Determining nanomaterials in food

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Determining nanomaterials in food Cristina Blasco, Yolanda Pico´ Nanotechnology has emerged as one of the most innovative technologies and has the potential to improve food quality and safety. However, there are a few studies demonstrating that nanomaterials (NMs) are not inherently benign. This review highlights some current applications of NMs in food, food additives and food-contact materials, and reviews analytical approaches suitable to address food-safety issues related to nanotechnology. We start with a preliminary discussion on the current regulatory situation with respect to nanotechnology in relation to foods. We cover sample preparation, imaging techniques (e.g., electron microscopy, scanning electron microscopy and X-ray microscopy), separation methods (e.g., field-flow fractionation and chromatographic techniques) and detection or characterization techniques (e.g., light scattering, Raman spectroscopy and mass spectrometry). We also show the first applications of the analysis of NMs in food matrices. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Consumer safety; Food; Food additives; Food analysis; Food matrix; Food packaging; Nanomaterial; Nanoparticle; Nanotechnology; Regulatory framework

Cristina Blasco, Yolanda Pico´* Laboratori de Nutricio´ i Bromatologia, Facultat de Farma`cia, Universitat de Vale`ncia, Av. Vicent Andre´s Estelle´s s/n, 46100 Burjassot, Vale`ncia, Spain

*

Corresponding author. Tel. +34 96 3543092; Fax: +34 96 3544954; E-mail: [email protected]

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1. Introduction A number of recent reports and reviews have identified current, short-term projected applications of nanomaterials (NMs) in the food industry, and for food and beverages [1–5]: (1) development of materials with novel functionality; (2) microscale and nanoscale processing; (3) new products development; and, (4) design of methods and instrumentation for food safety and biosecurity. Fig. 1 identifies these areas of application in the food-processing chain, grouped by target area. A complex set of engineering and scientific challenges in the food and bioprocessing industry in manufacturing high-quality, safe food with efficient, sustainable resources can be solved through nanotechnology. Among emerging applications of nanotechnology in the food industry are: (1) bacteria identification and food-quality monitoring using biosensors [6]; (2) intelligent, active, and smart foodpackaging systems [7]; and, (3) nanoencapsulation of bioactive food compounds (e.g., micelles, liposomes, nanoemulsion, biopolymeric nanoparticles, and cubosomes) [2,3]. Table 1 sets out some examples of NMs applied to food, divided into several categories including food, food additives and

food packaging, using many different types of materials [e.g., membrane, nanocapsule, nanoemulsion, liposomal nanovesicle, nanotube (NT), nanosphere, nanoceramic, nanoclay and nanowire]. According to a study from iRAP, Inc. [8], the total nano-enabled food and beverage packaging market in the year 2008 was $4.13bn, which grew in 2009 to $4.21bn and is forecast to grow to $7.3bn by 2014, at a compound annual growth rate of 11.65%. Active technology represents the largest share of the market, and will continue to do so in 2014 with $4.35bn in sales, and the intelligent segment will grow to $2.47bn. The US NM market, which totaled only $125m in 2000, is expected to reach $30bn by 2020, and packaging with nanotechnology is expected to grow at 11.65% from 2008 until 2013. While the majority of manufacturing and use of nanoscale materials is in USA, the European Union (EU), with its global share of the sector of around 30%, is not lagging far behind in this field [9]. Although the prospective beneficial effects of nanotechnologies are generally well described, studies assessing their potential toxicological effects and impacts are still limited [10,11]. However, the scientific community is concerned about this issue, and there is now a wider debate about the risks of the many manufactured NMs. Due to this, hundreds of in vitro

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Figure 1. Nano applications in food and the food industry.

toxicological studies have been reported, as well as numerous reviews and perspectives [12–18]. Recent examples in the literature show that engineered inorganic nanoparticles (NPs) and carbon nanostructures may incidentally or intentionally enter into contact with living organisms, may disrupt normal activity and may lead to malfunctioning and diseases [19]. NMs are able to cross biological membranes and access cells, tissues and organs that larger particles normally cannot [19]. NMs can also enter the blood stream via inhalation or ingestion, and some NMs penetrate the skin [20]. Then, once in the blood stream, NMs can be transported around the body and taken up by organs and tissues, including brain, heart, liver, kidneys, spleen, bone marrow and nervous system. Studies demonstrate the potential of NMs to cause DNA mutation and induce major structural damage to mitochondria, even resulting in cell death [21]. Size is a key factor in determining the potential toxicity of a particle [22], but there are also other contributing aspects (e.g., chemical composition, morphology or shape, surface structure, surface charge, aggregation and solubility, and the presence or absence of other chemical functional groups) [10]. It is difficult to generalize about health risks associated with exposure to NMs – each new NM must be individually

assessed, taking into account all material properties. As a consequence, international agencies and governments are paying attention to the study of the fate, transport, and health effects of NMs in food and the environment. Several reviews already present the latest research carried out to assess the risks of engineering NMs (ENMs) in the aquatic environment, including analytical methods and ecotoxicity assessments [23–28]. Outstandingly, the latest reviews, focusing on emerging contaminants in food and the environment, include NMs as one of the hottest topics in research today [29–35]. The prerequisite for toxicological, toxicokinetic, migration and exposure assessment is the development of analytical tools for detection and characterization of NPs in complex matrices [22]. Due to consumer safety, it is necessary to control the content of NMs in food [36]. To obtain this information, reliable quantitative methods of analysis are required to measure levels of NMs in a broad range of matrices. In food, there are natural NMs, intentionally added ENMs – derived from naturally occurring food components, or engineered using materials that are not endogenous to foods – and NMs resulting from contamination. Many food substances or ingredients have nanostructures in nature and are present at lm or nm in size: http://www.elsevier.com/locate/trac

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Table 1. Nanomaterials (NMs) in food, food additives and food packaging materials Product name

