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

1, 2011

Determining nanomaterials in food

Cristina Blasco, Yolanda Picó

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 micros-
copy), 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

1. Introduction food packaging, using many different types

Cristina Blasco, Yolanda Picó*
Laboratori de Nutrició i
of materials [e.g., membrane, nanocapsule,
Bromatologia, Facultat de A number of recent reports and reviews have nanoemulsion, liposomal nanovesicle,
Farmàcia, Universitat de identified current, short-term projected nanotube (NT), nanosphere, nanoceramic,
València, Av. Vicent Andrés applications of nanomaterials (NMs) in the nanoclay and nanowire].
Estellés s/n, 46100 Burjassot, food industry, and for food and beverages According to a study from iRAP, Inc. [8],
València, Spain
[1–5]: the total nano-enabled food and beverage
(1) development of materials with novel packaging market in the year 2008 was
functionality; $4.13bn, which grew in 2009 to $4.21bn
(2) microscale and nanoscale processing; and is forecast to grow to $7.3bn by 2014,
(3) new products development; and, at a compound annual growth rate of
(4) design of methods and instrumenta- 11.65%. Active technology represents the
tion for food safety and biosecurity. largest share of the market, and will con-
Fig. 1 identifies these areas of applica- tinue to do so in 2014 with $4.35bn in
tion in the food-processing chain, grouped sales, and the intelligent segment will grow
by target area. A complex set of engi- to $2.47bn. The US NM market, which to-
neering and scientific challenges in the taled only $125m in 2000, is expected to
food and bioprocessing industry in man- reach $30bn by 2020, and packaging with
ufacturing high-quality, safe food with nanotechnology is expected to grow at
efficient, sustainable resources can be 11.65% from 2008 until 2013. While the
solved through nanotechnology. Among majority of manufacturing and use of
emerging applications of nanotechnology nanoscale materials is in USA, the
in the food industry are: European Union (EU), with its global share
(1) bacteria identification and food-qual- of the sector of around 30%, is not lagging
ity monitoring using biosensors [6]; far behind in this field [9].
(2) intelligent, active, and smart food- Although the prospective beneficial
packaging systems [7]; and, effects of nanotechnologies are generally
(3) nanoencapsulation of bioactive food well described, studies assessing their po-
compounds (e.g., micelles, liposomes, tential toxicological effects and impacts
nanoemulsion, biopolymeric nano- are still limited [10,11]. However, the
*
particles, and cubosomes) [2,3]. scientific community is concerned about
Corresponding author.
Tel. +34 96 3543092;
Table 1 sets out some examples of NMs this issue, and there is now a wider debate
Fax: +34 96 3544954; applied to food, divided into several cate- about the risks of the many manufactured
E-mail: [email protected] gories including food, food additives and NMs. Due to this, hundreds of in vitro

84 0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.08.010
Trends in Analytical Chemistry, Vol. 30, No. 1, 2011 Trends

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

toxicological studies have been reported, as well as assessed, taking into account all material properties. As a
numerous reviews and perspectives [12–18]. Recent consequence, international agencies and governments
examples in the literature show that engineered inor- are paying attention to the study of the fate, transport, and
ganic nanoparticles (NPs) and carbon nanostructures health effects of NMs in food and the environment. Several
may incidentally or intentionally enter into contact with reviews already present the latest research carried out to
living organisms, may disrupt normal activity and may assess the risks of engineering NMs (ENMs) in the aquatic
lead to malfunctioning and diseases [19]. environment, including analytical methods and ecotox-
NMs are able to cross biological membranes and access icity assessments [23–28]. Outstandingly, the latest re-
cells, tissues and organs that larger particles normally views, focusing on emerging contaminants in food and the
cannot [19]. NMs can also enter the blood stream via environment, include NMs as one of the hottest topics in
inhalation or ingestion, and some NMs penetrate the skin research today [29–35]. The prerequisite for toxicological,
[20]. Then, once in the blood stream, NMs can be toxicokinetic, migration and exposure assessment is the
transported around the body and taken up by organs and development of analytical tools for detection and charac-
tissues, including brain, heart, liver, kidneys, spleen, terization of NPs in complex matrices [22].
bone marrow and nervous system. Studies demonstrate Due to consumer safety, it is necessary to control the
the potential of NMs to cause DNA mutation and induce content of NMs in food [36]. To obtain this informa-
major structural damage to mitochondria, even resulting tion, reliable quantitative methods of analysis are
in cell death [21]. Size is a key factor in determining the required to measure levels of NMs in a broad range of
potential toxicity of a particle [22], but there are also matrices. In food, there are natural NMs, intentionally
other contributing aspects (e.g., chemical composition, added ENMs – derived from naturally occurring food
morphology or shape, surface structure, surface charge, components, or engineered using materials that are not
aggregation and solubility, and the presence or absence endogenous to foods – and NMs resulting from con-
of other chemical functional groups) [10]. tamination. Many food substances or ingredients have
It is difficult to generalize about health risks associated nanostructures in nature and are present at lm or nm
with exposure to NMs – each new NM must be individually in size:

