A sensitive LC-MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow's milk and urine and application to farm milk samples from Germany.

Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow's milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89-106% and 85-104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites.

Detection and quantification of zearalenone and its modified forms in enzymatically treated oat and wheat flour.

An analytical method for the analysis of the mycotoxin zearalenone (ZEN) and its modified forms was developed. Sample preparation was performed based on a modified QuEChERS method combined with liquid chromatography coupled to a triple quadrupole mass spectrometry detection. The method was tested for linearity, precision, limits of detection and quantification and recoveries. The evaluation of the above-mentioned parameters was performed on oat flour. The method was applied to oat and wheat flours that were submitted to an amylolytic treatment (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process. A decrease in ß-zearalenol (ß-ZEL) and ß-ZEL-14-sulfate of approximately 40% after 90 min incubation was observed, the other analytes did not show any significant changes. To our knowledge, this is the first method that approaches the identification and assessment of ZEN-sulfate derivates in a cereal matrix. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05683-6.

Nano or Not Nano? A Structured Approach for Identifying Nanomaterials According to the European Commission's Definition.

Identifying nanomaterials (NMs) according to European Union legislation is challenging, as there is an enormous variety of materials, with different physico-chemical properties. The NanoDefiner Framework and its Decision Support Flow Scheme (DSFS) allow choosing the optimal method to measure the particle size distribution by matching the material properties and the performance of the particular measurement techniques. The DSFS leads to a reliable and economic decision whether a material is an NM or not based on scientific criteria and respecting regulatory requirements. The DSFS starts beyond regulatory requirements by identifying non-NMs by a proxy approach based on their volume-specific surface area. In a second step, it identifies NMs. The DSFS is tested on real-world materials and is implemented in an e-tool. The DSFS is compared with a decision flowchart of the European Commission's (EC) Joint Research Centre (JRC), which rigorously follows the explicit criteria of the EC NM definition with the focus on identifying NMs, and non-NMs are identified by exclusion. The two approaches build on the same scientific basis and measurement methods, but start from opposite ends: the JRC Flowchart starts by identifying NMs, whereas the NanoDefiner Framework first identifies non-NMs.

Results of an interlaboratory method performance study for the size determination and quantification of silver nanoparticles in chicken meat by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS).

Single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) promises fast and selective determination of nanoparticle size and number concentrations. While several studies on practical applications have been published, data on formal, especially interlaboratory validation of sp-ICP-MS, is sparse. An international interlaboratory study was organized to determine repeatability and reproducibility of the determination of the median particle size and particle number concentration of Ag nanoparticles (AgNPs) in chicken meat. Ten laboratories from the European Union, the USA, and Canada determined particle size and particle number concentration of two chicken meat homogenates spiked with polyvinylpyrrolidone (PVP)-stabilized AgNPs. For the determination of the median particle diameter, repeatability standard deviations of 2 and 5% were determined, and reproducibility standard deviations were 15 and 25%, respectively. The equivalent median diameter itself was approximately 60% larger than the diameter of the particles in the spiking solution. Determination of the particle number concentration was significantly less precise, with repeatability standard deviations of 7 and 18% and reproducibility standard deviations of 70 and 90%.

Analytical approaches for the characterization and quantification of nanoparticles in food and beverages.

Estimating consumer exposure to nanomaterials (NMs) in food products and predicting their toxicological properties are necessary steps in the assessment of the risks of this technology. To this end, analytical methods have to be available to detect, characterize and quantify NMs in food and materials related to food, e.g. food packaging and biological samples following metabolization of food. The challenge for the analytical sciences is that the characterization of NMs requires chemical as well as physical information. This article offers a comprehensive analysis of methods available for the detection and characterization of NMs in food and related products. Special attention was paid to the crucial role of sample preparation methods since these have been partially neglected in the scientific literature so far. The currently available instrumental methods are grouped as fractionation, counting and ensemble methods, and their advantages and limitations are discussed. We conclude that much progress has been made over the last 5 years but that many challenges still exist. Future perspectives and priority research needs are pointed out. Graphical Abstract Two possible analytical strategies for the sizing and quantification of Nanoparticles: Asymmetric Flow Field-Flow Fractionation with multiple detectors (allows the determination of true size and mass-based particle size distribution); Single Particle Inductively Coupled Plasma Mass Spectrometry (allows the determination of a spherical equivalent diameter of the particle and a number-based particle size distribution).

