Detection and Characterisation of Engineered Nanomaterials in Foods

Volker Gräf, Diana Behsnilian, Birgit Hetzer, Elke Walz and Ralf Greiner

Introduction

Together with natural nanoscale food components such as casein micelles in milk, engineered nanomaterials (ENMs) can be present in food as ingredients and additives or as contaminants from the environment and from food contact materials. The detection and characterisation of ENMs is not only essential for the assessment of their potential risks and benefits but also to guarantee an adequate and reliable labelling of food products containing ENMs. In the European Union the labelling of such products comes into force at the end of 2014 (European Union 2011).

Great efforts are undertaken towards the detection and characterisation of ENMs in foods. However, related key aspects such as the definition of ENMs (size based or size and physico-chemical properties), the stage analysis should occur (food ingredient and/or food ingredient in the food matrix) or the set of properties which need to be determined, are still under discussion.

Definition

Within different scopes a series of definitions has been proposed when referring to nanomaterials and ENMs. The European Commission recommended Member States, Union agencies and economic operators to use the following definition for ‘nanomaterial’ in the adoption and implementation of legislation and policy and research programmes concerning products of nanotechnologies: Nanomaterial means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50% (European Commission 2011). The International Organization for Standardization (ISO) defines a ‘nanomaterial’ as a material with any external dimension in the nanoscale or having internal structure or surface structure in the nanoscale, with the nanoscale being the size range from approximately 1 nm to 100 nm (ISO 2010).

Concerning food regulation topics, up to date there is no worldwide harmonised definition for ENMs. A very comprehensive overview about worldwide regulations regarding nanotechnology and food can be found in the OECD report “Regulatory Frameworks for Nanotechnology in Foods and Medical Products” (OECD 2013). For the purpose of the European regulation regarding consumer information (European Union 2011) ENMs are defined as any intentionally produced material that has one or more dimensions of the order of 100 nm or less or that is composed of discrete functional parts, either internally or at the surface, many of which have one or more dimensions of the order of 100 nm or less, including structures, agglomerates or aggregates, which may have a size above the order of 100 nm but retain properties that are characteristic of the nanoscale. Furthermore, the properties that are characteristic of the nanoscale include: (i) those related to the large specific surface area of the materials considered; and/or (ii) specific physico-chemical properties that are different from those of the non-nanoform of the same material.

In April 2012 the US Food and Drug Administration (FDA) distributed, for comment purposes, a draft guidance for the industry assessing the effect of manufacturing processes on the safety and regulatory status of food ingredients and food contact substances (FDA 2012). The FDA states herein that in the absence of a formal definition, when considering whether a FDA-regulated product contains nanomaterials or otherwise involves the application of nanotechnology, FDA will ask: (1) whether an engineered material or end product has at least one dimension in the nanoscale range (approximately 1 nm to 100 nm); or (2) whether an engineered material or end product exhibits properties or phenomena, including physical or chemical properties or biological effects, that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to one micrometre. Furthermore, it is stated that currently there are questions as to whether size alone should be the determining factor for the definition, or whether additional criteria such as shape, charge, the ratio of surface area to volume, or other physical or chemical properties should be included in a definition.

Characterisation

When analysing ENMs in food matrices, obtaining representative samples can be a very difficult task. The properties of the ENM can be affected and altered by a number of factors during sample preparation and analysis. Even a simple dilution with pure water changes the ionic strength and pH of the sample and can alter the particles properties. Figure 1 illustrates some important steps in the “life-time” of food related ENMs from manufacturing, processing and digestion until excretion. To investigate and understand all the interactions it is necessary to analyse samples from all those different steps. When the ENM is incorporated into the food matrix, changes in pH, ionic strength, ionic composition, cooling or heating as well as mechanical stress during food production and digestion can lead to the alteration of the ENM. In addition, food constituents can interact with nanoparticles and alter their properties. Proteins, for example, can form a so called “protein corona” around the nanoparticles leading to a shift of surface charge and particle size.

No single characterisation technique can provide all relevant information necessary for the characterisation of ENMs. Therefore, a combination of different analytical techniques is required. An overview of the techniques used or proposed for the characterisation of ENM is presented in Table 1.