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

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

Product name Manufacturer NMs Claim Web address or reference

Food packaging materials
Durethan KU 2-2601 Bayer Silica in a polymer- Nanoparticles of silica in the http://www.research.
based nanocomposite plastic prevent the penetration of bayer.com/edition_15/
oxygen and gas of the wrapping, 15_polyamides.pdfx
extending the product shelf life
Hite brewery beers: Honeywell HoneywellÕs Aegis OX http://www.packaging-
three-layer, 1.6 L beer nylon-based  Oxygen and carbon-dioxide gateway.com/features/
bottle nanocomposite barrier feature79/
 Clarity http://www51.honeywell.
 Recyclability com/sm/aegis/
 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: Nanocor Imperm nylon/ Imperm is a plastic imbedded http://www.nanocor.com/
 Lite nanocomposite barrier with clay nano-particles that applications.asp
 Genuine Draft technology produced by makes bottles less likely to http://www.forbes.com/
 Ice House Nanocor shatter and increases shelf life to investmentnewsletters/2005/
up to six months 08/09/nanotechnology-kraft-
hershey-
cz_jw_0810soapbox_
inl.html?partner=rss
Nano Plastic Wrap Songsing Nano zinc light catalyst Biodegradable after use http://www.physorg.com/
Nanotechnology Compostable to European news717488335.html
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
Constantia multifilm Constantia Multifilm Nanocomposite polymer A clear laminate with http://www.constantia-
N-COAT outstanding gas-barrier multifilm.com/
proprieties developed primarily
for the nuts, dry food and snack
markets
DuPont Light Stabilizer Du Pont Nano TiO2 UV-protected plastic food http://www2.dupont.com/
210 packaging Titanium_Technologies/
en_US/products/dls_210/
dls_210_landing.html
Adhesive form Ecosynthetix 50–150 nm starch The adhesive requires less water http://www.physorg.com/
MacDonaldÕs burger nanospheres as well as less time and energy to news71748835.htlm
containers dry
Food additives
AdNano Evonik (Degussa) Nano ZnO (food grade) www.advancednano
materials.com
Aerosil, Sipernat Evonik (Degussa) Silica (food grade) Free flow add for powdered www.aerosil.com
ingredients in the food industry
AquaNova NovaSol AquaNova Product micelle An optimum carrier system of http://www.aquanova.de/
(capsule) of lipophilic, hydrophobic substances for a product-micelle.htm
water-insoluble higher and faster intestinal and
substances dermal resorption and
penetration of active ingredients

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

Table 1 (continued)
Product name Manufacturer NMs Claim Web address or reference
Bioral Omega-3 nano- BioDelivery Sciences Nano- cochleates Effective means for the addition http://www.
cochleates International as small as 50 nm of omega-3 fatty acids for use in biodellveryscience.com/
cake, muffins, pasta, noodles, bioralnutrients.htlm
soup, cookies, cereals, chips and
candy bars
NanoCoQ10 Pharmanex Nano coQ10 Nano technology to deliver http://www.pharmanex.com/
highly bioavailable coenzyme intercom/
Q10, making it up to 10 times productDetall.do?prod1d-
more bioavailable than other 010036628.mkt1d-2031
forms of CoQ10
Nano self-assembled, Nutralease Nanomicelles for Improved bioavailability means http://www.nutralease.com/
structured liquids encapsulation of nutraceuticals are released into technology.asp
nutraceuticals membrane between the digestive
system and the blood
Solu E 200 BASF BASF Vitamin E nano-solution Solubilization of fat-soluble http://www.human-
using NovaSOl vitamins nutrition.basf
Synthetic Lycopene BASF LycoVit 10% http://www.human-
(< 200 nm synthetic nutrition.basf.com
lycopene