Physicochemical characterization of titanium dioxide pigments using various techniques for size determination and asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry.

Seven commercial titanium dioxide pigments and two other well-defined TiO2 materials (TiMs) were physicochemically characterised using asymmetric flow field flow fractionation (aF4) for separation, various techniques to determine size distribution and inductively coupled plasma mass spectrometry (ICPMS) for chemical characterization. The aF4-ICPMS conditions were optimised and validated for linearity, limit of detection, recovery, repeatability and reproducibility, all indicating good performance. Multi-element detection with aF4-ICPMS showed that some commercial pigments contained zirconium co-eluting with titanium in aF4. The other two TiMs, NM103 and NM104, contained aluminium as integral part of the titanium peak eluting in aF4. The materials were characterised using various size determination techniques: retention time in aF4, aF4 hyphenated with multi-angle laser light spectrometry (MALS), single particle ICPMS (spICPMS), scanning electron microscopy (SEM) and particle tracking analysis (PTA). PTA appeared inappropriate. For the other techniques, size distribution patterns were quite similar, i.e. high polydispersity with diameters from 20 to >700 nm, a modal peak between 200 and 500 nm and a shoulder at 600 nm. Number-based size distribution techniques as spICPMS and SEM showed smaller modal diameters than aF4-UV, from which mass-based diameters are calculated. With aF4-MALS calculated, light-scattering-based "diameters of gyration" (Øg) are similar to hydrodynamic diameters (Øh) from aF4-UV analyses and diameters observed with SEM, but much larger than with spICPMS. A Øg/Øh ratio of about 1 indicates that the TiMs are oblate spheres or fractal aggregates. SEM observations confirm the latter structure. The rationale for differences in modal peak diameter is discussed.

Regulatory aspects of nanotechnology in the agri/feed/food sector in EU and non-EU countries.

Nanotechnology has the potential to innovate the agricultural, feed and food sectors (hereinafter referred to as agri/feed/food). Applications that are marketed already include nano-encapsulated agrochemicals or nutrients, antimicrobial nanoparticles and active and intelligent food packaging. Many nano-enabled products are currently under research and development, and may enter the market in the near future. As for any other regulated product, applicants applying for market approval have to demonstrate the safe use of such new products without posing undue safety risks to the consumer and the environment. Several countries all over the world have been active in examining the appropriateness of their regulatory frameworks for dealing with nanotechnologies. As a consequence of this, different approaches have been taken in regulating nano-based products in agri/feed/food. The EU, along with Switzerland, were identified to be the only world region where nano-specific provisions have been incorporated in existing legislation, while in other regions nanomaterials are regulated more implicitly by mainly building on guidance for industry. This paper presents an overview and discusses the state of the art of different regulatory measures for nanomaterials in agri/feed/food, including legislation and guidance for safety assessment in EU and non-EU countries.

Local cholinergic interneurons modulate GABAergic inhibition in the chicken optic tectum.

The chicken optic tectum (TeO) and its mammalian counterpart, the superior colliculus, are important sensory integration centers. Multimodal information is represented in a topographic map, which plays a role in spatial attention and orientation movements. The TeO is organised in 15 layers with clear input and output regions, and further interconnected with the isthmic nuclei (NI), which modulate the response in a winner-takes-all fashion. While many studies have analysed tectal cell types and their modulation from the isthmic system physiologically, little is known about local network activity and its modulation in the tectum. We have recently shown with voltage-sensitive dye imaging that electrical stimulation of the retinorecipient layers results in a stereotypic response, which is under inhibitory control [S. Weigel & H. Luksch (2012) J. Neurophysiol., 107, 640-648]. Here, we analysed the contribution of acetylcholine (ACh) and the NI to evoked tectal responses using a pharmacological approach in a midbrain slice preparation. Application of the nicotinic ACh receptor (AChR) antagonist curarine increased the tectal response in amplitude, duration and lateral extent. This effect was similar but less pronounced when γ-aminobutyric acid(A) receptors were blocked, indicating interaction of inhibitory and cholinergic neurons. The muscarinic AChR antagonist atropine did not change the response pattern. Removal of the NI, which are thought to be the major source of cholinergic input to the TeO, reduced the response only slightly and did not result in a disinhibition. Based on the data presented here and the neuroanatomical literature of the avian TeO, we propose a model of the underlying local circuitry.