Manufacturer

Food packaging materials Durethan KU 2-2601 Bayer

Hite brewery beers: three-layer, 1.6 L beer bottle

Honeywell

NMs Silica in a polymerbased nanocomposite

HoneywellÕs Aegis OX nylon-based nanocomposite

Claim Nanoparticles of silica in the plastic prevent the penetration of oxygen and gas of the wrapping, extending the product shelf life         

Oxygen and carbon-dioxide barrier Clarity Recyclability Ease of Perform Processability Flavor/odor/aroma barrier Structural integrity Delamination resistance Aegis barrier nylon resins can be in a multitude of applications

Millar beers:  Lite  Genuine Draft  Ice House

Nanocor

Imperm nylon/ nanocomposite barrier technology produced by Nanocor

Imperm is a plastic imbedded with clay nano-particles that makes bottles less likely to shatter and increases shelf life to up to six months

Nano Plastic Wrap

Songsing Nanotechnology

Nano zinc light catalyst

Constantia multifilm N-COAT

Constantia Multifilm

Nanocomposite polymer

DuPont Light Stabilizer 210

Du Pont

Nano TiO2

Biodegradable after use Compostable to European standards EN13432 Made from renewable and sustainable resources (non-GM corn starch) Water dispersible, will not pollute local groundwater systems or waterways In use since 2002 A clear laminate with outstanding gas-barrier proprieties developed primarily for the nuts, dry food and snack markets UV-protected plastic food packaging

Adhesive form MacDonaldÕs burger containers Food additives AdNano

Ecosynthetix

50–150 nm starch nanospheres

Evonik (Degussa)

Nano ZnO (food grade)

Aerosil, Sipernat

Evonik (Degussa)

Silica (food grade)

AquaNova NovaSol

AquaNova

Product micelle (capsule) of lipophilic, water-insoluble substances

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The adhesive requires less water as well as less time and energy to dry

Free flow add for powdered ingredients in the food industry An optimum carrier system of hydrophobic substances for a higher and faster intestinal and dermal resorption and penetration of active ingredients

Web address or reference http://www.research. bayer.com/edition_15/ 15_polyamides.pdfx http://www.packaginggateway.com/features/ feature79/ http://www51.honeywell. com/sm/aegis/

http://www.nanocor.com/ applications.asp http://www.forbes.com/ investmentnewsletters/2005/ 08/09/nanotechnology-krafthersheycz_jw_0810soapbox_ inl.html?partner=rss http://www.physorg.com/ news717488335.html

http://www.constantiamultifilm.com/

http://www2.dupont.com/ Titanium_Technologies/ en_US/products/dls_210/ dls_210_landing.html http://www.physorg.com/ news71748835.htlm

www.advancednano materials.com www.aerosil.com http://www.aquanova.de/ product-micelle.htm

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Table 1 (continued) Product name

Manufacturer

NMs

Bioral Omega-3 nanocochleates

BioDelivery Sciences International

Nano- cochleates as small as 50 nm

NanoCoQ10

Pharmanex

Nano coQ10

Nano self-assembled, structured liquids

Nutralease

Nanomicelles for encapsulation of nutraceuticals

Solu E 200 BASF

BASF

Synthetic Lycopene

BASF

Vitamin E nano-solution using NovaSOl LycoVit 10% (< 200 nm synthetic lycopene

Shenzen Become Industry & Trading Co

Nanoparticles (160 nm)

Nano Slim

Nano Slim

Nanoceuticals Slim Shake Chocolate

RBC Lifescience

NAno-Diffuse Technology Nanodusters

Nanoceuticals Slim Shake Vanilla

RBC Lifescience

Nanodusters

Fortified fruit juice

High Vive.com

Daily Vitamin Boost

Jamba Juice Hawaii

300 nm iron (SunActive Fe) 300 nm iron (SunActive Fe)

Oat Chocolate Nutritional Drink Mix

Toddler Health

300 nm iron (SunActive Fe)

Oat Vanilla Nutritional Drink Mix

Toddler Health

300 nm iron (SunActive Fe)

Canola Active oil

Shemen

Nano-sized selfassembled structured liquid micelles

Food and beverages Nano Tea

Claim

Web address or reference

Effective means for the addition of omega-3 fatty acids for use in cake, muffins, pasta, noodles, soup, cookies, cereals, chips and candy bars Nano technology to deliver highly bioavailable coenzyme Q10, making it up to 10 times more bioavailable than other forms of CoQ10 Improved bioavailability means nutraceuticals are released into membrane between the digestive system and the blood Solubilization of fat-soluble vitamins

Patent No.: 0100033.3 – Threestep preparation method and its application for nanotea Patent No.:02100314.9/ 00244295.7 – Multi-layer, swinging nano-ball milling procedures Orosolic acid (derived from the Lagerstroemia spaciosa plant)

22 essential vitamins and minerals and 100%, or more of your daily needs of 18 of them! Toddler health is an all-natural balanced nutritional drink for children from 13 months to 5 years. One serving of Toddler Health helps little ones meet their daily requirements for vitamins, minerals and protein Toddler health is an all-natural balanced nutritional drink for children from 13 months to 5 years. One serving of Toddler Health helps little ones meet their daily requirements for vitamins, minerals and protein

http://www. biodellveryscience.com/ bioralnutrients.htlm

http://www.pharmanex.com/ intercom/ productDetall.do?prod1d010036628.mkt1d-2031 http://www.nutralease.com/ technology.asp

http://www.humannutrition.basf http://www.humannutrition.basf.com

http://www.369.comcn/Er/ nanotea.htm

http://www.nanoslim.con/ nanoslim-information.html http://www.rbdifesciences. com/Meal_Replacement_ Shakes.aspx http:// www.rbdifesciences.com/ Meal_ Replacement_Shakes.aspx http://www.highwive.com/ sunactiveiron.htm http:// jambajuicehawaii.com/vitaboost.asp http://www.toddlehealth.net/ OatChocolate.php