Food and beverages

Nano Tea Shenzen Become Nanoparticles Patent No.: 0100033.3 – Three- http://www.369.comcn/Er/
Industry & Trading Co (160 nm) step preparation method and its nanotea.htm
application for nanotea
Patent No.:02100314.9/
00244295.7 – Multi-layer,
swinging nano-ball milling
procedures
Nano Slim Nano Slim NAno-Diffuse Orosolic acid (derived from the http://www.nanoslim.con/
Technology Lagerstroemia spaciosa plant) nanoslim-information.html
Nanoceuticals Slim RBC Lifescience Nanodusters http://www.rbdifesciences.
Shake Chocolate com/Meal_Replacement_
Shakes.aspx
Nanoceuticals Slim RBC Lifescience Nanodusters http://
Shake Vanilla www.rbdifesciences.com/
Meal_
Replacement_Shakes.aspx
Fortified fruit juice High Vive.com 300 nm iron http://www.highwive.com/
(SunActive Fe) sunactiveiron.htm
Daily Vitamin Boost Jamba Juice Hawaii 300 nm iron 22 essential vitamins and http://
(SunActive Fe) minerals and 100%, or more of jambajuicehawaii.com/vita-
your daily needs of 18 of them! boost.asp
Oat Chocolate Toddler Health 300 nm iron (SunActive Toddler health is an all-natural http://www.toddlehealth.net/
Nutritional Drink Mix Fe) balanced nutritional drink for OatChocolate.php
children from 13 months to
5 years. One serving of Toddler
Health helps little ones meet
their daily requirements for
vitamins, minerals and protein
Oat Vanilla Nutritional Toddler Health 300 nm iron (SunActive Toddler health is an all-natural http://www.toddlehealth.net/
Drink Mix Fe) balanced nutritional drink for OatChocolate.php
children from 13 months to
5 years. One serving of Toddler
Health helps little ones meet
their daily requirements for
vitamins, minerals and protein
Canola Active oil Shemen Nano-sized http://www.shemen.co.il
selfassembled structured
liquid micelles

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

(1) food proteins, which are globular particles of 10s to formulation, environmental matrices and food mate-
100s of nm in size, are true NPs; rials [39], but it drew heavily on studies reporting
(2) linear polysaccharides with one-dimensional nano- characterization of NPs in raw products and environ-
structures are less than 1 nm in thickness; and, mental materials because limited work has been done
(3) starch polysaccharides have small 3-D crystalline to date on detection and characterization of NPs in
nanostructures that are only 10s of nm in thickness. food.
Although natural NMs in food are not considered This review takes a step forward by including the first
within the scope of this review, they contribute to the applications of NMs to food.
complexity of the analysis for two reasons:
(1) first, they should be distinguished from ENMs or
contaminating NMs [27]; and, 2. Regulations
(2) second, due to their specific physico-chemical prop-
erties, NPs could interact with proteins, lipids, car- The safety of nanoproducts has attracted attention in
bohydrates, nucleic acids, ions, minerals and line with their increasing use. Despite the rapid com-
water in food, feed and biological tissues. mercialization of nanotechnology, no specific nano reg-
It is important to characterize the effects and the inter- ulations exist anywhere in the world. Most regulatory
actions of ENMs in the relevant food matrix. Proteins and agencies remain in information-gathering mode, lacking
carbohydrates have large specific surface areas and a high the legal and scientific tools, information and resources
electrochemical surface charge that is likely to make them that they need to oversee the exponential market growth
interact with charged particles, like many engineering of nanotechnology adequately [1,40–44].
NPs (ENPs). These components also contain hydrophobic At present, international organizations are still
domains that are likely to interact with hydrophobic ENPs attempting to determine the current capacity to assess
[e.g., fullerenes and carbon nanotubes (CNTs)]. the health and safety risk associated with the use of
Food also contains natural colloids and dissolved ions. nanotechnology in food and food production and on
Dispersed colloids are particles in the ENP range (1– surfaces in contact with food.
200 nm) that are kept in a stable aqueous suspension Through its horizon-scanning activities, the Food and
[11]. They do not precipitate by gravitation due to their Agriculture Organization (FAO) has recognized the need
small size, a certain surface charge, electrostatic interac- for scientific advice on any food-safety implications that
tions, van der Waals forces and steric forces. Any changes may arise from the use of nanotechnologies in the food
(e.g., pH or ion concentrations) may destabilize the sus- and agriculture sectors.
pension. According to the classical double-layer and col- With the FAO, the World Health Organization (WHO)
loid-stability theories, particle stability is affected by the has published the report of a Joint Expert Meeting held in
concentration of cations (coagulants), meaning that, at June 2009 on the topic of Application of Nanotechnolo-
increasing salt concentrations, free NPs will start to gies in the Food and Agriculture Sectors: Potential Food
aggregate [20]. Releasing ENPs into such complex sys- Safety Implications [45]. This report presents an overview
tems is bound to lead to a range of interactions, and it is not of the wide range of current and projected nanotechnol-
evident whether a given ENP will be adsorbed to a surface ogy applications in food and agriculture (Fig. 2). Appli-
or if it will be stabilized by natural polymers so that it re- cations that may lead to human exposure to NPs through
mains mobile [37]. the environment to the food chain were not included.
Given the huge diversity of ENPs for use in the food The Council of the Organization for Economical
and feed sector (e.g., chemical composition, size, size Cooperation and Development (OECD) has established a
distribution, surface activity/modification) (see Table 1), Working Party on Manufactured NMs as a subsidiary
and their potential interaction with food-matrix com- body of its Chemicals Committee [46]. This working
ponents (e.g., proteins), the determination of NMs in food party was established to address human health and
is a challenging task requiring tailored solutions. As environmental safety aspects of manufactured NMs in
regulation for food nanotechnology moves forward, the the chemicals sector.
aim of this review is to address and to compare the Furthermore, in February 2009, the European Food
available analytical methods for determination of NMs in Safety Authority (EFSA) published its opinion on the po-
food. Two previous reviews underpin this one: tential risks arising from nanoscience and nanotechnolo-
(1) one provided a detailed description of food nano- gies in food and feed [47]. It considered, among other
delivery systems and considered the analytical tech- things, the suitability of current regulations relating to the
niques useful for identifying and characterizing use of nanotechnologies in the food sector. The report did
these systems in food [38]; and, not identify any major gaps in regulations, although it
(2) the other overviewed the different analytical tech- noted that there was uncertainty in some areas as to
niques available for detecting the ENPs in product whether applications of nanotechnologies would be picked