International interlaboratory study for sizing and quantification of Ag nanoparticles in food simulants by single-particle ICPMS.

This publication describes the first international intercomparison of particle-size determination by single-particle inductively coupled plasma mass spectrometry (sp-ICPMS). Concentrated monodisperse silver nanoparticle suspensions with particle diameters of 20, 40 and 100 nm and a blank solution were sent to 23 laboratories in Europe, the USA and Canada. Laboratories prepared eight nanoparticle preparations in two food simulants (distilled water; 10% ethanol) and reported median particle size, Ag particle mass concentration and Ag particle number concentrations. Average repeatability and reproducibility standard deviation (sr and sR) for the median particle diameter were 1 and 14 nm, respectively. Relative precision was worse for Ag particle number concentrations (RSD r = 11%; RSD R = 78%). While further improvements of the method, especially with respect to software tools for evaluation, hardware options for shorter dwell times, calibration standards for determining nebuliser efficiency and further experience by laboratories are certainly desirable, the results of this study demonstrate the suitability of sp-ICPMS for the detection and quantification of certain kinds of nanoparticles.

Development and validation of single particle ICP-MS for sizing and quantitative determination of nano-silver in chicken meat.

The application of nanomaterials is leading to innovative developments in industry, agriculture, consumer products, and food and related sectors. However, due to the special properties of these materials there are concerns about their safety, especially because of our limited knowledge of human health effects and the fact that constantly new nanomaterials and applications thereof are being produced. The development of analytical techniques is a key element to understand the benefits as well as the risks of the application of such materials. In this study, a method is developed and validated for sizing and quantifying nano-silver in chicken meat using single particle inductive coupled plasma mass spectrometry (ICP-MS). Samples are processed using an enzymatic digestion followed by dilution of the digest and instrumental analysis of the diluted digest using single particle ICP-MS. Validation of the method in the concentration of 5-25 mg/kg 60-nm silver nanoparticles showed good performance with respect to trueness (98-99% for size, 91-101% for concentration), repeatability (<2% for size, <11% for concentration), and reproducibility (<6% for size, <16% for concentration). The response of the method is linear, and a detection limit as low as 0.1 mg/kg can be obtained. Additional experiments showed that the method is robust and that digests are stable for 3 weeks at 4 °C. Once diluted for single particle ICP-MS analysis, the stability is limited. Finally, it was shown that nano-silver in chicken meat is not stable. Silver nanoparticles dissolved and were transformed into silver sulfide. While this has implications for the form in which nano-silver will be present in real-life meat samples, the developed method will be able to determine the presence and quantity of nanoparticle silver in such samples.

Quantification of sixteen cannabinoids in hemp seed edible oils and the influence of thermal processing on cannabinoid contents and profiles.

To investigate cannabinoid content and profiles, 16 cannabinoids were quantified in 30 commercial hemp seed edible oils. In addition, one hemp seed oil was subjected to thermal processing up to 200 °C for up to 60 min. UHPLC-MS/MS was used for analysis. The content of cannabinoids in the samples ranged from 9 to 279 mg kg-1 (sum) and for Δ9-tetrahydrocannabinol (Δ9-THC) from 0.2 to 6.7 mg kg-1. Three samples exceeded the EU Δ9-THC equivalent maximum levels of 7.5 mg kg-1 for hemp seed oils. Cannabinoid profiles can provide indications of different product characteristics (e.g. degree of processing, variety of plant material). Furthermore, intense thermal processing (200 °C, 60 min) led to 38% decrease in sum cannabinoid content (sum of all analysed cannabinoids in this study), 99% decrease in cannabinoid acids, and 22% increase in Δ9-THC.

Characterisation and quantification of liposome-type nanoparticles in a beverage matrix using hydrodynamic chromatography and MALDI-TOF mass spectrometry.

