Blank Feed Research Articles

Perfluoroalkyl and polyfluoroalkyl substances (PFASs): An optimized LC-MS/MS procedure for feed analysis

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are extremely stable highly fluorinated aliphatic compounds, widely used in chemical and industrial applications since 1950s. They are ubiquitously detected in the environment and diet is the main route for human exposure. To ensure human safety, it is necessary to follow the whole food production chain, including animal feed. Still PFASs are not regulated as undesirable substances in feed, although several studies have shown they can be transferred from feed to livestock and thereafter to food. This paper describes the development, optimization and the full validation of a sensitive and reliable analytical protocol enabling the quantification of 19 PFASs in animal feeds by liquid chromatography - mass spectrometry (LC-MS/MS). The method was optimized, assessing the possible matrix interferences, and submitted to comprehensive validation (55 independent spiking experiments). Validation experiments were conducted on blank fish feed samples (natural levels of PFASs < 0.10 ng g−1) spiked at five different concentrations (0.10, 0.50, 1.0, 5.0 and 10 ng g−1). Apparent recoveries (R%) were generally between 88 and 111%; R% < 80% were obtained only at the lower validation levels for those molecules not having the corresponding labelled analogues. Relative standard deviations in repeatability conditions (RSDr) and within-lab reproducibility conditions (RSDwR) were lower or equal to 11% and 22% respectively. Limits of quantification were set for most of the analytes at 0.10 ng g−1 (LOQs) and verified with repeated analysis on fortified samples (0.10 ng g−1). Limits of detection (LODs) were calculated as 1/3 LOQ. Finally the method was applied to 23 feed samples of different type (compound feed, vegetable feed material, complementary feed, pre-mixture and mineral feed) and all of them did not show PFAS above LOQ.

Applied Research Note: Alpha-monolaurin stimulates the antibody response elicited upon infectious bronchitis vaccination of broilers

Alpha-monolaurin has been demonstrated to have antipathogenic properties and is therefore used as feed additive for broilers to prevent infectious diseases and improve production performance. As its antiviral effect is thought to be exerted by disintegrating the viral envelope, α-monolaurin might counteract with the current vaccination programs used in poultry production that are based on administering live attenuated viral strains. In this study, the effect of a commercially available formulation of α-monolaurin (FRAC12 Dry) on infectious bronchitis ( IB ) vaccination was evaluated in Ross 308 broilers. In chickens orally vaccinated with live infectious bronchitis virus ( IBV ), supplementation of FRA C12 Dry did not seem to affect the uptake of vaccine IBV, though indication for enhanced viral clearance of this virus was seen after 30 d in the supplemented birds. Furthermore, anti-IBV antibody titer values were significantly higher in orally IBV-vaccinated animals receiving FRA C12 Dry compared with the control group receiving blank feed. Taken together, the results indicate that α-monolaurin does not influence oral IB vaccination efficacy but, in contrast, has the potency to stimulate the immune response that is elicited upon vaccination. This further supports the use of α-monolaurin as a feed additive for broilers. Follow-up studies are, however, necessary to evaluate if better protection can be established upon challenge with IBV and whether a similar effect can be observed for other pathogens.

Inter-laboratory study on the detection of bovine processed animal protein in feed by LC-MS/MS-based proteomics

Since the beginning of the feed ban regarding the use of processed animal protein (PAP) in feedstuffs, one of the main challenges has been to develop methods to detect and identify the type and origin of PAP or animal products. By now, in the context of a controlled relaxation of the ban, complementary methods are sought to characterize the product when positive responses are delivered by current official methods, with special attention on feed materials (whether or not authorized) originating from ruminants. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics was identified as the most promising method to meet this challenge. This study presents the first inter-laboratory test constructed to evaluate and compare the LC-MS/MS methods currently developed in six different laboratories from four countries. Nine samples consisting of blank feed matrix or feed adulterated with bovine processed animal proteins and/or milk products were analyzed using in-house protocols and the results obtained were discussed. This study confirms the potential of LC-MS/MS-based proteomics to resolve the current analytical gaps in the detection and differentiation of PAP and highlights the technical challenges that need to be addressed in future.