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 Trends in Analytical Chemistry, Vol. 30, No. 1, 2011
Nano-emulsions Spreads
S d
Surfactant micelles ayo a se
Mayonnaise
P
Procesed d
y
Emulsion bilayers, Cream
nanostructured in food Yoghurts
(NANOTEXTURES) Double or multiple emulsions
Reverse micelles ice creams

Vegetable oil
Nanomicelle-based carrier
Nanodeliver
N d li systems
t enriched in vitamins
vitamins,
system
p
based on encapsulated minerals and
Nanocluster delivery system
technology phytochemicals

Inorganic NMs (TiO 2, silver, silica, selenium, calcium, iron)

Nanomaterials relevant Surface functionalized NMs

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

NP reinforced materials (polymer

composites with nano-clays,
nano-clays nano- Nanoclay–polymer
N l l compositesit
Nano-enabled food metals or metal oxides, coating Oxygen
O yge dedetecting
ec g ink co
containing
a g
contact materials contained NPs and antimicrobial TiO2 NPs
(FCMs) and packaging nanoemulsions) Nanolayer of silver that react
y g sulfide
with hydrogen
IIntelligent
lli packaging
k i concepts based
b d
on nanosensors
http://www.elsevier.com/locate/trac

Natural biopolymer from yeast cell walls that bind

t i
mycotoxins
Nanotechnology in Animal feed Polystyrene
y y ((PS)) base,, p
polyethylene
y y g
glycol
y ((PEG)) linker,,
the agricultural sector Agrochemicals and mannose targeting biomolecule to bind E. coli
Slow or controlled
Slow- controlled-release
release fertilizers and pesticides

Figure 2. Nanomaterials relevant to food.

Trends
89
Trends Trends in Analytical Chemistry, Vol. 30, No. 1, 2011

Table 2. European Union legislation applicable to NMs in food

Legislation Comments Ref.

General for chemical compounds
REACH European Community legislation concerned with chemicals and their safe use and dealing with the [51]
Registration, Evaluation, Authorisation and restriction of CHemical substances.

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 [52,53]
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.

Food additives
Directive 89/107/EC and Only additives explicitly authorized may be used in food. In December 2008, a new Regulation was [54–56]
associated legislation 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.

Food-contact materials
Regulation EC/1935/2004 All materials that are intended to come into contact with foodstuffs, either directly or indirectly. The [57]
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.
Food supplements
Directive 2002/46/EC States that only vitamins and minerals on an approved list may be used as food supplements. New [58]
substances may be considered for inclusion on the list, but only after a safety assessment by EFSA.