This paper describes the characterisation of liposome-type nanoparticles (NPs) dispersed in a beverage matrix. Characterisation is based on a two-step procedure: first, liposomes are separated on the basis of size in the nanometre range by use of hydrodynamic chromatography (HDC); second, chemical characterisation is performed by use of MALDI-TOF mass spectrometry (MS). Characterisation of three types of Coatsome liposome, a commercially available type of empty liposome, was investigated. All three liposome types, Coatsome A = anionic, N = neutral, and C = cationic, gave single peaks in HDC, reflecting diameters of 153, 187, and 205 nm, respectively. Subsequent MALDI-TOF MS in positive mode furnished major signals at m/z = 734.5 ([M + H](+) adduct) and m/z = 756.6 ([M + Na](+) adduct) of L-(α)-dipalmitoylphosphatidylcholine (DPPC) monomer and dimeric adducts at m/z = 1468.1 and m/z = 1490.1, respectively. MALDI-TOF MS in negative mode gave a signal at m/z = 721.3 ([M - H](-) adduct) of L-(α)-dipalmitoylphosphatidylglycerol (DPPG), except for Coatsome C which lacks this phospholipid. After HDC separation of Coatsome A NPs the major DPPC and DPPG signals can be detected in the expected fractions by use of MALDI-TOF MS in positive and negative modes, respectively. Validation of the analytical strategy revealed linearity (R(2) > 0.99), repeatability (relative standard deviation <10 %), and reproducibility (relative standard deviation between days <10 %) were good, recovery was 61 ± 5 %, and the limit of quantification was 1 mg mL(-1) in this matrix. With 4 mg Coatsome A mL(-1) 20 out of 20 samples furnished the 734.5 and 756.6 signals typical of DPPC in MALDI-TOF MS characterisation.

Cannabinoid contents in hemp teas and estimation of their transfer into tea infusions.

This study focused on the investigation of cannabinoid profiles and contents of 23 different hemp teas and on the individual transfer of 16 cannabinoids from hemp teas into their tea infusions. The total cannabinoid content in the dry products averaged 14,960 mg kg-1, with CBD&CBDA (sum of cannabidiol (CBD) and cannabidiolic acid (CBDA)) being the major component, accounting for 87% of the total cannabinoid content. The Δ9-tetrahydrocannabinol (Δ9-THC) content ranged from 16 mg kg-1 to 935 mg kg-1 and was on average 221 mg kg-1. For each hemp tea, an infusion was prepared according to a standardized protocol issued by the German Standardisation body DIN and transfer rates per cannabinoid were estimated by comparing the contents in the dry material with the concentrations in the aqueous infusion. The limited water solubility of cannabinoids results in limited extraction efficiency for cannabinoids using boiling water to prepare a tea infusion and the average transfer rate of the psychoactive Δ9-THC was only 0.5%.

Detection of Hypoglycin A and MCPrG Metabolites in the Milk and Urine of Pasture Dairy Cows after Intake of Sycamore Seedlings.

Hypoglycin A (HGA), methylenecyclopropylglycine (MCPrG), hypoglycin B (HGB), and γ-glutamyl-α-(methylenecyclopropyl) glycine (γ-glutamyl-MCPrG) are secondary plant metabolites occurring in sycamore maple (Acer pseudoplatanus) as well as several other Sapindaceae (e.g., Blighia sapida). By interfering with energy metabolism, they may cause severe intoxication in humans and other species. However, to date, there is not enough data available concerning the intake, metabolism, or excretion of sycamore maple toxins in dairy cows. In May 2022, five cows were observed over four days, when they had first access to a pasture with two sycamore maples. Grazing of their seedlings that grew numerously in between the pasture plants was monitored by direct observation. Milk samples were drawn both from individual cows and from the bulk tank. Spontaneous urine samples were collected from all cows on day 3 after access to the pasture. Seedlings (100 g) were sampled on the pasture and analyzed, together with milk and urine samples, for sycamore toxins and their metabolites using liquid chromatography-tandem mass spectrometry and liquid chromatography-high-resolution mass spectrometry. Cows ingested sycamore seedlings while grazing. Values of HGA in milk were below the limit of quantification. However, metabolites of HGA and MCPrG were detected in individual milk samples already at the end of the first day of grazing. Urine samples of all five cows showed higher concentrations of conjugated HGA and MCPrG metabolites than in milk. Observations suggest that dairy cows may have a low susceptibility toward sycamore maple toxins. However, whether this could be attributed to foregut fermenting species in general requires further elucidation.