Feed-borne exposure to zearalenone impairs reproduction of rainbow trout

To assess the effects on rainbow trout reproduction of feed contamination with zearalenone (ZEN) at the current European guidance value (2 mg∙kg−1 feed), we performed a life-cycle feeding trial under aquaculture conditions. The trial began when the fish larvae commenced external feeding and continued for 96 weeks, up to the point when the fish reached sexual maturity. To compare the reproductive development of the exposed and control fish, histological and molecular observations and analyses were performed at 12, 72, and 96 weeks of the feeding trial. To investigate the effect of ZEN exposure on the fertility of male and female fish, gametes obtained from mature fish were fertilized, after which offspring mortality was tracked at the eyed egg, hatched, and swimming larva stages. The mortality data were also used to estimate the risk of offspring death depending on the parental exposure scenario. We observed that feed-borne exposure to ZEN resulted in abnormal development of the fishes' gonads, and the occurrence of intersex fish and sex-reversed (feminized) males. These observations suggest that ZEN could have interfered with sex differentiation of the exposed fish at an early stage of their reproductive development. Our data indicate that the feed-borne exposure to ZEN probably did not affect the timing of sexual maturation or the relative fecundity of the female fish, but they suggest it might have led to advanced ovarian development. Moreover, ZEN-exposed male fish had a higher mean sperm concentration and markedly higher plasma levels of vitellogenin than males in the control group. These findings suggest that males could be more susceptible to the detrimental effects of ZEN than females. Importantly, offspring of fish that were fed with ZEN-contaminated feed throughout their whole reproductive cycle had a higher mortality risk than offspring of fish that were fed blank feed. Moreover, the offspring of parents that were only exposed to ZEN during the last 17 weeks of feeding before spawning also had a markedly increased mortality risk. Together, these findings not only demonstrate that ZEN harms rainbow trout reproductive outcomes, but also indicate that fish at sexual maturation may be especially vulnerable to ZEN contamination in feed. Our findings raise concerns about the safety of the current recommended guidance values for ZEN in feed and feedstuff used in aquaculture and suggest a need to consider lowering the maximum allowable levels of this mycotoxin.

Molecularly Imprinted Polymer Based Chemiluminescence Method for Detection of Nitrofurans

In this study, a molecularly imprinted polymer capable of simultaneously recognising seven nitrofurans is synthesised. The polymer particles coated the wells of a conventional 96-well microplate as the recognition element. After sample loading, the analytes were absorbed and a highly sensitive imidazole-enhanced bis(2,4,6-trichlorophenyl) oxalate–H2O2 system was added to excite light emission. After optimisation of several parameters, the chemiluminescence method was used to determine the seven nitrofurans in animal feeds. Results showed that the method achieved ultrahigh sensitivity for the seven drugs with limits of detection of 5–12pgmL−1, and one assay was finished within 10min. In addition, the polymer-coated plate could be reused five times. The recoveries from the standard fortified blank feed samples were in the range of 74.8–97.4%. From a comparison with a high performance liquid chromatography method, the molecularly imprinted polymer based chemiluminescence method could be used as a simple, rapid, sensitive, and recyclable tool to monitor the abuse of nitrofurans in animal feeds.

Targeted approach for qualitative screening of pesticides in salmon feed by liquid chromatography coupled to traveling-wave ion mobility/quadrupole time-of-flight mass spectrometry

The current work reports the development and validation of a qualitative screening method for 156 pesticides in Atlantic salmon feeds based on the use of ultrahigh-performance liquid chromatography (UHPLC) coupled to traveling-wave ion mobility spectrometry (TWIMS)/quadrupole time-of-flight mass spectrometry (QTOFMS). This novel hyphenation allows a straightforward estimation of the collision cross section (CCS), a physicochemical property that recently has been proposed as an additional identification point for pesticides.In order to increase the overall efficiency of the screening workflow, data processing parameters and tolerances involved in the automated analyte detection were first optimized. The use of the CCS as an additional identification point greatly improved the method selectivity, decreasing the rate of false-positive detections in blank feed samples. Qualitative validation was then performed in accordance with the EU document SANTE/11945/2015 on analytical quality control and validation procedures for pesticide residues analysis in food and feed. Feed samples were spiked with 156 pesticides at three levels (0.01, 0.05 and 0.20 mg/kg) and analyzed by the proposed method. Under the optimized conditions, screening detection limits (SDLs) ≤0.05 mg/kg were achieved for 82% of the investigated pesticides.The applicability of this new approach for the screening of pesticides was finally proven by analyzing several commercial Atlantic salmon feed samples as well as various plant-based feed ingredients. Pirimiphos-methyl and ethoxyquin were among the most frequently detected analytes in the investigated samples.