up consistently. It concluded that ‘‘on the basis of current regulate NMs under several laws. The EPA could use
information, most potential uses of nanotechnologies that most of the environmental laws – Clean Water Act
could affect the food area would come under some form of (CWA), Clean Air Act (CAA), Comprehensive Environ-
approval process before being permitted for use’’. mental Response, Compensation and Liability Act
These international agencies identified a number of (CERCLA), Resource Conservation and Recovery Act
domains of interest as a starting point for this review, so (RCRA), Federal Insecticide, Fungicide and Rodenticide
that more research on NMs is needed to improve the basis Act (FIFRA), and Toxic Substances Control Act (TSCA).
of scientific knowledge in support of regulatory work: The US Food and Drug Administration (FDA) requires
– development of reliable measurement methods, ref- manufacturers to demonstrate that food and their
erence materials and materials characterization; ingredients are not dangerous to health, but there are no
– review and development of test methods for human specific rules for NPs because it regulates products, not
health, safety and the environment; technologies. Nevertheless, the FDA expects that many
– development of exposure information throughout nanotechnology products will come under its jurisdic-
the life-cycle of NMs; tion. The FDA regulates a wide range of products,
– review of existing risk-assessment methods; including foods, cosmetics, drugs, devices, and veteri-
– risk management for workersÕ protection; nary products, some of which contain NMs or are pro-
– networking existing and establishing new infra- duced nanotechnologically. The Acting Commissioner of
structures to examine health, safety and environ- the FDA initiated the Nanotechnology Task Force in
mental aspects of NMs. 2006 to help address questions regarding adequacy and
However, different countries are trying to include NMs application by regulatory authorities [48].
in their current regulations. In USA, the Environmental The European Commission (EC) aims to reinforce
Protection Agency (EPA) is already empowered to 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, Microscopy and microscopy-related (imaging): near field-scanning optical [22,23,26,27,38,39,59–62]
morphology or shape and microscopy (NSOM); scanning probe microscopy (SPM); confocal laser
aggregation state 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 [23,26,38,39,63]
Chromatography and related (separation): size exclusion chromatography [23,26,38,39,60]
(SEC); capillary electrophoresis (CE); hydrodynamic chromatography
(HDC); field flow fractionation (FFF)
Spectroscopy and related (characterization): static light scattering (SLS); [22,23,27,38,39,60,62,64]
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 [38,39,59]
Chemical characterization Analytical (spectroscopy) coupled to electron microscopy (imaging): TEM- [22,23,27,38,39,59–62]
dispersive 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; [22,23,27,38,39,60,64]
UV-Vis; infrared spectroscopy; NMR
Mass spectrometry (characterization): [27,39,65–67]
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)
Charge determination Laser droplet anemometry, Zeta potentiometer, CE [26,27,39]
Surface hydrophobicity Water contact angle measurements, rose bangle (dye) binding, [26,39]
hydrophobic interaction chromatography, X-ray photoelectron
spectroscopy
Chemical analysis of surface Spectroscopy and related (characterization): static X-ray spectroscopy [X- [22,38,39]
ray photoelectron (XPX); X-ray fluorescence (XRF); X-ray absorption
spectroscopy (XAS); and, X-ray diffraction (XRD)]
Mass spectrometry (characterization): [39,65–67]
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 time-
of-flight (QqTOF); static secondary ion mass spectrometry (SSIM)
Carrier-drug interaction Differential scanning calorimetry [39,68,69]
Nanoparticle-dispersion stability Critical flocculation temperature(CFT) [39,70,71]
Release profile In-vitro release characteristic under physiologic & sink condition [39]
Target-compound stability Bioassay of target compound extracted from NP, chemical analysis of the [39]
target compounds

for collaborative research and development (R&D) on the to meet a general safety requirement under the General
potential impact of nanotechnology on human health Principles of Food Law Regulation (EC/178/2002) [50].
and the environment via toxicological and ecotoxico- More specific legislation covers the use of novel foods,
logical studies. The EC is performing a regulatory food additives and food-contact materials (see Table 2).
inventory, covering EU regulatory frameworks that are
applicable to NMs (e.g., chemicals, worker protection,
environmental, and product-specific). The purpose of 3. Analytical approaches to characterize and
this inventory is to examine and, where appropriate, to determine NMs
propose adaptations of EU regulations in relevant sectors
[49]. This includes to apply existing food laws to food Recently, the problem of NM safety, once mainly limited
products using nanotechnology. All food products have to its chemical aspects, has been extended to possible