http://www.toddlehealth.net/ OatChocolate.php

http://www.shemen.co.il

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(1) food proteins, which are globular particles of 10s to 100s of nm in size, are true NPs; (2) linear polysaccharides with one-dimensional nanostructures are less than 1 nm in thickness; and, (3) starch polysaccharides have small 3-D crystalline nanostructures that are only 10s of nm in thickness. Although natural NMs in food are not considered within the scope of this review, they contribute to the complexity of the analysis for two reasons: (1) first, they should be distinguished from ENMs or contaminating NMs [27]; and, (2) second, due to their specific physico-chemical properties, NPs could interact with proteins, lipids, carbohydrates, nucleic acids, ions, minerals and water in food, feed and biological tissues. It is important to characterize the effects and the interactions of ENMs in the relevant food matrix. Proteins and carbohydrates have large specific surface areas and a high electrochemical surface charge that is likely to make them interact with charged particles, like many engineering NPs (ENPs). These components also contain hydrophobic domains that are likely to interact with hydrophobic ENPs [e.g., fullerenes and carbon nanotubes (CNTs)]. Food also contains natural colloids and dissolved ions. Dispersed colloids are particles in the ENP range (1– 200 nm) that are kept in a stable aqueous suspension [11]. They do not precipitate by gravitation due to their small size, a certain surface charge, electrostatic interactions, van der Waals forces and steric forces. Any changes (e.g., pH or ion concentrations) may destabilize the suspension. According to the classical double-layer and colloid-stability theories, particle stability is affected by the concentration of cations (coagulants), meaning that, at increasing salt concentrations, free NPs will start to aggregate [20]. Releasing ENPs into such complex systems is bound to lead to a range of interactions, and it is not evident whether a given ENP will be adsorbed to a surface or if it will be stabilized by natural polymers so that it remains mobile [37]. Given the huge diversity of ENPs for use in the food and feed sector (e.g., chemical composition, size, size distribution, surface activity/modification) (see Table 1), and their potential interaction with food-matrix components (e.g., proteins), the determination of NMs in food is a challenging task requiring tailored solutions. As regulation for food nanotechnology moves forward, the aim of this review is to address and to compare the available analytical methods for determination of NMs in food. Two previous reviews underpin this one: (1) one provided a detailed description of food nanodelivery systems and considered the analytical techniques useful for identifying and characterizing these systems in food [38]; and, (2) the other overviewed the different analytical techniques available for detecting the ENPs in product

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formulation, environmental matrices and food materials [39], but it drew heavily on studies reporting characterization of NPs in raw products and environmental materials because limited work has been done to date on detection and characterization of NPs in food. This review takes a step forward by including the first applications of NMs to food.

2. Regulations The safety of nanoproducts has attracted attention in line with their increasing use. Despite the rapid commercialization of nanotechnology, no specific nano regulations exist anywhere in the world. Most regulatory agencies remain in information-gathering mode, lacking the legal and scientific tools, information and resources that they need to oversee the exponential market growth of nanotechnology adequately [1,40–44]. At present, international organizations are still attempting to determine the current capacity to assess the health and safety risk associated with the use of nanotechnology in food and food production and on surfaces in contact with food. Through its horizon-scanning activities, the Food and Agriculture Organization (FAO) has recognized the need for scientific advice on any food-safety implications that may arise from the use of nanotechnologies in the food and agriculture sectors. With the FAO, the World Health Organization (WHO) has published the report of a Joint Expert Meeting held in June 2009 on the topic of Application of Nanotechnologies in the Food and Agriculture Sectors: Potential Food Safety Implications [45]. This report presents an overview of the wide range of current and projected nanotechnology applications in food and agriculture (Fig. 2). Applications that may lead to human exposure to NPs through the environment to the food chain were not included. The Council of the Organization for Economical Cooperation and Development (OECD) has established a Working Party on Manufactured NMs as a subsidiary body of its Chemicals Committee [46]. This working party was established to address human health and environmental safety aspects of manufactured NMs in the chemicals sector. Furthermore, in February 2009, the European Food Safety Authority (EFSA) published its opinion on the potential risks arising from nanoscience and nanotechnologies in food and feed [47]. It considered, among other things, the suitability of current regulations relating to the use of nanotechnologies in the food sector. The report did not identify any major gaps in regulations, although it noted that there was uncertainty in some areas as to whether applications of nanotechnologies would be picked

Nanodeliver N d li systems t p based on encapsulated technology

Nanomicelle-based carrier system Nanocluster delivery system

Spreads S d ayo a se Mayonnaise Cream Yoghurts ice creams

Vegetable oil enriched in vitamins vitamins, minerals and phytochemicals

Inorganic NMs (TiO 2, silver, silica, selenium, calcium, iron) Nanomaterials relevant to food applications

Nano-enabled food contact materials (FCMs) and packaging

Surface functionalized NMs o o u e e es, Organic NMs (synthetic nanosized a os ed form of lycopene, ycope e, fullerenes, carbon nanotubes)

NP reinforced materials (polymer composites with nano-clays, nano-clays nanometals or metal oxides, coating contained NPs and antimicrobial nanoemulsions)

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IIntelligent lli packaging k i concepts based b d on nanosensors

Nanotechnology in the agricultural sector

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P Procesed d nanostructured in food (NANOTEXTURES)

Nano-emulsions Surfactant micelles y Emulsion bilayers, Double or multiple emulsions Reverse micelles

Animal feed Agrochemicals

Nanoclay–polymer N l l composites it Oxygen O yge de detecting ec g ink co containing a g TiO2 NPs Nanolayer of silver that react y g sulfide with hydrogen

Natural biopolymer from yeast cell walls that bind t i mycotoxins Polystyrene y y ((PS)) base,, p polyethylene y y g glycol y ((PEG)) linker,, and mannose targeting biomolecule to bind E. coli Slow or controlled Slowcontrolled-release release fertilizers and pesticides

Figure 2. Nanomaterials relevant to food.

