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Trends in Analytical Chemistry, Vol. 30, No. 1, 2011

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

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


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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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toxicokinetic. bone marrow and nervous system. heart. and some NMs penetrate the skin [20]. As a consequence. but there are also other contributing aspects (e. 2011 Trends Figure 1. The prerequisite for toxicological.Trends in Analytical Chemistry. reliable quantitative methods of analysis are required to measure levels of NMs in a broad range of matrices. NMs can also enter the blood stream via inhalation or ingestion. kidneys. there are natural NMs. including analytical methods and ecotoxicity assessments [23–28]. and health effects of NMs in food and the environment. morphology or shape. taking into account all material properties. may disrupt normal activity and may lead to malfunctioning and diseases [19]. To obtain this information. NMs can be transported around the body and taken up by organs and tissues. Many food substances or ingredients have nanostructures in nature and are present at lm or nm in size: http://www. Several reviews already present the latest research carried out to assess the risks of engineering NMs (ENMs) in the aquatic environment. transport. spleen.elsevier. Studies demonstrate the potential of NMs to cause DNA mutation and induce major structural damage to mitochondria. Due to consumer safety. In food. NMs are able to cross biological membranes and access cells. Size is a key factor in determining the potential toxicity of a particle [22]. aggregation and solubility. No. Nano applications in food and the food industry. chemical composition. focusing on emerging contaminants in food and the environment. including brain. 1. Vol. 30. as well as numerous reviews and perspectives [12–18]. migration and exposure assessment is the development of analytical tools for detection and characterization of NPs in complex matrices [22]. and the presence or absence of other chemical functional groups) [10]. it is necessary to control the content of NMs in food [36]. surface charge. Then. liver. or engineered using materials that are not endogenous to foods – and NMs resulting from contamination. It is difficult to generalize about health risks associated with exposure to NMs – each new NM must be individually assessed. international agencies and governments are paying attention to the study of the fate. tissues and organs that larger particles normally cannot [19]. even resulting in cell death [21]. include NMs as one of the hottest topics in research today [29–35]. toxicological studies have been reported.g.. the latest reviews. surface structure. intentionally added ENMs – derived from naturally occurring food components. once in the blood stream. 85 . Recent examples in the literature show that engineered inorganic nanoparticles (NPs) and carbon nanostructures may incidentally or intentionally enter into contact with living organisms.

com/edition_15/ feature79/ http://www51. 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 Web address or reference http://www.htlm Evonik (Degussa) Evonik (Degussa) AquaNova Nano ZnO (food grade) Silica (food grade) Product micelle (capsule) of lipophilic. .elsevier.advancednano materials. news71748835. 2011 Table 1. com/sm/aegis/ Food packaging materials Durethan KU 2-2601 Bayer Hite brewery beers: three-layer.html http://www.aquanova.html?partner=rss http://www. food additives and food packaging materials Product name Manufacturer NMs Silica in a polymerbased nanocomposite Claim Nanoparticles of silica in the plastic prevent the penetration of oxygen and gas of the wrapping.htm 86 http://www. Vol. will not pollute local groundwater systems or waterways In use since 2002 A clear laminate with outstanding gas-barrier proprieties developed primarily for the Adhesive form MacDonaldÕs burger containers Food additives AdNano Aerosil. Nanomaterials (NMs) in investmentnewsletters/2005/ 08/09/nanotechnology-krafthersheycz_jw_0810soapbox_ inl.physorg. Titanium_Technologies/ en_US/products/dls_210/ dls_210_landing.aerosil.html http://www.asp http://www.Trends Trends in Analytical www. water-insoluble substances 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 http://www. news717488335.6 L beer bottle Honeywell HoneywellÕs Aegis OX nylon-based nanocomposite 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. dry food and snack markets UV-protected plastic food packaging http://www. 1. Sipernat AquaNova NovaSol Ecosynthetix 50–150 nm starch nanospheres The adhesive requires less water as well as less time and energy to dry http://www2.