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

toxicity associated with NPs as physical entities [22], so throughput when using these techniques for analysis of
the analysis of NMs commonly requires two types of NPs in food.
method: For CNTs, Raman spectroscopy is a very important
(1) those for characterizing and/or detecting NPs or technique and could be very useful for finding CNTs and
NMs; and, fullerenes. This technique is cheap, non-destructive and
(2) those for determining their chemical composition. not time consuming. It has also the advantage of being
However, the line that divides these types is very thin, used in in situ monitoring. Several reviews highlight the
taking into account that the chemical composition of power of Raman spectroscopy for measuring CNTs
NMs is one of their properties. As outlined in Table 3, [72,73].
there are a number of analytical tools for qualitative and The second group of techniques possesses a lower
quantitative categorization of NMs: analytical ambition but is best suited to routine analysis.
(1) single-particle techniques; These approaches are based on the chemical character-
(2) techniques characterizing the ensemble of NMs; ization of NMs, without generating information on their
and, physical state. Hence, the metal content of NMs can be
(3) techniques to determine their chemical composi- quantified by analytical-chemistry tools [e.g., induc-
tion. tively-coupled plasma atomic emission spectroscopy
For a wide discussion on the advantages or the (ICP-AES) or mass spectrometry (ICP-MS)], or by radio-
drawbacks of each technique, we refer the reader to analysis after appropriate neutron irradiation. Generally,
excellent reviews by Tiede et al. [39] and Luykx et al. sample preparation includes acidic sample digestion
[38], who accurately explain their advantages and lim- before analysis. The limitations of chemical analysis
itations, so there is no need for a repeat in this article. result from artificial losses during the preparatory steps,
The main problem of analyzing NMs in food is that most analytical detection limits and the inability to charac-
of these analytical systems have been used to charac- terize carbon NPs (e.g., polymeric NPs, fullerenes, and
terize the NM themselves and only few are applicable to CNTs). In the case of organic NMs, detection or quanti-
the analysis of more complex samples. Food is a very fication of the chemical may be possible, where a test for
intricate material, and, probably, for sufficient charac- the species exists, but a focus on characteristic structures
terization, the NMs need to be separated from the food may be needed to determine whether it is still in nano-
matrix, as Luykx et al. [38] discussed for nanodelivery form.
systems. However, the tendency is to reduce sample So far, only very limited work has been done on the
preparation as much as possible because it is important detection of organic NPs in food. However, the need to
to measure the NMs in the relevant matrix, as their determine organic NMs and nano-delivery systems led
properties may depend on the surrounding matrix and scientists to imagine what techniques could also be
be affected by processing as well as by the extraction applicable to their characterization in food, even though
procedure. This is usually much more demanding than they have not yet been used. There are a few recent
to analyze NMs in simpler or model matrices. examples of NP-enabled MS that, though not explicitly
With nanoscale metals or semiconductors containing linked to NP determination in food, demonstrated the
NMs, these can be detected even in rather complex potential of this approach. For example, matrix-assisted
matrices (e.g., food, feed and biological tissues) by means laser desorption/ionization (MALDI)-time-of-flight (TOF)-
of electron microscopy (EM) coupled with chemical MS and laser desorption/ionization (LDI)-TOF-MS were
analytical tools. However, detection by EM is only pos- useful for characterization of ultrasmall NPs of TiO2
sible if the number of NMs is sufficiently high in the [67]. The size distributions of TiO2 NPs obtained from
matrix to localize them, since high magnification is re- MALDI-TOF-MS and LDI-TOF-MS were in good agree-
quired due the small size of NMs. As a result, the ment with parallel TEM observations.
investigation of NM distribution in food is generally ex- TOF secondary-ion MS (SIMS), fluorescence micros-
tremely time consuming. We need to mention that copy and scanning EM (SEM) were employed to monitor
transmission EM (TEM) has so far provided the most the immobilization of biotinylated shell-crosslinked NPs
detailed information regarding NM location by allowing on biotin/streptavidin-functionalized, UV-photo pat-
both visualization of the location within food, and, in terned self-assembled monolayers [74].
conjunction with spectroscopic methods, characteriza- There are also some studies reporting quantification of
tion of the composition of the internalized NPs. NMs in a related field (e.g., environmental analysis),
Visualization is most easily accomplished with elec- where much more information is available. Isaacson and
tron-dense NMs (e.g., metal NPs). However, the tech- Bouchard [75] reported the first methods for the asym-
nique is also suitable for any other NMs, including metric flow field-flow fractionation (AF4) size separation
organic compounds (e.g., fullerenes and CNTs). How- of aqueous C60 aggregates in deionized water without
ever, the long time required for both sample preparation use of mobile-phase modifiers coupled with in-line dy-
and image analysis greatly limits the analytical namic light scattering (DLS) and off-line with liquid