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Table 2. European Union legislation applicable to NMs in food Legislation

Comments

General for chemical compounds REACH European Community legislation concerned with chemicals and their safe use and dealing with the Registration, Evaluation, Authorisation and restriction of CHemical substances. Novel foods regulation Regulation (EC) No 258/97

Food additives Directive 89/107/EC and associated legislation

Food-contact materials Regulation EC/1935/2004

Food supplements Directive 2002/46/EC

[51]

Novel foods are foods and food ingredients that have not been used for human consumption to a significant degree in the EC before 15 May 1997, and the Regulation subjects all novel foods and foods manufactured using novel processes to a mandatory pre-market approval system. In January 2008, the European Commission published a proposal to revise and update the Novel Foods Regulation. Various proposals have been discussed by the Commission, Parliament and Council (The draft Regulation is currently going through the co-decision procedure). A definition of NMs has been introduced at the request of the European Parliament, and supported by the Council. Discussions are continuing on how to bring nanotechnologies specifically into the revised Regulation.

[52,53]

Only additives explicitly authorized may be used in food. In December 2008, a new Regulation was passed (Regulation EC/1333/2008), which set out a common authorization procedure for additives, enzymes and flavorings. From early 2010, a list of approved additives, including vitamins and minerals, came into force. Inclusion of additives on the list was decided by the Commission on the basis of an Opinion from the EFSA. Those included often had limits set on their use, for example, restrictions on the quantities permitted for use. The new regulations also specify that, where the starting material used, or the process by which an additive is produced, is significantly different (for example, through a change in particle size), it must go through a fresh authorization process, including a new safety evaluation.

[54–56]

All materials that are intended to come into contact with foodstuffs, either directly or indirectly. The Commission or Member States may request the EFSA to conduct a safety evaluation of any substance or compound used in the manufacture of a food contact material. Certain materials, including plastics, are subject to additional measures. The Commission has proposed updating the Regulation governing food-contact plastics to specify that a deliberately-altered particle size should not be used, even behind a migration barrier, without specific authorization.

[57]

States that only vitamins and minerals on an approved list may be used as food supplements. New substances may be considered for inclusion on the list, but only after a safety assessment by EFSA.

[58]

up consistently. It concluded that ‘‘on the basis of current information, most potential uses of nanotechnologies that could affect the food area would come under some form of approval process before being permitted for use’’. These international agencies identified a number of domains of interest as a starting point for this review, so that more research on NMs is needed to improve the basis of scientific knowledge in support of regulatory work: – development of reliable measurement methods, reference materials and materials characterization; – review and development of test methods for human health, safety and the environment; – development of exposure information throughout the life-cycle of NMs; – review of existing risk-assessment methods; – risk management for workersÕ protection; – networking existing and establishing new infrastructures to examine health, safety and environmental aspects of NMs. However, different countries are trying to include NMs in their current regulations. In USA, the Environmental Protection Agency (EPA) is already empowered to 90

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regulate NMs under several laws. The EPA could use most of the environmental laws – Clean Water Act (CWA), Clean Air Act (CAA), Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), Resource Conservation and Recovery Act (RCRA), Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), and Toxic Substances Control Act (TSCA). The US Food and Drug Administration (FDA) requires manufacturers to demonstrate that food and their ingredients are not dangerous to health, but there are no specific rules for NPs because it regulates products, not technologies. Nevertheless, the FDA expects that many nanotechnology products will come under its jurisdiction. The FDA regulates a wide range of products, including foods, cosmetics, drugs, devices, and veterinary products, some of which contain NMs or are produced nanotechnologically. The Acting Commissioner of the FDA initiated the Nanotechnology Task Force in 2006 to help address questions regarding adequacy and application by regulatory authorities [48]. The European Commission (EC) aims to reinforce nanotechnology and, at the same time, enhance support

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Table 3. Different parameters and characterization methods for NMs Parameters

Characterization methods

Ref.

Particle size & size distribution, morphology or shape and aggregation state

Microscopy and microscopy-related (imaging): near field-scanning optical microscopy (NSOM); scanning probe microscopy (SPM); confocal laser scanning microscopy (CLSM); scanning electron microscopy (SEM); transmission electron microscopy (TEM); scanning transmission electron microscopy (STEM); X-ray microscopy (XRM); scanning transmission X-ray microscopy (STXM); and, atomic force microscopy (AFM) Centrifugation and filtration Chromatography and related (separation): size exclusion chromatography (SEC); capillary electrophoresis (CE); hydrodynamic chromatography (HDC); field flow fractionation (FFF) Spectroscopy and related (characterization): static light scattering (SLS); dynamic light scattering (DLS); neutron scattering (NS); SLS-DLS; SLS-FFF; small-angle X-ray scattering (SAXS); laser-induced breakdown detection (LIBD); Raman spectroscopy; laser-induced fluorescence (LIF); nuclear magnetic resonance (NMR); photon correlation spectroscopy; mercury porositometry; and, laser diffractrometry Analytical (spectroscopy) coupled to electron microscopy (imaging): TEMdispersive X-ray spectroscopy (EDS); SEM-EDS; TEM-electron energy loss spectroscopy (EELS); TEM-selected area electron diffraction (SAED); and, AFM-chemical force microscopy (CFM) Spectroscopy and related (characterization): Raman spectroscopy; LIF; UV-Vis; infrared spectroscopy; NMR Mass spectrometry (characterization): Sources – electrospray ionization (ESI); matrix-assisted laser desorption/ ionization (MALDI); laser desorption/ionization (LDI); and, inductively coupled plasma (ICP) Mass analyzers – time-of-flight (TOF); quadrupole linear ion-trap (QqLIT); ion trap (IT); single quadrupole; triple quadrupole (QqQ); and, quadruple time-of-flight (QqTOF) Laser droplet anemometry, Zeta potentiometer, CE Water contact angle measurements, rose bangle (dye) binding, hydrophobic interaction chromatography, X-ray photoelectron spectroscopy Spectroscopy and related (characterization): static X-ray spectroscopy [Xray photoelectron (XPX); X-ray fluorescence (XRF); X-ray absorption spectroscopy (XAS); and, X-ray diffraction (XRD)] Mass spectrometry (characterization): Sources – electrospray ionization (ESI); matrix-assisted laser desorption/ ionization (MALDI)); laser desorption/ionization (LDI); and, inductively coupled plasma (ICP) Mass analyzers – time-of-flight (TOF); quadrupole linear ion-trap (QqLIT); ion trap (IT); single quadrupole; triple quadrupole (QqQ); quadruple timeof-flight (QqTOF); static secondary ion mass spectrometry (SSIM) Differential scanning calorimetry Critical flocculation temperature(CFT) In-vitro release characteristic under physiologic & sink condition Bioassay of target compound extracted from NP, chemical analysis of the target compounds