noodles.toddlehealth. 2011 Table 1 (continued) Product name Bioral Omega-3 nanocochleates Manufacturer BioDelivery Sciences International NMs Nano. minerals and protein Toddler health is an all-natural balanced nutritional drink for children from 13 months to 5 years.html http://www. 0100033.369. One serving of Toddler Health helps little ones meet their daily requirements for Jamba Juice Hawaii 300 nm iron (SunActive Fe) 300 nm iron (SunActive Fe) 300 nm iron (SunActive Fe) Oat Chocolate Nutritional Drink Mix Toddler Health Oat Vanilla Nutritional Drink Mix Toddler Health 300 nm iron (SunActive Fe) 22 essential vitamins and minerals and 100%. biodellveryscience.highwive.3 – Threestep preparation method and its application for nanotea Patent No.pharmanex. One serving of Toddler Health helps little ones meet their daily requirements for vitamins.nutralease. chips and candy bars Nano technology to deliver highly bioavailable coenzyme intercom/ productDetall. cereals. pasta.php Nano self-assembled.htlm NanoCoQ10 Pharmanex Nano coQ10 http://www.7 – Multi-layer.toddlehealth.htm http:// Food and beverages Nano Tea Shenzen Become Industry & Trading Co Nanoparticles (160 nm) Nano Slim Nanoceuticals Slim Shake Chocolate Nanoceuticals Slim Shake Vanilla Nano Slim RBC Lifescience NAno-Diffuse Technology Nanodusters Patent No. No.cochleates as small as 50 nm Claim Effective means for the addition of omega-3 fatty acids for use in cake. http://www.php Canola Active oil Shemen Nano-sized selfassembled structured liquid micelles http://www.comcn/Er/ nanotea.con/ nanoslim-information. technology. 1.9/ 00244295. swinging nano-ball milling procedures Orosolic acid (derived from the Lagerstroemia spaciosa plant) http://www.htm RBC Lifescience Nanodusters Fortified fruit juice Daily Vitamin Boost High 87 . minerals and protein http://www. structured liquids Nutralease Nanomicelles for encapsulation of nutraceuticals Vitamin E nano-solution using NovaSOl LycoVit 10% (< 200 nm synthetic lycopene Solu E 200 BASF Synthetic Lycopene BASF BASF sunactiveiron. 30.Trends in Analytical Meal_ Replacement_Shakes. 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.shemen. Vol.aspx http:// www.rbdifesciences.basf http://www.rbdifesciences.mkt1d-2031 http://www.humannutrition.asp bioralnutrients.nanoslim. com/Meal_Replacement_ OatChocolate. 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 Trends Web address or reference http://www.

and. NPs could interact with proteins. particle stability is affected by the concentration of cations (coagulants). minerals and water in food. . van der Waals forces and steric forces. among other things. 1. Through its horizon-scanning activities. 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]. although it noted that there was uncertainty in some areas as to whether applications of nanotechnologies would be picked 88 http://www. feed and biological tissues. This report presents an overview of the wide range of current and projected nanotechnology applications in food and agriculture (Fig. information and resources that they need to oversee the exponential market growth of nanotechnology adequately [1. This review takes a step forward by including the first applications of NMs to food. the determination of NMs in food is a challenging task requiring tailored solutions. at increasing salt concentrations. 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].elsevier. no specific nano regulations exist anywhere in the world.g. They do not precipitate by gravitation due to their small size. (3) starch polysaccharides have small 3-D crystalline nanostructures that are only 10s of nm in thickness. 2. proteins).. like many engineering NPs (ENPs). nucleic acids. lipids. As regulation for food nanotechnology moves forward. Any changes (e.. lacking the legal and scientific tools. ions. No. 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]. 30. which are globular particles of 10s to 100s of nm in size. This working party was established to address human health and environmental safety aspects of manufactured NMs in the chemicals sector. At present.g. It is important to characterize the effects and the interactions of ENMs in the relevant food matrix. they should be distinguished from ENMs or contaminating NMs [27]. (2) second. a certain surface charge. the aim of this review is to address and to compare the available analytical methods for determination of NMs in food.. electrostatic interactions. size distribution. Despite the rapid commercialization of nanotechnology. Releasing ENPs into such complex systems is bound to lead to a range of interactions. With the FAO. free NPs will start to aggregate [20]. Food also contains natural colloids and dissolved ions. 2011 (1) food proteins. Most regulatory agencies remain in information-gathering mode. 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. size. (2) linear polysaccharides with one-dimensional nanostructures are less than 1 nm in thickness. Regulations The safety of nanoproducts has attracted attention in line with their increasing use. carbohydrates.g.Trends Trends in Analytical Chemistry. 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].g. and. Furthermore. meaning that. the European Food Safety Authority (EFSA) published its opinion on the potential risks arising from nanoscience and nanotechnologies in food and feed [47]. in February 2009. It considered. are true NPs. These components also contain hydrophobic domains that are likely to interact with hydrophobic ENPs [e. Given the huge diversity of ENPs for use in the food and feed sector (e. The report did not identify any major gaps in regulations. 2). surface activity/modification) (see Table 1). 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. Proteins and carbohydrates have large specific surface areas and a high electrochemical surface charge that is likely to make them interact with charged particles.40–44]. Dispersed colloids are particles in the ENP range (1– 200 nm) that are kept in a stable aqueous suspension [11]. fullerenes and carbon nanotubes (CNTs)]. chemical composition. and their potential interaction with food-matrix components (e. 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. environmental matrices and food materials [39]. Vol. due to their specific physico-chemical properties. According to the classical double-layer and colloid-stability theories. (2) the other overviewed the different analytical techniques available for detecting the ENPs in product formulation. pH or ion concentrations) may destabilize the suspension. Applications that may lead to human exposure to NPs through the environment to the food chain were not included. 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.. the suitability of current regulations relating to the use of nanotechnologies in the food sector.