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

chromatography with atmospheric pressure photo-ioni- quality-control material (IRMM-304) of silica NPs. There
zation MS (LC-APPI-MS). are also available gold NPs (NIST RM 8011, 8012 and
Meanwhile, Farre et al. [76] gave details of the first 8013) and polystyrene spheres (NIST SRM 1963a and
determination of C60 and C70 fullerenes and N-methyl- 1964) from the National Institute of Standards and
fulleropyrrolidine C60 on the suspended material of Technology (NIST) [77].
wastewater effluents by LC hybrid quadrupole linear ion Furthermore, interlaboratory studies, which are nec-
trap tandem MS. As we go deeper into the analysis of essary for validation of protocols and generating preci-
NPs, we need analytical procedures to account for ex- sion and bias statements for measurement standards, are
plicit information, including molecular weight and the almost non-existent.
number and the identity of functional groups. Sensitive Of the difficulties encountered in conducting inter-
and mass-selective detection, as offered for MS combined laboratory studies on these emerging contaminants, the
with optimal extraction procedures, shows great poten- major problem is the wide range of analytical techniques
tial to achieve this goal. applicable to these compounds and the different purposes
There are some additional complications not yet of the methods developed [78,79]. There is much work
solved in the analysis of NMs, such as the fact that some yet to be done to show how far determination of NMs in
ENMs cannot be distinguished from naturally-occurring food can go.
variants of the same {e.g., nanoscale engineered silicon
dioxide (SiO2) or endogenous lipids used in capsule
membranes}. Detection may also be hindered by inter- 4. Applications to food analysis
actions with solutes or food components that obscure
clear analytical signals. Despite some successes in this Applications of the analytical methodology reported in
field, its evolution seems to be hampered by the limited the previous section to the analysis of NMs in food are
information available. This will be a growing area within still very scarce. Table 4 lists the results of the literature
NM analysis. search. Various approaches suggested for use in studies
Moreover, there remain several obstacles to obtaining of NP bioaccumulation are included because they can be
adequate characterization and quantification of NMs in applied to its determination in food, since many of the
food. Foremost among them are those presented by the target organisms are also edible.
lack of analytical standards, relevant reference materials We need to mention that methods to determine inor-
and internationally standardized practices, protocols, and ganic NPs in food, especially aquatic organisms, are
procedures for testing the preparation of food, NP quite developed. There are two main types of procedure:
measurement, and data analysis. The OECD has estab- (1) those combining microscopy and spectroscopy to
lished a list of prioritized materials that takes into account identify and to characterize the NMs as well as to
those materials that are already in production (or close to detect their chemical composition; and,
commercial use), as well as considerations of production (2) those looking at their chemical composition only.
volume, the likely availability of materials for testing and For inorganic NPs, the latter methods are based
the existing information [46]. The OECD list comprises: mainly on wet digestion with a strong acid (e.g., nitric or
 fullerenes (e.g., C60); perchloric) followed by ICP optical emission spectroscopy
 single-walled and multi-walled CNTs (SWCNTs (ICP-OES) or ICP-MS. For example, several methods to
and MWCNTs, respectively); measure gold NPs have been developed. These methods
 carbon black; are based only on chemical analysis, since we may as-
 polystyrene; sume that all the gold present in the samples is from NPs,
 dendrimers; since gold is not abundant in the environment. Con-
 nanoclays; and, trarily, other metal NMs {e.g., unmodified commercial
 NPs of Ag, Fe, TiO2, Al2O3, CeO2, ZnO, and SiO2 nanoscale metal oxides, zinc oxide (ZnO), cerium dioxide
[46]. (CeO2) and titanium dioxide (TiO2)} were characterized
Internal standard quantification using stable isotope- by TEM and environmental scanning EM (ESEM) with
labeled internal standard is de rigueur for trace-level energy dispersive X-ray analysis (EDX) elemental anal-
analysis of contaminants in food. Currently, there is ysis to establish their structure as NPs [81].
commercially available only a single, stable-isotope la- Also, fish samples were digested with acid and ana-
beled fullerene internal standard, 13C60. The current lyzed by ICP-MS. Definitive uptake from the water
limited number of standardized reference materials for column and location of TiO2 NPs in gills was demon-
engineered NMs is another brake on precise, reproduc- strated for the first time by using coherent anti-Stokes
ible detection and quantification of engineered NMs in Raman scattering (CARS) microscopy. CARS imaging of
food and feed. rainbow-trout gill tissues clearly showed large aggre-
The Joint Research Centre, Institute of Reference gates of TiO2 (up to 3 lm) on the surface of the gill
Materials and Measurements, has recently released a epithelium following 24–96 h exposure (Fig. 3).

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

Table 4. Selected applications for detection, characterization and/or quantification of NMs in food or food like matrices

Problem Nanomaterial Matrix Techniques Comment Ref.

Food and beverages
Investigation of the Inorganic Bread and ESEM/EDS Detection of organic and [80]
presence of microsized NMs biscuits inorganic microscale and
and nanosized nanoscale contaminants: ESEM
contaminants Identification of their chemical
composition: EDS
Bioavailability of TiO2, CeO2 Rainbow trout TEM Characterization of size, particle [81]
nanoscale metal oxides and ZnO ESEM/EDS shape/morphology and
to fish ICP-MS qualitative aggregation: TEM and
ICP-OES ESEM/EDS
CARS Analysis of elements content :
wet digestion and ICP-MS, ICP-
OES
Confirmation of the presence of
the NP in fish: CARS
Bioaccumulation of gold Au NPs Mytilus edulis ICP-OES Analysis of Au content: wet [82,83]
NPs in fish digestion with nitric acid and
hydrogen peroxide and ICP-OES
In vivo toxicity studies Fullerenes Embryo zebra LC-MS2 Digestion with glacial acetic acid [84]
C70 C98 fish and toluene
150 mm · 2 mm Targa C18
column and toluene/methanol
(55:45) isocratic mobile phase
Only molecular ions were
obtained
Accumulation of NMs in Multi-walled Wheat tissues TPEM coupled chemical TPEM combined with [85]
plants carbon nanotubes autofluorescence autofluorescence can be used to
TiO2, CeO2 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.