[22,23,26,27,38,39,59–62]

Chemical characterization

Charge determination Surface hydrophobicity

Chemical analysis of surface

Carrier-drug interaction Nanoparticle-dispersion stability Release profile Target-compound stability

for collaborative research and development (R&D) on the potential impact of nanotechnology on human health and the environment via toxicological and ecotoxicological studies. The EC is performing a regulatory inventory, covering EU regulatory frameworks that are applicable to NMs (e.g., chemicals, worker protection, environmental, and product-specific). The purpose of this inventory is to examine and, where appropriate, to propose adaptations of EU regulations in relevant sectors [49]. This includes to apply existing food laws to food products using nanotechnology. All food products have

[23,26,38,39,63] [23,26,38,39,60]

[22,23,27,38,39,60,62,64]

[38,39,59] [22,23,27,38,39,59–62]

[22,23,27,38,39,60,64] [27,39,65–67]

[26,27,39] [26,39]

[22,38,39]

[39,65–67]

[39,68,69] [39,70,71] [39] [39]

to meet a general safety requirement under the General Principles of Food Law Regulation (EC/178/2002) [50]. More specific legislation covers the use of novel foods, food additives and food-contact materials (see Table 2).

3. Analytical approaches to characterize and determine NMs Recently, the problem of NM safety, once mainly limited to its chemical aspects, has been extended to possible http://www.elsevier.com/locate/trac

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toxicity associated with NPs as physical entities [22], so the analysis of NMs commonly requires two types of method: (1) those for characterizing and/or detecting NPs or NMs; and, (2) those for determining their chemical composition. However, the line that divides these types is very thin, taking into account that the chemical composition of NMs is one of their properties. As outlined in Table 3, there are a number of analytical tools for qualitative and quantitative categorization of NMs: (1) single-particle techniques; (2) techniques characterizing the ensemble of NMs; and, (3) techniques to determine their chemical composition. For a wide discussion on the advantages or the drawbacks of each technique, we refer the reader to excellent reviews by Tiede et al. [39] and Luykx et al. [38], who accurately explain their advantages and limitations, so there is no need for a repeat in this article. The main problem of analyzing NMs in food is that most of these analytical systems have been used to characterize the NM themselves and only few are applicable to the analysis of more complex samples. Food is a very intricate material, and, probably, for sufficient characterization, the NMs need to be separated from the food matrix, as Luykx et al. [38] discussed for nanodelivery systems. However, the tendency is to reduce sample preparation as much as possible because it is important to measure the NMs in the relevant matrix, as their properties may depend on the surrounding matrix and be affected by processing as well as by the extraction procedure. This is usually much more demanding than to analyze NMs in simpler or model matrices. With nanoscale metals or semiconductors containing NMs, these can be detected even in rather complex matrices (e.g., food, feed and biological tissues) by means of electron microscopy (EM) coupled with chemical analytical tools. However, detection by EM is only possible if the number of NMs is sufficiently high in the matrix to localize them, since high magnification is required due the small size of NMs. As a result, the investigation of NM distribution in food is generally extremely time consuming. We need to mention that transmission EM (TEM) has so far provided the most detailed information regarding NM location by allowing both visualization of the location within food, and, in conjunction with spectroscopic methods, characterization of the composition of the internalized NPs. Visualization is most easily accomplished with electron-dense NMs (e.g., metal NPs). However, the technique is also suitable for any other NMs, including organic compounds (e.g., fullerenes and CNTs). However, the long time required for both sample preparation and image analysis greatly limits the analytical 92

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throughput when using these techniques for analysis of NPs in food. For CNTs, Raman spectroscopy is a very important technique and could be very useful for finding CNTs and fullerenes. This technique is cheap, non-destructive and not time consuming. It has also the advantage of being used in in situ monitoring. Several reviews highlight the power of Raman spectroscopy for measuring CNTs [72,73]. The second group of techniques possesses a lower analytical ambition but is best suited to routine analysis. These approaches are based on the chemical characterization of NMs, without generating information on their physical state. Hence, the metal content of NMs can be quantified by analytical-chemistry tools [e.g., inductively-coupled plasma atomic emission spectroscopy (ICP-AES) or mass spectrometry (ICP-MS)], or by radioanalysis after appropriate neutron irradiation. Generally, sample preparation includes acidic sample digestion before analysis. The limitations of chemical analysis result from artificial losses during the preparatory steps, analytical detection limits and the inability to characterize carbon NPs (e.g., polymeric NPs, fullerenes, and CNTs). In the case of organic NMs, detection or quantification of the chemical may be possible, where a test for the species exists, but a focus on characteristic structures may be needed to determine whether it is still in nanoform. So far, only very limited work has been done on the detection of organic NPs in food. However, the need to determine organic NMs and nano-delivery systems led scientists to imagine what techniques could also be applicable to their characterization in food, even though they have not yet been used. There are a few recent examples of NP-enabled MS that, though not explicitly linked to NP determination in food, demonstrated the potential of this approach. For example, matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)MS and laser desorption/ionization (LDI)-TOF-MS were useful for characterization of ultrasmall NPs of TiO2 [67]. The size distributions of TiO2 NPs obtained from MALDI-TOF-MS and LDI-TOF-MS were in good agreement with parallel TEM observations. TOF secondary-ion MS (SIMS), fluorescence microscopy and scanning EM (SEM) were employed to monitor the immobilization of biotinylated shell-crosslinked NPs on biotin/streptavidin-functionalized, UV-photo patterned self-assembled monolayers [74]. There are also some studies reporting quantification of NMs in a related field (e.g., environmental analysis), where much more information is available. Isaacson and Bouchard [75] reported the first methods for the asymmetric flow field-flow fractionation (AF4) size separation of aqueous C60 aggregates in deionized water without use of mobile-phase modifiers coupled with in-line dynamic light scattering (DLS) and off-line with liquid