silica. silver. 2011 P Procesed d nanostructured in food (NANOTEXTURES) Nano-emulsions Surfactant micelles y Emulsion bilayers.p polyethylene y y g glycol y ( (PEG) ) Nanotechnology in the agricultural sector Animal feed Agrochemicals Natural biopolymer from yeast cell walls that bind t i mycotoxins Polystyrene y y ( (PS) ) base. minerals and phytochemicals Inorganic NMs (TiO 2.elsevier. Vol. Nanomaterials relevant to food.Trends in Analytical Chemistry. . calcium. Double or multiple emulsions Reverse micelles Spreads S d ayo a se Mayonnaise Cream Yoghurts ice creams Nanodeliver N d li systems t p based on encapsulated technology Nanomicelle-based carrier system Nanocluster delivery system Vegetable oil enriched in vitamins vitamins. ycope e. selenium. fullerenes. . No. 1. coli Slow or controlled Slowcontrolled-release release fertilizers and pesticides Figure 2. coating contained NPs and antimicrobial nanoemulsions) Intelligent I lli packaging k i concepts based b d on nanosensors 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 http://www. Trends 89 . iron) Nanomaterials relevant to food applications Surface functionalized NMs o o u e e es. and mannose targeting biomolecule to bind E. Organic NMs (synthetic nanosized a os ed form of lycopene. carbon nanotubes) Nano-enabled food contact materials (FCMs) and packaging NP reinforced materials (polymer composites with nano-clays. nano-clays nanometals or metal oxides. 30.

the European Commission published a proposal to revise and update the Novel Foods Regulation. are subject to additional measures. including vitamins and minerals. 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]. including a new safety regulate NMs under several laws. came into force. enzymes and flavorings. – networking existing and establishing new infrastructures to examine health. European Union legislation applicable to NMs in food Legislation Comments Ref. From early 2010. a list of approved additives. Novel foods regulation Regulation (EC) No 258/97 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. In USA. a new Regulation was passed (Regulation EC/1333/2008). The FDA regulates a wide range of products. for example. devices. A definition of NMs has been introduced at the request of the European Parliament. safety and environmental aspects of NMs. States that only vitamins and minerals on an approved list may be used as food supplements. Various proposals have been discussed by the Commission. is significantly different (for example. It concluded that ‘‘on the basis of current information. or the process by which an additive is produced. some of which contain NMs or are produced nanotechnologically. The new regulations also specify that. – review and development of test methods for human health. Vol. Federal Insecticide. safety and the environment. including plastics. Resource Conservation and Recovery Act (RCRA). which set out a common authorization procedure for additives. Those included often had limits set on their use. New substances may be considered for inclusion on the list. Discussions are continuing on how to bring nanotechnologies specifically into the revised Regulation. [52. not technologies. restrictions on the quantities permitted for use. – risk management for workersÕ protection. The European Commission (EC) aims to reinforce nanotechnology and. Inclusion of additives on the list was decided by the Commission on the basis of an Opinion from the EFSA.Trends Trends in Analytical Chemistry. Authorisation and restriction of CHemical substances. Certain materials. 1. drugs. but there are no specific rules for NPs because it regulates products. In December 2008. In January 2008. No. Fungicide and Rodenticide Act (FIFRA). it must go through a fresh authorization process. Nevertheless.elsevier. Comprehensive Environmental Response. The Commission has proposed updating the Regulation governing food-contact plastics to specify that a deliberately-altered particle size should not be used. The EPA could use most of the environmental laws – Clean Water Act (CWA). Clean Air Act (CAA). reference materials and materials characterization. either directly or indirectly. where the starting material used. [54–56] Food-contact materials Regulation EC/1935/2004 All materials that are intended to come into contact with foodstuffs. 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. at the same time. different countries are trying to include NMs in their current regulations. the FDA expects that many nanotechnology products will come under its jurisdiction. including foods. – development of exposure information throughout the life-cycle of NMs. [57] Food supplements Directive 2002/46/EC [58] up consistently. enhance support . through a change in particle size). – review of existing risk-assessment methods. but only after a safety assessment by EFSA. cosmetics. [51] General for chemical compounds REACH European Community legislation concerned with chemicals and their safe use and dealing with the Registration. without specific authorization. most potential uses of nanotechnologies that could affect the food area would come under some form of approval process before being permitted for use’’. 30. Parliament and Council (The draft Regulation is currently going through the co-decision procedure). and the Regulation subjects all novel foods and foods manufactured using novel processes to a mandatory pre-market approval system. 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. and supported by the Council. However. Compensation and Liability Act (CERCLA).53] Food additives Directive 89/107/EC and associated legislation Only additives explicitly authorized may be used in food. The US Food and Drug Administration (FDA) requires manufacturers to demonstrate that food and their ingredients are not dangerous to health. and veterinary products. Evaluation. and Toxic Substances Control Act (TSCA). These international agencies identified a number of domains of interest as a starting point for this review. 2011 Table 2. even behind a migration barrier. the Environmental Protection Agency (EPA) is already empowered to 90 http://www.

