Challenges in Chemical Food Contaminants and Residue Analysis … · 2013. 12. 20. · 1.1 The food chain: A global issue 3 1.2 Issues according to the Rapid Alert System for Food

CHAPTER 1Challenges in Chemical FoodContaminants and Residue Analysis

Michel W.F. Nielen and Hans J.P. Marvin

Contents 1. Introduction 2

1.1 The food chain: A global issue 3

1.2 Issues according to the Rapid Alert System for Food and Feed 4

1.3 Issues according to the feed and food industry 6

1.4 The consumer perception 6

2. Emerging Contaminants 7

2.1 Brominated flame retardants 8

2.2 Perfluorinated organic substances 8

2.3 Endocrine disruptors 9

2.4 Metal speciation 10

2.5 Alkaloid biotoxins 10

2.6 Peptide and protein growth promoters 11

2.7 Genetically modified organisms (GMO) 12

2.8 Nanoparticles 13

3. Masked Contaminants 14

4. Unknown Bioactive Contaminants 15

4.1 Bioactivity-directed identification of emerging unknown

contaminants 15

4.2 Metabolomics approach to the identification of emerging

unknown contaminants 17

4.3 In silico prediction approach to the identification of unknowns 17

5. Emerging Technologies 18

5.1 Omics 18

5.2 Bioassays 19

5.3 (Bio)nanotechnology 20

5.4 Ambient mass spectrometry 20

6. Validation and QA/QC Challenges 22

6.1 Biochemical or biological rapid screening assays 22

6.2 Comprehensive instrumental analysis methods 23

Comprehensive Analytical Chemistry, Volume 51 r 2008 Elsevier B.V.ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00001-9 All rights reserved.

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7. Conclusions and Future Trends 24

Acknowledgements 25

References 25

1. INTRODUCTION

Nowadays food is produced and distributed in a global market leading tostringent legislation and regulation for food quality and safety in order to protectconsumers and ensure fair trade. Despite these efforts, food safety incidentsoccasionally occur and originate from both microbial and chemical contamina-tion. Pesticide and veterinary drug residues, endocrine disruptors, food additivesand packaging materials, environmental contaminants (including dioxins andheavy metals) and contaminants of natural origin (including mycotoxins andmarine toxins) are of particular concern. As a consequence of the introduction offood commodities containing ingredients produced by modern biotechnologyand resulting legal requirements of safety and labeling, a strong additionaldemand for adequate methods of analysis has occurred. Risk analysis provides aframework for regulatory authorities to protect the consumer from potential foodsafety hazards and is performed in an iterative manner by food safety managers(regulatory authorities), risk assessors (scientists) and stakeholders (i.e.,consumers, industry, non-governmental organizations). The assessment of foodsafety is a scientific exercise performed by scientists and consists of hazardidentification, hazard characterization, exposure assessment and risk character-ization. An important prerequisite for performing risk assessment adequately isthe presence of data generated by reliable and fit-for-purpose analytical methodsto estimate the level of exposure and intake of the consumer to contaminants andresidues. Hence, the accuracy of risk assessment will benefit from the availabilityof comprehensive quantitative monitoring and consumption data. However, costand time considerations of food safety managers (in regulatory institutions andindustry) favour the development and implementation of inexpensive and rapidscreening methods having a limited scope and providing qualitative or semi-quantitative ‘‘on-off’’ data only. Global food production practices and thechanging climate showed that new unexpected food safety hazards and risksmay appear in the food and feed production chain stressing the need foranalytical tools capable of early warning for such emerging risks. Some of thesepotential food safety hazards and methodologies capable of detecting known andunknown emerging contaminants are discussed and related challenges defined.It is argued that monitoring programmes should anticipate these newconspicuous threats. In this context a key role is proposed for bioactivity-basedscreening concepts and bioactivity-directed identification tools.

The development of both rapid screening methods and comprehensive toolscovering as many contaminants as possible including emerging and evenunknown contaminants is justified by the different needs from food safety

2 Michel W.F. Nielen and Hans J.P. Marvin

stakeholders. The rapid screening developments are facing the challenge ofmultiplex detection in order to extend their scope, the comprehensive methodson the other hand are facing major challenges in generic sample preparation andadvanced data evaluation.