Research on Flange State during Spinning of AZ31 Magnesium Alloy Rotators

The stress-strain condition and flange state during spinning of magnesium alloy rotators was researched. The preliminary conclusion is obtained: There are three states during spinning of rotators, which appropriate "forward" and "straight" is conducive to forming, but the "backward" should be avoided. Based on ABAQUS/Explicit platform to analyze flange state and forming quality of these rotators under various load conditions. FE analysis shows: blank diameter, roller structure and feed ratio are key factors of formability. Most favorable process parameters are blank diameter 160mm, feed ratio 0.8r/mm, while using combination roller. Finally, the role of the main process parameters on flange state was verified by experiment method.

Stamp Forming of Polypropylene Based Polymer-Metal Laminates: The Effect of Process Parameters on Spring Back

This paper investigates the effect of process parameters such as Blank Holder Force (BHF) and Feed Rate, on the spring back behavior of a polymer metal laminate (PML) system comprised of aluminum and polypropylene. Specimens were formed over a hemispherical punch in stamp forming process. A novel real time strain measuring system, ARAMIS, was employed to capture the strain evolution during forming. The results of this work indicate that both BHF and feed-rate exert influence in PML spring back behavior. Fundamental correlation between strain evolution during spring back and the shape of the finished part will be presented. A major finding from this work is that aluminum dominates the spring back behavior of PML in stamp forming.

Chronic exposure to the mycotoxin T‐2 promotes oral absorption of chlortetracycline in pigs

The aim of this study was to investigate whether T-2 toxin, a potent Fusarium mycotoxin, affects the oral absorption of the antibiotic chlortetracycline in pigs. Animals were allocated to blank feed without T-2 toxin (controls), feed containing 111μg T-2/kg feed, T-2-contaminated feed supplemented with a yeast-derived feed additive, or blank feed supplemented solely with the feed additive, respectively. After 21days, an intragastric bolus of chlortetracycline was given to assess potential alterations in the pharmacokinetics of this commonly used antibiotic. A significantly higher area under the plasma concentration-time curve and maximal plasma concentration of chlortetracycline was observed after intake of T-2-contaminated feed compared with control. Thus, exposure to T-2-contaminated feed can influence the oral bioavailability of chlortetracycline. This effect could have consequences for the withdrawal time of the drug and the occurrence of undesirable residues in edible tissues.

A hybrid of theory and numerical simulation research for virtual rolling of double-groove ball rings

Double-groove ring rolling (DGRR) is a type of abnormal section-rolling process capable of diameter increasing and profile forming at the same time. This brings difficulties in analyzing the behavior of the rolling process thoroughly due to many influence factors. In this paper, the process parameters, including outer radii growth principle, controlling mode of guide roller, design method for ring blank dimension, and reasonable range of feed velocity, are studied theoretically. Then, a 3D elastic–plastic finite element model is developed using the dynamic explicit code ABAQUS/Explicit, based on the theoretical results, and the optimum blank size and feed velocity are determined. Finally, experiments are carried out that show a good agreement with the simulation results. Thus, the feasibility of the proposed method has been demonstrated. The research results provide valuable guidelines for the selection of blank design and velocity in the actual DGRR production.

DEVELOPMENT OF A BIOMIMETIC ENZYME-LINKED IMMUNOSORBENT ASSAY METHOD BASED ON A HYDROPHILIC MOLECULARLY IMPRINTED POLYMER FILM FOR DETERMINATION OF OLAQUINDOX IN CHICK FEED SAMPLES

In this study, a new way to substitute the biology antibody was introduced by using a hydrophilic molecularly imprinted film, which was directly prepared on the well surface of MaxiSorp polystyrene 96-well plate by the bulk polymerization technique. This imprinted film exhibited good recognition and fast adsorption-desorption dynamics toward olaquindox. Using it as the recognition element, a fast and new direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) method for the determination of olaquindox in chick feed was developed. This BELISA method had low cross-reactivities of 6.2% and 12% for two analogues. Under the optimal conditions, the sensitivity (IC50) and the limit of detection (IC15) were 700 ± 60 µg L−1 and 17.0 ± 1.6 µg L−1, respectively. The blank chick feed samples spiked with olaquindox at three levels were determined by this developed method with recoveries ranging from 82.0–96.0%.