Vol.23.elsevier. matrix-assisted laser desorption/ ionization (MALDI)).39.27.60. mercury porositometry. laser desorption/ionization (LDI).39. scanning electron microscopy (SEM). UV-Vis.27. has been extended to possible http://www.59] [22. inductively coupled plasma (ICP) Mass analyzers – time-of-flight (TOF). chemical analysis of the target compounds Ref. the problem of NM safety. scanning probe microscopy (SPM). worker protection. covering EU regulatory frameworks that are applicable to NMs (e. The purpose of this inventory is to examine and.39.g. and. The EC is performing a regulatory inventory. atomic force microscopy (AFM) Centrifugation and filtration Chromatography and related (separation): size exclusion chromatography (SEC). laser diffractrometry Analytical (spectroscopy) coupled to electron microscopy (imaging): TEMdispersive X-ray spectroscopy (EDS).38.26.59–62] [22. triple quadrupole (QqQ).23. food additives and food-contact materials (see Table 2). X-ray fluorescence (XRF). X-ray absorption spectroscopy (XAS).38. dynamic light scattering (DLS). Zeta potentiometer.68. AFM-chemical force microscopy (CFM) Spectroscopy and related (characterization): Raman spectroscopy. Different parameters and characterization methods for NMs Parameters Particle size & size distribution.39. NMR Mass spectrometry (characterization): Sources – electrospray ionization (ESI). 30. field flow fractionation (FFF) Spectroscopy and related (characterization): static light scattering (SLS).60] [22.70.69] [39. single quadrupole. 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. LIF. and. scanning transmission X-ray microscopy (STXM). TEM-electron energy loss spectroscopy (EELS). ion trap (IT).64] [27. and. CE Water contact angle measurements. quadruple timeof-flight (QqTOF). X-ray microscopy (XRM). X-ray diffraction (XRD)] Mass spectrometry (characterization): Sources – electrospray ionization (ESI).38.39. matrix-assisted laser desorption/ ionization (MALDI).38. and product-specific). to propose adaptations of EU regulations in relevant sectors [49].Trends in Analytical Chemistry. 2011 Trends Table 3. photon correlation spectroscopy.38.60. This includes to apply existing food laws to food products using nanotechnology. small-angle X-ray scattering (SAXS).26. and.23. laser-induced breakdown detection (LIBD). SLS-FFF.65–67] Carrier-drug interaction Nanoparticle-dispersion stability Release profile Target-compound stability [39. infrared spectroscopy. All food products have to meet a general safety requirement under the General Principles of Food Law Regulation (EC/178/2002) [50]. once mainly limited to its chemical aspects. 1. morphology or shape and aggregation state Characterization methods Microscopy and microscopy-related (imaging): near field-scanning optical microscopy (NSOM). where appropriate.64] Chemical characterization [38.59–62] [23. inductively coupled plasma (ICP) Mass analyzers – time-of-flight (TOF). single quadrupole.62. and.39. scanning transmission electron microscopy (STEM).27. environmental. nuclear magnetic resonance (NMR). Raman spectroscopy.39] [26. [22.65–67] Charge determination Surface hydrophobicity [26.27. TEM-selected area electron diffraction (SAED).com/locate/trac 91 .71] [39] [39] for collaborative research and development (R&D) on the potential impact of nanotechnology on human health and the environment via toxicological and ecotoxicological studies. quadruple time-of-flight (QqTOF) Laser droplet anemometry.39. quadrupole linear ion-trap (QqLIT). More specific legislation covers the use of novel foods. ion trap (IT). chemicals.39] [39. and.27. SEM-EDS. neutron scattering (NS).23.63] [23.38. hydrophobic interaction chromatography. confocal laser scanning microscopy (CLSM). laser-induced fluorescence (LIF). quadrupole linear ion-trap (QqLIT).38. capillary electrophoresis (CE). transmission electron microscopy (TEM). triple quadrupole (QqQ).26.. Analytical approaches to characterize and determine NMs Recently. No. hydrodynamic chromatography (HDC). rose bangle (dye) binding. SLS-DLS.39] Chemical analysis of surface [22. laser desorption/ionization (LDI). and. 3.39. X-ray photoelectron spectroscopy Spectroscopy and related (characterization): static X-ray spectroscopy [Xray photoelectron (XPX).