Table 1: Important parameters and analysis methods for the characterisation of nanomaterials (adapted from EFSA 2011)

A number of analytical methods can be used for the characterisation of ENMs in simple matrices such as aqueous solutions. Some of these methods are even standardised, e.g. ASTM E2490-09 (Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by PCS), ASTM E2834-12 (Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by NTA), ISO 22412 (Particle size analysis – DLS), ISO 13321 (Particle size analysis – PCS).

However, foods are generally very complex matrices containing many different structures and substances and/or even a mixture of different ENMs. Although a few characterisation methods can be successfully applied directly, generally some kind of sample preparation is necessary prior to analysis. Thereby the main challenge is the separation of the EMN from the food matrix without affecting the size/size distribution of the nanoparticles. Generally, application of sample preparation methods such as filtration, centrifugation or field-flow fractionation is inevitable.

Inorganic nanomaterials such as titanium dioxide and silicon dioxide can be isolated by acid, alkalis, solvents or enzymic digestion from foods and biological samples. By applying some of those digestion methods it is not possible to remove the food components completely. Therefore, further purification and separation steps (destruction of residual protein, multiple centrifugation, etc) are necessary before the particles can finally be characterised by, for example, field-flow fractionation.

To develop and validate new and existing characterisation methods and to calibrate analysis systems, standard and reference materials are essential. Up to now such materials are scarce, especially for the food sector. An overview of reference materials and representative test materials can be found in Roebben et al. (2013).

As seen above, the current state of the art is a size-based definition for ENMs. Hence, particle size/size distribution is probably the only parameter that needs to be determined when characterising ENMs. The European Food Safety Authority (EFSA) claims that particle size/size distribution should always be determined by at least two independent methods (one being electron microscopy) as the results obtained from different measurement techniques may differ because the physical principles of the techniques are different (EFSA 2011, Domingos et al. 2009). The term ‘‘size of a particle’’ is meaningless without specification of the type of size (e.g. hydrodynamic diameter) and the method used to obtain this size information (Linsinger et al. 2013). Therefore, it is essential to specify the method and the system used, all parameters used for the analysis itself (e.g. system, temperature, refractive index, wavelength, etc.) and for manipulation of the data (e.g. mathematical model used) beside the presentation of the determined parameters itself.

Another important aspect concerning ENMs in food is the characterisation of the chemical composition in order to distinguish between engineered and natural occurring nanoparticles. Therefore qualitative analysis techniques (e.g. energy-dispersive x-ray microanalysis coupled to electron microscopy, ICP-MS) are necessary. However, even with these methods it is quite difficult or even impossible to distinguish between natural nanoparticles and ENMs if the particles consist of the same material and have similar or the same properties. At present, one promising approach is the coupling of field-flow fractionation and ICP-MS for successive quantitative and qualitative ENM analysis. For the simultaneous characterisation of size and chemical composition of inorganic nanoparticles the so-called single particle-ICP-MS arose as a very promising technique. It was first described by Delgueldre et al. (2012) around 10 years ago and has been further developed and refined in the past three years particularly because of the further technical improvement of the ICP-MS instrumentations (e.g. sample introduction, time resolution of detection and data acquisition). This method is based on the direct introduction of highly diluted nanoparticle-containing samples into the ICP-MS system. Both the coupling of field-flow fractionation with ICP-MS, as well as SP-ICP-MS, have their assets and drawbacks (e.g. detection limit, sample preparation, time and effort, type of ENM etc.), which were recently summarised by Ulrich et al. (2012) and have to be weighed against each other depending on the current application.

The European NanoLyse project (www.nanolyse.eu) focuses on the development of validated methods and reference materials for the analysis of ENMs in food and beverages. It is planned that the developed methods cover all relevant classes of ENP with reported or expected food and food contact material applications, i.e. metal, metal oxide/silicate, surface functionalised and organic encapsulated engineered nanoparticles. Some of the main achievements reported until now are the development of sample preparation methods, enzymic digestion for silver nanoparticles in meat (Loeschner et al. 2013) and acid digestion for silica nanoparticles in tomato soup; the development and validation of a quantification method for fullerenes in vegetable oil (at ppb levels) based on liquid chromatography – mass spectrometry (LC/MS). The results are promising, but only a limited number of ENM-food matrix combinations are being the object of study within the framework of the project.