NanoDefiner Framework and e-Tool Revisited According to the European Commission's Nanomaterial Definition 2022/C 229/01.

The new recommended definition of a nanomaterial, 2022/C 229/01, adopted by the European Commission in 2022, will have a considerable impact on European Union legislation addressing chemicals, and therefore tools to implement this new definition are urgently needed. The updated NanoDefiner framework and its e-tool implementation presented here are such instruments, which help stakeholders to find out in a straightforward way whether a material is a nanomaterial or not. They are two major outcomes of the NanoDefine project, which is explicitly referred to in the new definition. This work revisits the framework and e-tool, and elaborates necessary adjustments to make these outcomes applicable for the updated recommendation. A broad set of case studies on representative materials confirms the validity of these adjustments. To further foster the sustainability and applicability of the framework and e-tool, measures for the FAIRification of expert knowledge within the e-tool's knowledge base are elaborated as well. The updated framework and e-tool are now ready to be used in line with the updated recommendation. The presented approach may serve as an example for reviewing existing guidance and tools developed for the previous definition 2011/696/EU, particularly those adopting NanoDefine project outcomes.

Root uptake and metabolization of Alternaria toxins by winter wheat plants using a hydroponic system.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants.

Spatiotemporal analysis of electrically evoked activity in the chicken optic tectum: a VSDI study.

The midbrain is an important processing area for sensory information in vertebrates. The optic tectum and its mammalian counterpart, the superior colliculus, receive multimodal, topographic information and contain a sensory map that plays a role in spatial attention and orientation movements. Many studies have investigated the tectal circuitry by cytochemistry and by characterization of particular cell types. However, only a few studies have investigated network activation throughout the depth of the tectum. Our study provides the first data on spatiotemporal activity profiles in the depth and width of the avian optic tectum. We used an optical imaging approach with voltage-sensitive dyes to investigate population responses at a high temporal and spatial resolution. With the necessary caution due to cell extension across several layers, we can thus link our findings tentatively with the general layout of the avian optic tectum. Single electrical stimuli in the retinorecipient layers 1-4 evoked a complex optical response pattern with two components: a short, strong transient response and a weaker persistent response that lasted several hundred milliseconds. The response started in layer 5 and spread within this layer before it propagated into deeper layers. This is in line with neuroanatomical and earlier physiological data. Analysis of temporal sequence and pharmacological manipulations revealed that these responses were mainly driven by postsynaptic activation. Thus tectal network responses to patterned input can be studied by voltage-sensitive dye imaging.

Quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 µg kg(-1), oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 µg kg(-1) and for qualitative confirmatory analysis of arbidol at levels below 1 µg kg(-1).

Development of surface plasmon resonance-based sensor for detection of silver nanoparticles in food and the environment.

Silver nanoparticles are recognized as effective antimicrobial agents and have been implemented in various consumer products including washing machines, refrigerators, clothing, medical devices, and food packaging. Alongside the silver nanoparticles benefits, their novel properties have raised concerns about possible adverse effects on biological systems. To protect consumer's health and the environment, efficient monitoring of silver nanoparticles needs to be established. Here, we present the development of human metallothionein (MT) based surface plasmon resonance (SPR) sensor for rapid detection of nanosilver. Incorporation of human metallothionein 1A to the sensor surface enables screening for potentially biologically active silver nanoparticles at parts per billion sensitivity. Other protein ligands were also tested for binding capacity of the nanosilver and were found to be inferior to the metallothionein. The biosensor has been characterized in terms of selectivity and sensitivity towards different types of silver nanoparticles and applied in measurements of real-life samples-such as fresh vegetables and river water. Our findings suggest that human MT1-based SPR sensor has the potential to be utilized as a routine screening method for silver nanoparticles, that can provide rapid and automated analysis dedicated to environmental and food safety monitoring.

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit.

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 µg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.