The second group of techniques possesses a lower analytical ambition but is best suited to routine analysis. 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.g. 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. the tendency is to reduce sample preparation as much as possible because it is important to measure the NMs in the relevant matrix. 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.. there are a number of analytical tools for qualitative and quantitative categorization of NMs: (1) single-particle techniques. the investigation of NM distribution in food is generally extremely time consuming. This technique is cheap. With nanoscale metals or semiconductors containing NMs. (2) techniques characterizing the ensemble of NMs.elsevier.Trends Trends in Analytical Chemistry. The size distributions of TiO2 NPs obtained from MALDI-TOF-MS and LDI-TOF-MS were in good agreement with parallel TEM observations. these can be detected even in rather complex matrices (e. so there is no need for a repeat in this article. the long time required for both sample preparation and image analysis greatly limits the analytical 92 http://www.g. without generating information on their physical state. However. including organic compounds (e.g. but a focus on characteristic structures may be needed to determine whether it is still in nanoform. Food is a very intricate material. These approaches are based on the chemical characterization of NMs. Visualization is most easily accomplished with electron-dense NMs (e. feed and biological tissues) by means of electron microscopy (EM) coupled with chemical analytical tools.g. detection by EM is only possible if the number of NMs is sufficiently high in the matrix to localize them. and CNTs). taking into account that the chemical composition of NMs is one of their properties. Hence. 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]. Raman spectroscopy is a very important technique and could be very useful for finding CNTs and fullerenes.. The limitations of chemical analysis result from artificial losses during the preparatory steps. [38] discussed for nanodelivery systems. There are a few recent examples of NP-enabled MS that. characterization of the composition of the internalized NPs. (3) techniques to determine their chemical composition. as their properties may depend on the surrounding matrix and be affected by processing as well as by the extraction procedure. only very limited work has been done on the detection of organic NPs in food. or by radioanalysis after appropriate neutron irradiation.. However. inductively-coupled plasma atomic emission spectroscopy (ICP-AES) or mass spectrometry (ICP-MS)]. However. UV-photo patterned self-assembled monolayers [74].com/locate/trac throughput when using these techniques for analysis of NPs in food. since high magnification is required due the small size of NMs. so the analysis of NMs commonly requires two types of method: (1) those for characterizing and/or detecting NPs or NMs. It has also the advantage of being used in in situ monitoring. fullerenes and CNTs). and. food. for sufficient characterization. non-destructive and not time consuming. 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 . For a wide discussion on the advantages or the drawbacks of each technique. and. even though they have not yet been used.g. However.g. environmental analysis). analytical detection limits and the inability to characterize carbon NPs (e. However. In the case of organic NMs. [39] and Luykx et al. (2) those for determining their chemical composition. 1. where a test for the species exists. Several reviews highlight the power of Raman spectroscopy for measuring CNTs [72. However. we refer the reader to excellent reviews by Tiede et al. and. Generally. metal NPs). the metal content of NMs can be quantified by analytical-chemistry tools [e. though not explicitly linked to NP determination in food. As outlined in Table 3.73]. who accurately explain their advantages and limitations. No.. the technique is also suitable for any other NMs. demonstrated the potential of this approach. [38]. For example. This is usually much more demanding than to analyze NMs in simpler or model matrices.. TOF secondary-ion MS (SIMS). As a result. There are also some studies reporting quantification of NMs in a related field (e. detection or quantification of the chemical may be possible. sample preparation includes acidic sample digestion before analysis. For CNTs. as Luykx et al. Vol. 2011 toxicity associated with NPs as physical entities [22]. So far. fluorescence microscopy and scanning EM (SEM) were employed to monitor the immobilization of biotinylated shell-crosslinked NPs on biotin/streptavidin-functionalized. 30. where much more information is available. polymeric NPs. fullerenes. in conjunction with spectroscopic methods.. the NMs need to be separated from the food matrix. and. the line that divides these types is very thin. probably.

