for diclazuril was very low when implementing this method and performed experiments demonstrating that the indicated sorbent mass of the solid-phase extraction (SPE) was too low. Objective: Therefore, the paper presents a modified method protocol and the results of an interlaboratory study, performed on two compound feedingstuffs containing diclazuril around the legal limit. Methods: The official method was modified by using a higher SPE sorbent mass and was further subjected to validation. Results: The obtained values for the relative standard deviation for repeatability were 4.5 and 11.2%, and the corresponding values for the relative standard deviation for reproducibility were 14.3 and 18.1%; the calculated HorRat values were 0.95 and 1.14. Furthermore, acceptable mean recovery values of 98 and 111% were obtained for the two test materials, respectively. Conclusions: Based on the obtained performance profile, it was concluded that the modified official method was fit for purpose. In consequence, the official EU method will be corrected accordingly. Highlights: The highlights of this work are reflected by the following terms, namely Diclazuril, correction of EU official method, and interlaboratory study.

Spectroscopy applied to feed additives of the European Union Reference Laboratory: a valuable tool for traceability.

Feed additives need to be authorised to be placed on the market according to Regulation (EU) No. 1831/2003. Next to laying down the procedural requirements, the regulation creates the European Union Reference Laboratory for Feed Additives (EURL-FA) and requires that applicants send samples to the EURL-FA. Once authorised, the characteristics of the marketed feed additives should correspond to those deposited in the sample bank of the EURL-FA. For this purpose, the submitted samples were subjected to near-infrared (NIR) and Raman spectroscopy for spectral characterisation. These techniques have the valuable potential of characterising the feed additives in a non-destructive manner without any complicated sample preparation. This paper describes the capability of spectroscopy for a rapid characterisation of products to establish whether specific authorisation criteria are met. This study is based on the analysis of feed additive samples from different categories and functional groups, namely products containing (1) selenium, (2) zinc and manganese, (3) vitamins and (4) essential oils such as oregano and thyme oil. The use of chemometrics turned out to be crucial, especially in cases where the differentiation of spectra by visual inspection was very difficult.

Development and validation of a multi-analyte method for the regulatory control of carotenoids used as feed additives in fish and poultry feed.

Carotenoids are used in animal nutrition mainly as sensory additives that favourably affect the colour of fish, birds and food of animal origin. Various analytical methods exist for their quantification in compound feed, reflecting the different physico-chemical characteristics of the carotenoid and the corresponding feed additives. They may be natural products or specific formulations containing the target carotenoids produced by chemical synthesis. In this study a multi-analyte method was developed that can be applied to the determination of all 10 carotenoids currently authorised within the European Union for compound feedingstuffs. The method functions regardless of whether the carotenoids have been added to the compound feed via natural products or specific formulations. It is comprised of three steps: (1) digestion of the feed sample with an enzyme; (2) pressurised liquid extraction; and (3) quantification of the analytes by reversed-phase HPLC coupled to a photodiode array detector in the visible range. The method was single-laboratory validated for poultry and fish feed covering a mass fraction range of the target analyte from 2.5 to 300 mg kg-1. The following method performance characteristics were obtained: the recovery rate varied from 82% to 129% and precision expressed as the relative standard deviation of intermediate precision varied from 1.6% to 15%. Based on the acceptable performance obtained in the validation study, the multi-analyte method is considered fit for the intended purpose.

A review of the work of the EU Reference Laboratory supporting the authorisation process of feed additives in the EU. [corrected].

This paper describes the operation of the European Union Reference Laboratory for Feed Additives (EURL) and its role in the authorisation procedure of feed additives in the European Union. Feed additives are authorised according to Regulation (EC) No. 1831/2003, which introduced a completely revised authorisation procedure and also established the EURL. The regulations authorising feed additives contain conditions of use such as legal limits of the feed additives, which require the availability of a suitable method of analysis for official control purposes under real world conditions. It is the task of the EURL to evaluate the suitability of analytical methods as proposed by the industry for this purpose. Moreover, the paper shows that one of the major challenges is the huge variety of the methodology applied in feed additive analysis, thus requiring expertise in quite different analytical areas. In order to cope with this challenge, the EURL is supported by a network of national reference laboratories (NRLs) and only the merged knowledge of all NRLs allows for a scientifically sound assessment of the analytical methods.

Differentiation of coccidiostats-containing feed additives by mid and near infrared microscopy.

Coccidiostats belong to the group of feed additives authorised within the European Union exclusively for specific preparations. These preparations not only contain one or more coccidiostats as active substance(s) but also various ingredients such as the carrier, which are included in the European legislation authorising the product. In order to allow the full traceability of the use of feed additives and to check for compliance with legal provisions, there is a strong need for analytical methods that enable the rapid characterisation of these products. This paper describes the applicability of non-destructive techniques such as mid infrared (MIR) and near infrared (NIR) microscopy supported by multivariate analysis for the characterisation of coccidiostats-containing feed additives. The application of these methods demonstrated that different feed additives could be distinguished from each other even when containing the same active substance. The use of chemometrics turned out to be crucial especially in cases where the differentiation of spectra by visual inspection was very difficult.

Intra- and inter-laboratory validation of a dipstick immunoassay for the detection of tropane alkaloids hyoscyamine and scopolamine in animal feed.

Tropane alkaloids (TAs) are toxic secondary metabolites produced by plants of, inter alia, the genera Datura (thorn apple) and Atropa (deadly nightshade). The most relevant TAs are (-)-L-hyoscyamine and (-)-L-scopolamine, which act as antagonists of acetylcholine muscarinic receptors and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning). The European Union has regulated the presence of seeds of Datura sp. in animal feeds, specifying that the content should not exceed 1000 mg kg(-1) (Directive 2002/32/EC). For materials that have not been ground, visual screening methods are often used to comply with these regulations, but these cannot be used for ground materials and compound feeds. Immunological assays, preferably in dipstick format, can be a simple and cost-effective approach to monitor feedstuffs in an HACCP setting in control laboratories. So far no reports have been published on immunoassays that are capable of detecting both hyoscyamine and scopolamine with equal sensitivity and that can be used, preferably in dipstick format, for application as a fast screening tool in feed analysis. This study presents the results obtained for the in-house and inter-laboratory validation of a dipstick immunoassay for the detection of hyoscyamine and scopolamine in animal feed. The target level was set at 800 µg kg(-1) for the sum of both alkaloids. By using a representative set of compound feeds during validation and a robust study design, a reliable impression of the relevant characteristics of the assay could be obtained. The dipstick test displayed similar sensitivity towards the two alkaloids and it could be concluded that the test has a very low probability of producing a false-positive result at blank level or a false-negative result at target level. The assay can be used for monitoring of TAs in feedstuffs, but has also potential as a quick screening tool in food- or feed-related poisonings.

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed.

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 µg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n = 146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 µg/kg by ELISA which correlated to >10 µg/kg by LC-MS/MS.