Concluding remarks

In food matrices nanoparticles must be detected along with larger particles. Therefore a suitable sample preparation (appropriate extraction and preparation method) is needed to extract the interesting nanoparticles from the food matrix and separate them from larger particles.

The discrimination between natural and engineered nanomaterials should be possible. However, because of the chemical nature of food (huge amount of natural organic nanoparticles, e.g. macromolecular structures as proteins) it is difficult to identify engineered organic structures in food.

At the present time only a few preparation and analysis methods for the determination of (number based) particle size distributions in complex matrices like food have been developed and validated, mainly in the framework of a European research project. Though model food materials, spiked with well-known nanoparticles were used for all of the studies, the development of analysis methods was complicated because it had to be ensured, that the preparation methods used did not affect the particle size distribution of the nanoparticles.

Last but not least, detection and characterisation of nanomaterial in complex matrices such as food will continue to be a challenge, especially when real samples need to be analysed (no information about the presence of nanomaterials and their nature). This is essentially important in respect to the fact that the EU regulation regarding the labelling of ENMs used as ingredients or additives in food products will come into force at the end of 2014.

References

Degueldre, C and Favarger, PY (2003) Colloid analysis by single particle inductively coupled plasma-mass spectroscopy: a feasibility study. Colloids Surfaces –Physicochem. Eng. Asp. 217: 137-142.

Domingos, RF, Baalousha, MA, Ju-Nam, Y, Reid, MM, Tufenkji, N, Lead, JR, Leppard, GG and Wilkinson, KJ (2009) Characterizing manufactured nanoparticles   in the environment: Multimethod determination of particle sizes. Environ. Sci. Technol. 43: 7277-7284.

EFSA Scientific Committee (2011) Scientific Opinion: Guidance on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain.  EFSA J. 9(5): 2140

European Commission (2011) Recommendation of 18 October 2011 on the definition of nanomaterial (2011/696/EU).

European Union (2011) Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers.

Food and Drug Administration (2012) Draft Guidance for Industry - Assessing the Effects of Significant Manufacturing Process Changes, Including Emerging Technologies, on the Safety and Regulatory Status of Food Ingredients and Food Contact Substances, Including Food Ingredients that are Color Additives. Washington, DC: US FDA.

ISO (2010) ISO/TS 80004-1 (2010): Nanotechnologies  ̶  Vocabulary  ̶  Part 1: Core terms. Geneva: International Standards Organization: Linsinger, TPJ, Chaudhry, Q, Dehalu, V, Delahaut, P Dudkiewicz, A, Grombe, R, Von Der Kammer, F, Larsen, EH, Legros, S, Loeschner, K, Peters, R, Ramsch, R, Roebben, G, Tiede, K and Weigel, S (2013) Validation of methods for the detection and quantification of engineered nanoparticles in food. Food Chem. 138: 1959-1966.

Loeschner, K, Navratilova, J, Købler, C, Mølhave, K, Wagner, S, von der Kammer, F and Larsen, EH (2013) Detection and characterization of silver nanoparticles in chicken meat by asymmetric flow field flow fractionation with detection by conventional or single particle ICP-MS. Anal. Bioanal. Chem. 405(25): 8185-8195.

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Roebben, G, Rasmussen, K, Kestens, V., Linsinger, TPJ, Rauscher, H, Emons, H and Stamm, H (2013) Reference materials and representative test materials: the nanotechnology case. J. Nanoparticle Res. 15: 1-13.

Ulrich, A, Losert, S, Bendixen, N, Al-Kattan, A, Hagendorfer, H, Nowack, B, Adlhart, C, Ebert, J, Lattuada, M and Hungerbuhler, K (2012) Critical aspects of sample handling for direct nanoparticle analysis and analytical challenges using asymmetric field flow fractionation in a multi-detector approach. J. Anal. Atomic Spectrom. 27: 1120-1130.

Drs Volker Gräf, Diana Behsnilian, Birgit Hetzer, Elke Walz and Ralf Greiner are with the  Max-Rubner Institut, Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany; E-mails: volker.graef@mri.bund.de; diana.behsnilian@mri.bund.de; birgit.hetzer@mri.bund.de; elke.walz@mri.bund.de; ralf.greiner@mri.bund.de