the latter methods are based mainly on wet digestion with a strong acid (e. The OECD list comprises:  fullerenes (e. 2011 Trends chromatography with atmospheric pressure photo-ionization MS (LC-APPI-MS). http://www.elsevier. There are some additional complications not yet solved in the analysis of NMs. Fe. As we go deeper into the analysis of NPs. has recently released a quality-control material (IRMM-304) of silica NPs.g. reproducible detection and quantification of engineered NMs in food and feed. and. the likely availability of materials for testing and the existing information [46]. there remain several obstacles to obtaining adequate characterization and quantification of NMs in food. including molecular weight and the number and the identity of functional groups.  NPs of Ag.79].  nanoclays. Sensitive and mass-selective detection... Meanwhile. [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. other metal NMs {e.  polystyrene. 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. 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). Vol. ZnO. The current limited number of standardized reference materials for engineered NMs is another brake on precise. respectively). 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].. interlaboratory studies. Various approaches suggested for use in studies of NP bioaccumulation are included because they can be applied to its determination in food. 13C60. 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. 8012 and 8013) and polystyrene spheres (NIST SRM 1963a and 1964) from the National Institute of Standards and Technology (NIST) [77]. since we may assume that all the gold present in the samples is from NPs. For inorganic NPs. protocols. 3). relevant reference materials and internationally standardized practices. 4. 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. zinc oxide (ZnO). stable-isotope labeled fullerene internal standard. Despite some successes in this field. Internal standard quantification using stable isotopelabeled internal standard is de rigueur for trace-level analysis of contaminants in food. There are also available gold NPs (NIST RM 8011. nanoscale engineered silicon dioxide (SiO2) or endogenous lipids used in capsule membranes}. and data analysis. C60). Also. TiO2. since many of the target organisms are also edible. and SiO2 [46]. Furthermore. For example. Currently. several methods to measure gold NPs have been developed.g.  dendrimers.Trends in Analytical Chemistry. CeO2. NP measurement. are quite developed. Al2O3. 1.  single-walled and multi-walled CNTs (SWCNTs and MWCNTs. are almost non-existent. there is commercially available only a single.g. the major problem is the wide range of analytical techniques applicable to these compounds and the different purposes of the methods developed [78. such as the fact that some ENMs cannot be distinguished from naturally-occurring variants of the same {e. 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.. 30. Contrarily. No. The Joint Research Centre. Detection may also be hindered by interactions with solutes or food components that obscure clear analytical signals. nitric or perchloric) followed by ICP optical emission spectroscopy (ICP-OES) or ICP-MS. Moreover. These methods are based only on chemical analysis. Farre et al.  carbon black. especially aquatic organisms. shows great potential to achieve this goal. fish samples were digested with acid and analyzed by ICP-MS. Of the difficulties encountered in conducting interlaboratory studies on these emerging contaminants. Institute of Reference Materials and Measurements. which are necessary for validation of protocols and generating precision and bias statements for measurement standards. its evolution seems to be hampered by the limited information available. as offered for MS combined with optimal extraction procedures. and. Foremost among them are those presented by the lack of analytical standards. as well as considerations of production volume.g. we need analytical procedures to account for explicit information. This will be a growing area within NM analysis. unmodified commercial nanoscale metal oxides. since gold is not abundant in the environment. Table 4 lists the results of the literature search. (2) those looking at their chemical composition only. and procedures for testing the preparation of food. There is much work yet to be done to show how far determination of NMs in food can 93 . We need to mention that methods to determine inorganic NPs in food.