Validation of a recombinant cell bioassay for the detection of (gluco)corticosteroids in feed

Use of hormones for fattening purposes is forbidden in the animal production in Europe (European Commission. 1996. Council Directive EC/96/22 (replacement of 88/146/EC). Off J Eur Commun. L125:3–9; European Commission. 1996. Council Directive EC/96/23. Off J Eur Commun. L125:10–32). Moreover, Regulation (EC) 178/2002 (European Commission. 2002. Regulation EC No 178/2002. Off J Eur Commun. L31:1–24) and Regulation (EC) 882/2004 (European Commission. 2004. Regulation EC No 882/2004. Off J Eur Commun. L165:1–135) oblige the member states to identify emerging risks and use validated and accredited methods for control analysis. Only combinations of bioassay activity screening with chemical identification are suited to uphold all laws. No such combination is described for the detection of (gluco)corticoids. In the present study, the GR-CALUX bioassay was validated as a qualitative screening method for the determination of glucocorticoid activity in feed. This validation was performed according to EC Decision 2002/657/EC (European Commission. 2002. Commission Decision 2002/657/EC from Directive 96/23. Off J Eur Commun. L221:8–36). Twenty-two representative blank feed samples were selected and spiked with 50 ng g−1 of dexamethasone, 100 ng g−1 of betamethasone or 500 ng g−1 of triamcinolone. All blank and spiked feed samples fulfilled the CCα and CCβ criteria; the method was specific and robust and glucocorticoids in feed were stable for at least 88 days.

Influence of Mycotoxins and a Mycotoxin Adsorbing Agent on the Oral Bioavailability of Commonly Used Antibiotics in Pigs

It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health.

Development of an on‐line molecularly imprinted chemiluminescence sensor for determination of trace olaquindox in chick feeds

Olaquindox, as one of the antimicrobial growth accelerants, is usually used in livestock production to improve feed efficiency. Due to health concerns over possible carcinogenic, mutagenic and photoallergenic effects of olaquindox on animals, the development of simple, rapid and sensitive analytical method for determination of olaquindox is crucial and necessary. In this study, a surface molecularly imprinted polymer was prepared by a molecular imprinting technique in combination with a sol-gel process using activated silica gel as a support material. This imprinted material exhibited with good recognition and selective ability, and fast adsorption-desorption dynamics toward olaquindox. Using it as the recognition element, a new on-line molecularly imprinted solid phase extraction coupled with chemiluminescence sensor for the determination of olaquindox was developed. The factors affecting preconcentration of the analytes and sensitivity of the method were all investigated. Under the optimal condition, the linear range of the calibration graph was between 2 × 10(-8) and 1 × 10(-6) g mL(-1) , and the detection limit of this method was 7 × 10(-9) g mL(-1) . The blank chick feed samples spiked with olaquindox at 0.3, 0.9 and 1.5 µg g(-1) levels were extracted and determined by this presented method with recoveries ranging from 87% to 94%. This method was validated by high-performance liquid chromatography and the results correlated well with those obtained by both methods. Moreover, this method was quantitatively analysed with two contaminated chick feed samples. This study will provide a sensitive and fast method for the monitoring of olaquindox residues in foods.

Molecularly imprinted solid-phase extraction combined with high-performance liquid chromatography for analysis of trace olaquindox residues in chick feeds