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chromatography with atmospheric pressure photo-ionization MS (LC-APPI-MS). Meanwhile, Farre et al. [76] gave details of the first determination of C60 and C70 fullerenes and N-methylfulleropyrrolidine C60 on the suspended material of wastewater effluents by LC hybrid quadrupole linear ion trap tandem MS. As we go deeper into the analysis of NPs, we need analytical procedures to account for explicit information, including molecular weight and the number and the identity of functional groups. Sensitive and mass-selective detection, as offered for MS combined with optimal extraction procedures, shows great potential to achieve this goal. There are some additional complications not yet solved in the analysis of NMs, such as the fact that some ENMs cannot be distinguished from naturally-occurring variants of the same {e.g., nanoscale engineered silicon dioxide (SiO2) or endogenous lipids used in capsule membranes}. Detection may also be hindered by interactions with solutes or food components that obscure clear analytical signals. Despite some successes in this field, its evolution seems to be hampered by the limited information available. This will be a growing area within NM analysis. Moreover, there remain several obstacles to obtaining adequate characterization and quantification of NMs in food. Foremost among them are those presented by the lack of analytical standards, relevant reference materials and internationally standardized practices, protocols, and procedures for testing the preparation of food, NP measurement, and data analysis. The OECD has established a list of prioritized materials that takes into account those materials that are already in production (or close to commercial use), as well as considerations of production volume, the likely availability of materials for testing and the existing information [46]. The OECD list comprises:  fullerenes (e.g., C60);  single-walled and multi-walled CNTs (SWCNTs and MWCNTs, respectively);  carbon black;  polystyrene;  dendrimers;  nanoclays; and,  NPs of Ag, Fe, TiO2, Al2O3, CeO2, ZnO, and SiO2 [46]. Internal standard quantification using stable isotopelabeled internal standard is de rigueur for trace-level analysis of contaminants in food. Currently, there is commercially available only a single, stable-isotope labeled fullerene internal standard, 13C60. The current limited number of standardized reference materials for engineered NMs is another brake on precise, reproducible detection and quantification of engineered NMs in food and feed. The Joint Research Centre, Institute of Reference Materials and Measurements, has recently released a

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quality-control material (IRMM-304) of silica NPs. There are also available gold NPs (NIST RM 8011, 8012 and 8013) and polystyrene spheres (NIST SRM 1963a and 1964) from the National Institute of Standards and Technology (NIST) [77]. Furthermore, interlaboratory studies, which are necessary for validation of protocols and generating precision and bias statements for measurement standards, are almost non-existent. Of the difficulties encountered in conducting interlaboratory studies on these emerging contaminants, the major problem is the wide range of analytical techniques applicable to these compounds and the different purposes of the methods developed [78,79]. There is much work yet to be done to show how far determination of NMs in food can go.

4. Applications to food analysis Applications of the analytical methodology reported in the previous section to the analysis of NMs in food are still very scarce. Table 4 lists the results of the literature search. Various approaches suggested for use in studies of NP bioaccumulation are included because they can be applied to its determination in food, since many of the target organisms are also edible. We need to mention that methods to determine inorganic NPs in food, especially aquatic organisms, are quite developed. There are two main types of procedure: (1) those combining microscopy and spectroscopy to identify and to characterize the NMs as well as to detect their chemical composition; and, (2) those looking at their chemical composition only. For inorganic NPs, the latter methods are based mainly on wet digestion with a strong acid (e.g., nitric or perchloric) followed by ICP optical emission spectroscopy (ICP-OES) or ICP-MS. For example, several methods to measure gold NPs have been developed. These methods are based only on chemical analysis, since we may assume that all the gold present in the samples is from NPs, since gold is not abundant in the environment. Contrarily, other metal NMs {e.g., unmodified commercial nanoscale metal oxides, zinc oxide (ZnO), cerium dioxide (CeO2) and titanium dioxide (TiO2)} were characterized by TEM and environmental scanning EM (ESEM) with energy dispersive X-ray analysis (EDX) elemental analysis to establish their structure as NPs [81]. Also, fish samples were digested with acid and analyzed by ICP-MS. Definitive uptake from the water column and location of TiO2 NPs in gills was demonstrated for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging of rainbow-trout gill tissues clearly showed large aggregates of TiO2 (up to 3 lm) on the surface of the gill epithelium following 24–96 h exposure (Fig. 3). http://www.elsevier.com/locate/trac

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Table 4. Selected applications for detection, characterization and/or quantification of NMs in food or food like matrices Problem Food and beverages Investigation of the presence of microsized and nanosized contaminants

Nanomaterial

Matrix

Techniques

Inorganic NMs

Bread and biscuits

ESEM/EDS

Bioavailability of nanoscale metal oxides to fish

TiO2, CeO2 and ZnO

Rainbow trout

TEM ESEM/EDS ICP-MS ICP-OES CARS

Bioaccumulation of gold NPs in fish

Au NPs

Mytilus edulis

ICP-OES

In vivo toxicity studies

Fullerenes C70 C98

Embryo zebra fish

LC-MS2

Accumulation of NMs in plants

Multi-walled carbon nanotubes TiO2, CeO2

Wheat tissues

TPEM coupled chemical autofluorescence

Migration and stability studies with food-packaging materials Detect clay NPs and Biopolymer polylactide 95% ethanol as characterize their size with 5% Cloisite food simulant 30B as filler