FA4. 2011 Table 4. [86] Stability of NPs during heat treatment Chitosan NPs for l-ascorbic acid Aqueous solutions Zeta potential PDI Ultracentrifugation [87] CARS. Two-photon excitation microscopy. TEM Chemical characterization of elements: ICP-MS Separation and size determination of clays: centrifugation. MALS. 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. Transmission electron microscopy. PDI. TPEM. Environmental scanning electron microscopy/X-ray microprobe of an energy dispersive system. 94 http://www. zeta potential.83] Fullerenes C70ÀC98 Embryo zebra fish LC-MS2 [84] Accumulation of NMs in plants Multi-walled carbon nanotubes TiO2. 1. CeO2 Wheat tissues TPEM coupled chemical autofluorescence [85] 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 XRD TEM Centrifugation FA-MALS ICP-MS FA4-MALS-ICP-MS Characterization of polylactide/ Cloisite 30 B: XRD. X-ray diffraction. 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 Bioavailability of nanoscale metal oxides to fish Nanomaterial Inorganic NMs Matrix Bread and biscuits Techniques ESEM/EDS Comment Detection of organic and inorganic microscale and nanoscale contaminants: ESEM Identification of their chemical composition: EDS Characterization of size. encapsulation efficiency (EE). [80] TiO2. ICP-MS. Coherent anti-Stokes Raman scattering microscopy. Selected applications for detection. Polydispersity index. No. XRD. CeO2 and ZnO Rainbow trout TEM ESEM/EDS ICP-MS ICP-OES CARS [81] Bioaccumulation of gold NPs in fish In vivo toxicity studies Au NPs Mytilus edulis ICP-OES [82. Liquid chromatography-tandem mass spectroscopy. 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. TEM. Multi-angle light-scattering detection. LC-MS2. 30. particle shape/morphology and qualitative aggregation: TEM and ESEM/EDS Analysis of elements content : wet digestion and ICP-MS. ICP-OES. Vol.elsevier. Inductively coupled plasma optical emission . FA4-MALS and clay aspect ratio Migration study FA-MALS-ICPMS Characteristics of AA-loaded CS nanoparticles: particle size.Trends Trends in Analytical Chemistry. ESEM/EDS. Asymmetrical flow field-flow fractionation. Inductively coupled plasma mass spectrometry.

An aggregate of NPs can be seen occupying the space between the pillar cells. (Reproduced from [81] with permission. iron. it is important to characterize migrates from nanocomposites containing clay as fillers. sulfur. EDX was employed to identify their chemical composition. Biopolymer nanocomposites are a field of emerging interest. Fig. [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. 1. Oncorhynchus mykiss. Vol. (A) Gill tissue. following a 28-day exposure. which contained cadmium. this coupled instrumentation alone did not provide any information on the identity of the NPs occurring in the simulated food. From a food-safety point of view. calcium. Gatti et al. The results indicated that 40% of the samples analyzed contained foreign bodies (e. ceramic and metallic debris. composed of pillar cells (PCs) and pavement cells (PVs).com/locate/trac 95 .g. 4 shows other debris found in a biscuit. A cluster of NPs can be seen in the region of the marginal channel (MC).Trends in Analytical Chemistry. since such materials can exhibit improved mechanical and barrier properties. The two adjacent sub-panels specifically locate the NPs inside the tissue near the surface of the MC. silver. The analytical system was applied to characterize migrates http://www. probably of environmental or industrial origin). The red blood cells are effectively separated from the lamellae cells by forward detection of the CH2 vibration (shown in blue). aluminum. cobalt and copper in particles. 2011 Trends Figure 3. [80] investigated the presence of inorganic micro-sized and nano-sized contaminants in bread and biscuits from 14 different countries by ESEM. 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. was obtained by epidetection of the CH2 vibration (shown in green). following water-borne exposure to titanium dioxide (TiO2) nanoparticles (NPs).elsevier. Schmidt et al. The cellular structure of the primary (PL) and secondary (SL) gill lamellae. No. However. (D) Multi-planar view of the same exposure. (C) Projection of a 300 · 100 lm 3D data set of gill tissue following a 14-day exposure.. 30. (B) The same NP aggregate under a 3· increase in magnification. Coherent anti-Stokes Raman scattering (CARS) microscopy images of the gill tissue of rainbow trout. and they can be more suitable for a wider range of food-packaging applications. ª 2010 American Chemical Society). tungsten.