Olaquindox, one of the antimicrobial growth accelerants, is usually used as a feed additive in livestock production to improve feed efficiency. Due to health concerns over possible carcinogenic, mutagenic and photoallergenic effects of olaquindox on animals, the development of a simple, rapid and sensitive analytical method for determination of olaquindox is crucial and necessary. In this paper, a novel and hydrophilic functionalised material of olaquindox-imprinted polymer was synthesised in aqueous solution by a surface molecular imprinting in combination with a sol-gel process. This imprinted material was characterised by Fourier transform infrared, scanning electron microscopy, and static and kinetic adsorption experiments, and results showed that it had good recognition and selective ability, and fast adsorption-desorption dynamics for olaquindox. Applying the prepared material as sorbent, a method of molecularly imprinted solid-phase extraction (MISPE) for separation and analysis of olaquindox residues in feeds coupled with HPLC was presented. Under the selected MISPE condition, the detection limit (S/N = 3) for olaquindox was 68.0 ng L(-1), the RSD for five replicate extractions of 50 µg L(-1) olaquindox was 9.8%. The blank chick feed samples spiked with olaquindox at 0.0025 and 0.010 mg g(-1) levels were extracted and determined by the developed method, with recoveries ranging from 90% to 96%. This method was applied for enrichment and analysis of olaquindox in animal feed samples with good accuracy and repeatability. This study will provide a sensitive and fast method for the monitoring of olaquindox residues in foods.

Analysis of Fluoroquinolones in Animal Feed Based on Microwave-Assisted Extraction by LC–MS–MS Determination

An environmentally friendly method for the determination of fluoroquinolone (FQ) antibiotics including enrofloxacin, ciprofloxacin, levofloxacin, fleroxacin and sparfloxacin in four feeds is proposed. Disodium ethylenediaminetetraacetate (EDTA)–Mcllvaine buffer (0.1 mol L−1, pH 4.0) was used as an extracting solvent and the extraction process was accelerated by microwave irradiation. No organic solvents were used in the extraction procedure. The extract obtained was then cleaned up and concentrated by an Oasis hydrophilic–lipophilic balance (HLB) solid-phase extraction cartridge. The relative intra- and inter-day standard deviations obtained were in the range of 3.7–9.1 and 2.1–11.4%, respectively. In the three fortified levels of blank feed sample (30, 100 and 500 ng g−1), recoveries of FQs ranging from 61.1 to 97.9% were obtained. The analytes desorbed from HLB were analyzed by liquid chromatography–tandem mass spectrometry. The limit of detection was in the range of 5.0–9.1 ng g−1. The method presented here can be considered a promising alternative to traditional techniques using shaking or stirring for extraction, being more effective, and producing less pollution.

Sulfadimethoxine residues in rabbit muscle after extended oral treatment at therapeutic dosage

Sulfadimethoxine is extensively used in rabbit breeding for preventive and curative purpose and residues are sometimes observed in carcasses at slaughter. It has been suggested this is due to dosage and/or duration of treatment not being in compliance with the manufacturer?s recommendations, which probably induces residue levels in the meat above the maximum residue limit (MRL) value of 100 ?g/kg. In order to test this hypothesis, a study was carried out on gravid rabbits and their progeny. The animals were subjected to an extended treatment with sulfadimethoxine at therapeutic level in the feed. The feed was supplemented before pelleting with a commercial veterinary product containing 20 g of trimethoprim and 93 g of sulfadimethoxine per kg. On the basis of the dosage indicated for this commercial veterinary product, the incorporation level in the feed was 5 kg/ton (i.e. 465 g of sulfadimethoxine/ton), providing oral daily therapeutic treatment of the animals of ca. 12.5 to 50 mg of sulfadimethoxine per kg bodyweight. The mothers were treated during the last 21 d of pregnancy and during the whole period of lactation (35 d). The animals were sacrificed after a wash-out period of 12 d with blank feed. The young rabbits received the supplemented feed after weaning during the first 40 d of the fattening period. These animals were also sacrifi ced after a wash-out period of 8, 12, 15 or 20 d, respectively, with a blank feed. A sample of the leg muscle was taken for analysis. An HPLC analytical method was used to determine the sulfadimethoxine concentrations in tissue, with a LLOQ (Lower Limit Of Quantification) of 50 ?g/kg of muscle (trimethoprim was not considered in this study). Sulfadimethoxine concentrations above the MRL value of 100 ?g/kg were registered only in muscle from 1 out of 8 mothers and in 2 out of 8 young rabbits sacrificed 12 d after cessation of the treatment. For other young rabbits sacrificed on the 8th, 15th or 20th d after cessation of treatment, Sulphonamide concentrations in muscle always remained below the MRL value (8 animals per slaughtering time). These results show that oral treatment of rabbits with veterinary products containing sulfadimethoxine administered for a long period at the daily therapeutic level of 12.5 to 50 mg/kg does not seem to induce the accumulation of this molecule in muscle.