Stability of NPs during heat treatment

Chitosan NPs for l-ascorbic acid

Aqueous solutions

Comment Detection of organic and inorganic microscale and nanoscale contaminants: ESEM Identification of their chemical composition: EDS Characterization of size, particle shape/morphology and qualitative aggregation: TEM and ESEM/EDS Analysis of elements content : wet digestion and ICP-MS, ICPOES Confirmation of the presence of the NP in fish: CARS Analysis of Au content: wet digestion with nitric acid and hydrogen peroxide and ICP-OES Digestion with glacial acetic acid and toluene 150 mm · 2 mm Targa C18 column and toluene/methanol (55:45) isocratic mobile phase Only molecular ions were obtained TPEM combined with autofluorescence can be used to detect NMs interacting with vegetation and TPEM can be used simultaneously to detect and to monitor the interactions of MWCNTs and PAHs in vivo in roots.

Ref. [80]

[81]

[82,83]

[84]

[85]

XRD TEM Centrifugation FA-MALS ICP-MS FA4-MALS-ICP-MS

Characterization of polylactide/ Cloisite 30 B: XRD, TEM Chemical characterization of elements: ICP-MS Separation and size determination of clays: centrifugation, FA4-MALS and clay aspect ratio Migration study FA-MALS-ICPMS

[86]

Zeta potential PDI Ultracentrifugation

Characteristics of AA-loaded CS nanoparticles: particle size, zeta potential, encapsulation efficiency (EE), and release effects Stability of AA-loaded CS nanoparticles with the changes of physico-chemical properties and release rate before and after heat processing in aqueous solutions at various temperatures.

[87]

CARS, Coherent anti-Stokes Raman scattering microscopy; ESEM/EDS, Environmental scanning electron microscopy/X-ray microprobe of an energy dispersive system; FA4, Asymmetrical flow field-flow fractionation; ICP-MS, Inductively coupled plasma mass spectrometry; ICP-OES, Inductively coupled plasma optical emission spectroscopy; LC-MS2, Liquid chromatography-tandem mass spectroscopy; MALS, Multi-angle light-scattering detection; PDI, Polydispersity index; TEM, Transmission electron microscopy; TPEM, Two-photon excitation microscopy, XRD, X-ray diffraction.

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Figure 3. Coherent anti-Stokes Raman scattering (CARS) microscopy images of the gill tissue of rainbow trout, Oncorhynchus mykiss, following water-borne exposure to titanium dioxide (TiO2) nanoparticles (NPs). The cellular structure of the primary (PL) and secondary (SL) gill lamellae, composed of pillar cells (PCs) and pavement cells (PVs), was obtained by epidetection of the CH2 vibration (shown in green). The red blood cells are effectively separated from the lamellae cells by forward detection of the CH2 vibration (shown in blue). (A) Gill tissue, following a 28-day exposure. An aggregate of NPs can be seen occupying the space between the pillar cells. (B) The same NP aggregate under a 3· increase in magnification. (C) Projection of a 300 · 100 lm 3D data set of gill tissue following a 14-day exposure. A cluster of NPs can be seen in the region of the marginal channel (MC). (D) Multi-planar view of the same exposure. The two adjacent sub-panels specifically locate the NPs inside the tissue near the surface of the MC. (Reproduced from [81] with permission, ª 2010 American Chemical Society).

Gatti et al. [80] investigated the presence of inorganic micro-sized and nano-sized contaminants in bread and biscuits from 14 different countries by ESEM. EDX was employed to identify their chemical composition. The results indicated that 40% of the samples analyzed contained foreign bodies (e.g., ceramic and metallic debris, probably of environmental or industrial origin). Fig. 4 shows other debris found in a biscuit, which contained cadmium, silver, tungsten, aluminum, sulfur, calcium, iron, cobalt and copper in particles. Biopolymer nanocomposites are a field of emerging interest, since such materials can exhibit improved mechanical and barrier properties, and they can be more suitable for a wider range of food-packaging applications.

From a food-safety point of view, it is important to characterize migrates from nanocomposites containing clay as fillers. Schmidt et al. [86] demonstrated that AF4 with multi-angle light scattering (MALS) detection was useful for characterizing the size of NPs contained in migrates from nanocomposites of polylactide and organomodified montmorillonite clay as filler. However, this coupled instrumentation alone did not provide any information on the identity of the NPs occurring in the simulated food. This limitation was overcome by coupling AF4-MALS to the element-selective ICP-MS detector, which provided additional information on trace elements known to be naturally present in the clay. The analytical system was applied to characterize migrates http://www.elsevier.com/locate/trac

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Figure 4. Environmental scanning electron microscopy (ESEM) image (A) of debris found in a sample of bread from South Italy made with hard wheat with its energy-dispersive system (EDS) spectrum and the semi-quantitative concentrations of the elements (B). It contains micro-scale and nano-scale cadmium-tungsten-silver-cobalt contamination. (Reproduced from [80] with permission, ª 2009 Taylor and Francis Group).