96 Isaacson et al. The average recovery of C60 from fish extracts was 90% and http://www. Vol. including C60 (2 lg/L in zebrafish homogenate matrix). C88 (2. Additional fullerenes in a higher-order mixture not shown. Selected chromatograms after liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS) with methanol/toluene (80:20) unless otherwise noted. [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. and C98 (0. 30. but ICP-MS signals corresponding to clay minerals were absent. 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). No.5 lg/L).4 lg/L).elsevier. 2011 Figure . ª 2007 American Chemical Society). C70 (10 lg/L). (Reproduced from [80] with permission.Trends Trends in Analytical Chemistry. (Reproduced from [84] with permission. ª 2009 Taylor and Francis Group). C60. Figure 5. C82 (3. 1. during a toxicological assay using zebrafish embryos and aqueous-exposure solutions.4 lg/L). 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. It contains micro-scale and nano-scale cadmium-tungsten-silver-cobalt contamination. 13C60 (10 lg/L in zebrafish homogenate matrix). 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. B. as indicated by the relative standard deviation. M. Diaz-Garcia. Vol. as an extension of those already existing for environmental samples.L.S. p. we can highlight the rapid http://www. D.aspx?id=68&pg=107& rcd=FT-102&pd=7/1/2009).S. Sozer. but. [14] G. [10] K. Calzon. bioavailability. Pereira de Abreu. [7] D. C. [9] A. A.Trends in Analytical Chemistry. Kokini. Corio. Photochem. Tran. N.W.g. L. Lins. feed and biological tissues. in these cases. Kokini. Butz. J. but most often not to establish their presence in nanoform. EU. because of the background occurrence of NMs. The most abundant ions formed under ESI-MS conditions were molecular ions. ten Voorde. V. Pradie. The potential of TPEM coupled with autofluorescence in visualizing MWCNTs and their interactions with in vivo cellular systems is both extensive and diverse. Nature (London) 444 (2006) 267.C.G. Tinkle. and targeted drug delivery in systems from plants and bacteria to skin or synthetics. C. Toxicology 269 (2010) 92. TiO2. References [1] G. Academic Press. No. EFSA.R. progress that represents a major step forward and lays the foundation to develop quantitative methods for the analysis of NM residues in foods. Summarizing. K. The assessment of the risk to consumers needs to be addressed as regulations for food nanotechnology move forward.G. Two-photon excitation microscopy (TPEM) combined with autofluorescence was used to detect NMs (MWCNTs. P. Donaldson. J.C. L. G. Nano-enabled packaging for the food and beverage industry . P. Keller. Nano Today 5 (2010) 97 . 30. CA. C. M.A. 2011 Trends precision. due to the enormous variety of NMs.J. Y. is still in its earliest infancy. 32 (2010) 137. the results are promising.J. [6] M. only in exceptional cases is it possible both to detect specifically and to measure particular ENMs. At present. N. A 211 (2010) 99. H.R. the situation is divided. interactions and behavior. [2] N. Bouwmeester. Valdes. Conclusions and future trends Analysis of NMs in food. J. Experience with the combination of TPEM and autofluorescence is very scarce. However. Seaton. was 2%. do Nascimento. the methods to quantify them are still rare.J. J. in: B. L. 2009. it is not usually possible to establish the presence of ENMs. T. Philbert.Y. 5. Dogan. San Diego. I.A global technology industry and market analysis. Noordam.P. S. Oberdorster. J.B.V. The mobile phase (55:45 toluene/methanol) provided chromatographic separation of the fullerene analytes using a conventional C18 analytical column. Paul (Editors). [4] P. Hagens. Savolainen.A. LC-ESI-MS detection was used to identify and to quantify C60–C98 fullerenes. A. H. [3] M. However. P. Huang.M.E.C. 1. Photobiol. Ryan. 43 (2007) 2229.G. Trends Biotechnol. On the one hand. Aitken. Polym. [12] X.G. that is feasible thanks to the combination of a number of analytical techniques. At present. de Oliveira. Sanguansri.M. Alenius.40 lg/kg. S. 5). R. chemical carriage. N. [13] H. Appetite 51 (2008) 283. TPEM combined with autofluorescence provides a non-intrusive tool for the in vivo visualization of NM fate. G. Chang. Walter. and US EPA) arising from the possible toxic effects of NMs. Delgado. so a combination of techniques is usually necessary. Microchim. 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. W. A. S. R. M. and highlights the techniqueÕs potential for use with other NMs. Summers.R. J. It may also provide a method for looking at the purity of NM manufacture. There are a growing number of these quantitative analytical methods.P. there are methods to analyze the chemicals in specific ENMs. Regul. USA. Technol. M.A. Q. S. H. Stampfli. Kasper.. An important driver for their development is the concern for the food safety of international agencies and organizations (FAO/WHO. T. Chemosphere 78 (2010) 209. The method quantification limit was 0. P. Trends Food Sci. but.C. Losada. Kastenholz. M. Di Mascio. 53 (2009) 52. Gonzalez. Y.A. J.S. Bulder. Maynard. G. P. de Heer. Tuomi. Eur. as has been widely remarked upon in the literature.innoresearch.M. Sips. and we expect impressive applications within food safety. Dekkers.J. Alan. Pharm. Martinez.D. Dresselhaus. David. even though there are methods that have proved their effectiveness in detecting and characterizing NMs. Cruz. 17 (2006) 547. Global Issues in Food Science and Technology.P. Acta 166 (2009) 1. 27 (2009) 82. Augustin. Chen. Zhu. S. Wijnhoven. H. A. if the results compiled in this review are compared with the data presented in other previous treatments of this topic. [11] H. [8] Innovative Research and Products (iRAP). On the other hand. there are methods to analyze the structural form of NMs in food. food quality and food analysis in the very near future. again. Worfel. Moraru.E. Stone. there are many different ways to analyze particles and there is no best technique for all situations. Colvin. Future applications may include studies on NM environmental fate. H. Walker. Technol. 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