Migration and stability studies with food-packaging materials

Detect clay NPs and Biopolymer polylactide 95% ethanol as XRD Characterization of polylactide/ [86]
characterize their size with 5% Cloisite food simulant TEM Cloisite 30 B: XRD, TEM
30B as filler Centrifugation Chemical characterization of
FA-MALS elements: ICP-MS
ICP-MS Separation and size
FA4-MALS-ICP-MS determination of clays:
centrifugation, FA4-MALS and
clay aspect ratio
Migration study FA-MALS-ICP-
MS

Stability of NPs during Chitosan NPs for Aqueous Zeta potential PDI Characteristics of AA-loaded CS [87]
heat treatment l-ascorbic acid solutions Ultracentrifugation 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.

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

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 From a food-safety point of view, it is important to
micro-sized and nano-sized contaminants in bread and characterize migrates from nanocomposites containing
biscuits from 14 different countries by ESEM. EDX was clay as fillers. Schmidt et al. [86] demonstrated that AF4
employed to identify their chemical composition. The with multi-angle light scattering (MALS) detection was
results indicated that 40% of the samples analyzed useful for characterizing the size of NPs contained in
contained foreign bodies (e.g., ceramic and metallic migrates from nanocomposites of polylactide and
debris, probably of environmental or industrial origin). organomodified montmorillonite clay as filler. However,
Fig. 4 shows other debris found in a biscuit, which this coupled instrumentation alone did not provide any
contained cadmium, silver, tungsten, aluminum, sulfur, information on the identity of the NPs occurring in the
calcium, iron, cobalt and copper in particles. simulated food. This limitation was overcome by cou-
Biopolymer nanocomposites are a field of emerging pling AF4-MALS to the element-selective ICP-MS detec-
interest, since such materials can exhibit improved tor, which provided additional information on trace
mechanical and barrier properties, and they can be more elements known to be naturally present in the clay. The
suitable for a wider range of food-packaging applications. analytical system was applied to characterize migrates

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

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/tol-
uene (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 Isaacson et al. [84] developed and validated an ana-
organomodified Cloisite 30B montmorillonite clay used lytical method to quantify a suite of fullerenes and then
as filler. The results demonstrated that NPs of 50– apply the analytical method to determine the behavior of
800 nm radius indeed migrated from the nanocompos- a single fullerene, C60, during a toxicological assay using
ite, but ICP-MS signals corresponding to clay minerals zebrafish embryos and aqueous-exposure solutions. The
were absent. average recovery of C60 from fish extracts was 90% and

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

precision, as indicated by the relative standard deviation, progress that represents a major step forward and lays
was 2%. The method quantification limit was 0.40 lg/kg. the foundation to develop quantitative methods for the
LC-ESI-MS detection was used to identify and to quantify analysis of NM residues in foods. There are a growing
C60–C98 fullerenes. The most abundant ions formed under number of these quantitative analytical methods, and we
ESI-MS conditions were molecular ions. The mobile phase expect impressive applications within food safety, food
(55:45 toluene/methanol) provided chromatographic quality and food analysis in the very near future, as an
separation of the fullerene analytes using a conventional extension of those already existing for environmental
C18 analytical column. Chromatographic analysis indi- samples. An important driver for their development is
cated co-elution of C60 with the 13C60 internal standard the concern for the food safety of international agencies
with the retention times of the larger fullerenes increasing and organizations (FAO/WHO, EU, EFSA, and US EPA)
in order of increasing carbon number (Fig. 5). arising from the possible toxic effects of NMs. The
Two-photon excitation microscopy (TPEM) combined assessment of the risk to consumers needs to be ad-
with autofluorescence was used to detect NMs dressed as regulations for food nanotechnology move
(MWCNTs, TiO2, and CeO2) interacting with vegetation forward.
and used simultaneously to detect and to monitor the Summarizing, due to the enormous variety of NMs,
interactions of MWCNTs and polycyclic aromatic there are many different ways to analyze particles and
hydrocarbons in vivo in roots [85]. The potential of there is no best technique for all situations, so a com-
TPEM coupled with autofluorescence in visualizing bination of techniques is usually necessary.
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 Acknowledgments
provide a method for looking at the purity of NM man- The authors thank the Spanish Ministry of Education
ufacture, where artifacts (e.g., catalysts from the man- and Science and the European Regional Development
ufacturing process) can be identified rapidly from their Funds (ERDF) (Project CGL2007-66687-C02-01/BOS)
autofluorescence signals. TPEM combined with auto- for financial support.
fluorescence provides a non-intrusive tool for the in vivo
visualization of NM fate, interactions and behavior.
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