Residue Depletion and Tissue-Plasma Correlation of Methyl-3-quinoxaline-2-carboxylic Acid after Dietary Administration of Olaquindox in Pigs

A residue depletion study was performed to investigate the tissue kinetics and tissue-plasma correlation of methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residue of olaquindox (OLA), in pigs. Twenty-five pigs were randomly divided into a test and a control group. The former group was treated with 100 mg/kg OLA in its feed for 30 consecutive days, and the latter was given blank feed for the same period. One control and four treated animals were slaughtered at 0.5, 3, 10, 17, and 28 days post-medication. Muscle, liver, kidney, fat, and plasma samples were collected and analyzed using the validated high-performance liquid chromatography method (HPLC). Results showed that the tissue concentration of MQCA in the liver > kidney > fat > muscle, at almost all time points. The half-lives of MQCA in the muscle, liver, kidney, fat, and plasma were 12, 8, 15, 8, and 6 days, respectively. A withdrawal period of 38 days was calculated using the statistical method recommended by the European Medical Evaluation Agency (EMEA). Good correlations between tissue and plasma MQCA levels were found in the present study with correlation coefficients of more than 0.92. These correlations would be helpful in the routine monitoring of OLA in porcine tissues, without sacrificing the animals.

Coupled thermo-mechanical FE simulation of the hot splitting spinning process of magnesium alloy AZ31

Abstract Magnesium alloy AZ31 shows excellent ductility and formability at elevated temperatures, and using hot splitting spinning it can be formed into a structural component subjected to impact loadings, such as, a wheel hub of aero undercarriage or kinds of light whole wheels. In this paper, based on the analysis of microstructures and deformation characteristics of magnesium alloy AZ31, a reasonable deformation temperature range is obtained during the thermoplastic forming process. Adopting a flow stress model of magnesium alloy AZ31 considering the dynamic recrystallization (DRX), a 3D elastic–plastic FE model of hot splitting spinning of magnesium alloy AZ31 is established based on the FEM software platform of ABAQUS/Explicit and a developed 3D-FE model of cold splitting spinning, and its reliability is validated. Furthermore, field distributions of deformed component, including temperature fields, stress fields and strain fields, and variations of different nodal temperatures are obtained. And the influencing laws of different initial temperatures of disk blank and different feed rates of splitting roller on forming quality of deformed flanges are investigated, consequently the optimal forming temperature is approximate 300 °C and the optimal feed rate of splitting roller ranges from 1 to 3 mm/s during the hot splitting spinning process of magnesium alloy AZ31. The results may help to understand the forming characteristics and optimum design of the hot splitting spinning process.

Novel approach for interlaboratory transfer of real-time PCR methods: detecting bovine meat and bone meal in feed

The availability of robust methods for the species-specific detection of meat and bone meal (MBM) in compound feedingstuffs is an important prerequisite to enforce current and upcoming European legislation on the use of processed animal proteins in animal nutrition. Among possible methods, those based on DNA turned out to be a reliable tool for this aim, since DNA is a quite thermostable molecule able to resist severe heat treatments applied in the manufacturing of animal meals. The application of such methods by control laboratories implies that the method has been validated including an assessment of its robustness. Successful transferability between laboratories is considered an important robustness criterion of the method. However, corresponding guidelines regarding the design of such a study relevant to this field are missing. Here, we demonstrate the feasibility of an alternative concept that was applied to check for the transferability of a qualitative assay for the detection of banned MBM in feedingstuffs at trace level based on real-time PCR. The concept was based on an experimental nested design applying analysis of variance (ANOVA) that was conducted independently in two laboratories and which allows for establishing major factors influencing the result of analysis. Statistical assessment of the results confirmed the importance of the DNA extraction/purification step utilised, whereas the PCR step turned out to be a minor factor regarding the overall variability of the results. Furthermore, blind samples comprised of compound feed adulterated with MBM at 0.1% and blank compound feed were correctly classified as "positive" or "negative" samples, thus confirming fitness of purpose for the method. This approach can be of interest for other research groups working in the development of real-time PCR methods and in their use by control laboratories.