Figure 5. Selected chromatograms after liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS) with methanol/toluene (80:20) unless otherwise noted, including C60 (2 lg/L in zebrafish homogenate matrix), 13C60 (10 lg/L in zebrafish homogenate matrix), C70 (10 lg/L), C82 (3.4 lg/L), C88 (2.5 lg/L), and C98 (0.4 lg/L). Additional fullerenes in a higher-order mixture not shown. (Reproduced from [84] with permission, ª 2007 American Chemical Society).

from nanocomposite films of polylactide and the organomodified Cloisite 30B montmorillonite clay used as filler. The results demonstrated that NPs of 50– 800 nm radius indeed migrated from the nanocomposite, but ICP-MS signals corresponding to clay minerals were absent. 96

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Isaacson et al. [84] developed and validated an analytical method to quantify a suite of fullerenes and then apply the analytical method to determine the behavior of a single fullerene, C60, during a toxicological assay using zebrafish embryos and aqueous-exposure solutions. The average recovery of C60 from fish extracts was 90% and

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precision, as indicated by the relative standard deviation, was 2%. The method quantification limit was 0.40 lg/kg. LC-ESI-MS detection was used to identify and to quantify C60–C98 fullerenes. The most abundant ions formed under ESI-MS conditions were molecular ions. The mobile phase (55:45 toluene/methanol) provided chromatographic separation of the fullerene analytes using a conventional C18 analytical column. Chromatographic analysis indicated co-elution of C60 with the 13C60 internal standard with the retention times of the larger fullerenes increasing in order of increasing carbon number (Fig. 5). Two-photon excitation microscopy (TPEM) combined with autofluorescence was used to detect NMs (MWCNTs, TiO2, and CeO2) interacting with vegetation and used simultaneously to detect and to monitor the interactions of MWCNTs and polycyclic aromatic hydrocarbons in vivo in roots [85]. The potential of TPEM coupled with autofluorescence in visualizing MWCNTs and their interactions with in vivo cellular systems is both extensive and diverse, and highlights the techniqueÕs potential for use with other NMs. It may also provide a method for looking at the purity of NM manufacture, where artifacts (e.g., catalysts from the manufacturing process) can be identified rapidly from their autofluorescence signals. TPEM combined with autofluorescence provides a non-intrusive tool for the in vivo visualization of NM fate, interactions and behavior. Future applications may include studies on NM environmental fate, bioavailability, ecotoxicology, chemical carriage, and targeted drug delivery in systems from plants and bacteria to skin or synthetics. Experience with the combination of TPEM and autofluorescence is very scarce, but, again, the results are promising.

5. Conclusions and future trends Analysis of NMs in food, as has been widely remarked upon in the literature, is still in its earliest infancy, even though there are methods that have proved their effectiveness in detecting and characterizing NMs. However, the methods to quantify them are still rare. At present, the situation is divided. On the one hand, there are methods to analyze the structural form of NMs in food, feed and biological tissues, but, because of the background occurrence of NMs, it is not usually possible to establish the presence of ENMs. On the other hand, there are methods to analyze the chemicals in specific ENMs, but most often not to establish their presence in nanoform. At present, only in exceptional cases is it possible both to detect specifically and to measure particular ENMs, and, in these cases, that is feasible thanks to the combination of a number of analytical techniques. However, if the results compiled in this review are compared with the data presented in other previous treatments of this topic, we can highlight the rapid

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progress that represents a major step forward and lays the foundation to develop quantitative methods for the analysis of NM residues in foods. There are a growing number of these quantitative analytical methods, and we expect impressive applications within food safety, food quality and food analysis in the very near future, as an extension of those already existing for environmental samples. An important driver for their development is the concern for the food safety of international agencies and organizations (FAO/WHO, EU, EFSA, and US EPA) arising from the possible toxic effects of NMs. The assessment of the risk to consumers needs to be addressed as regulations for food nanotechnology move forward. Summarizing, due to the enormous variety of NMs, there are many different ways to analyze particles and there is no best technique for all situations, so a combination of techniques is usually necessary. Acknowledgments The authors thank the Spanish Ministry of Education and Science and the European Regional Development Funds (ERDF) (Project CGL2007-66687-C02-01/BOS) for financial support. References [1] G.C. Delgado, Technol. Soc. 32 (2010) 137. [2] N. Sozer, J.L. Kokini, Trends Biotechnol. 27 (2009) 82. [3] M. Siegrist, N. Stampfli, H. Kastenholz, C. Keller, Appetite 51 (2008) 283. [4] P. Sanguansri, M.A. Augustin, Trends Food Sci. Technol. 17 (2006) 547. [5] C. Moraru, Q. Huang, P. Takhistov, H. Dogan, J. Kokini, in: B. Gustavo, M. Alan, L. David, S. Walter, B. Ken, C. Paul (Editors), Global Issues in Food Science and Technology, Academic Press, San Diego, CA, USA, 2009, p. 369. [6] M.G. Valdes, A.C.V. Gonzalez, J.A.G. Calzon, M.E. Diaz-Garcia, Microchim. Acta 166 (2009) 1. [7] D.A. Pereira de Abreu, P.P. Losada, I. Angulo, J.M. Cruz, Eur. Polym. J. 43 (2007) 2229. [8] Innovative Research and Products (iRAP), Nano-enabled packaging for the food and beverage industry - A global technology industry and market analysis, FT-102 (2009) 1–107 (http:// www.innoresearch.net/report_summary.aspx?id=68&pg=107& rcd=FT-102&pd=7/1/2009). [9] A.D. Maynard, R.J. Aitken, T. Butz, V. Colvin, K. Donaldson, G. Oberdorster, M.A. Philbert, J. Ryan, A. Seaton, V. Stone, S.S. Tinkle, L. Tran, N.J. Walker, D.B. Warheit, Nature (London) 444 (2006) 267. [10] K. Savolainen, H. Alenius, H. Norppa, L. Pylkkenen, T. Tuomi, G. Kasper, Toxicology 269 (2010) 92. [11] H. Bouwmeester, S. Dekkers, M.Y. Noordam, W.I. Hagens, A.S. Bulder, C. de Heer, S.E.C.G. ten Voorde, S.W.P. Wijnhoven, H.J.P. Marvin, A.J.A.M. Sips, Regul. Toxicol. Pharm. 53 (2009) 52. [12] X. Zhu, Y. Chang, Y. Chen, Chemosphere 78 (2010) 209. [13] H. Summers, Nano Today 5 (2010) 83. [14] G.M. do Nascimento, R.C. de Oliveira, N.A. Pradie, P.R.G. Lins, P.R. Worfel, G.R. Martinez, P. Di Mascio, M.S. Dresselhaus, P. Corio, J. Photochem. Photobiol. A 211 (2010) 99.

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