1.1 The food chain: A global issue

The food chain as schematically represented by Figure 1 is rather complex andmany factors worldwide play a role in the final issue of food quality and safety.

Raw materials for feed and food production come from all over the worldwith very different local climate, harvesting and storage conditions, all having animpact on the occurrence of microbiological and chemical contaminants such asmycotoxins, pesticide residues, environmental pollution and packagingmigrants. The feed producer will mix different raw materials according to itsspecifications and add additives such as stabilizers but in some cases alsomedication. Medicated feed production can cause drug residues in non-medicated feed produced at the same facility due to carry-over. Next, the feedis transported to the first consumer level, i.e., the farm animals. The increasedawareness of animal welfare might put a stronger demand on feed-related risk-benefit issues. At the farm stage again additives, but also pesticides, veterinarydrugs or even illegal hormones might be applied, which residues and/ormetabolites can again build up in the food chain. The food industry producesfood products and/or food ingredients but they also yield a waste stream whichis at least partly recycled and used as feed ingredient. Packaging into smaller

Rawmaterial

Feed

Farmers+animals

Industry,processing

Retailers Consumers

Figure 1 Simplified representation of the food chain.

Challenges in Chemical Food Contaminants and Residue Analysis 3

pieces might increase the migration issue of chemicals from the packaging intothe food commodity. Following transport the final products come to the retailerswhere a storage issue might influence the final contamination load. Productsbeing beyond the storage limit date might be recycled and end up in the feedstream again. Finally the consumer buys food that must be stored and preparedfor cooking, actions known for their potential introduction of contaminants ifhygiene guidelines are not followed. During cooking, food processing con-taminants, such as acrylamide, heterocyclic amines and polycyclic hydrocarbons,are introduced but some of the contaminants present might be degraded (orbioactivated) so the real load of dietary intake of contaminants is not so easy todetermine. The occurrence of residues from intentionally added chemicalssomewhere in the food chain can in theory be avoided, but very much dependson the attitude and behaviour of the actors in the food production chain.Retrospective studies on recent food safety incidents have shown that the humanfactor (unawareness, fraudulent and illegal actions) plays an important role inthe development of food safety incidents [1]. Globalization of food trade,changing climate conditions and agricultural practices, changing food consump-tion patterns and environmental pollution are all drivers of food safety risks andshould be taken into account in systems aimed at identifying emerging foodsafety hazards and risks. Control of food safety standards, monitoring ofcontaminants and knowledge about the fate of food contaminants through theentire food chain is needed thus requiring the availability of analysis methodsdedicated to the different parts and their actors within the chain.

1.2 Issues according to the Rapid Alert System for Food and Feed

The Rapid Alert System for Food and Feed (RASFF) is primarily a tool forexchange of information between food and feed authorities in the EuropeanUnion (EU) member states in cases where a risk to human health has beenidentified and measures have been taken, such as withholding, recalling, seizureor rejection of the products concerned. The European Commission (EC) publishesweekly overviews of RASFF alert and information notifications on its website,and the summarizing annual reports provide an overview of the numbers ofnotifications and the categories of food products and hazards that they pertainedto [2]. These annual reports also highlight conspicuous developments within theparticular year. Kleter et al. [3] have explored the utility of notifications filedthrough RASFF to identify emerging trends in food safety issues. To this endRASFF information and alert notifications published in the four-year period ofJuly 2003–June 2007 amounting to a total of 11,403 notifications were dividedinto categories and analysed. The breakdown per hazard category is given inFigure 2.

The major categories included chemical (44%), mycotoxins (29%) andmicrobiological hazards (17%), which together accounted for the majority of thenotifications (90%). Within the chemical hazard category, contaminants inproducts from seafood (30%) and spices and condiments (15%) were the mostcommonly reported. The most frequently reported are allergens (e.g., sulfite and


histamine), heavy metals (e.g., cadmium, mercury and lead), veterinaryantibiotics (e.g., the nitrofurans; furazolidone and nitrofurazone, as well aschloramphenicol), dyes (e.g., Sudan 1 and 4) and pesticides (e.g., dimethoate,isophenfos-methyl and omethoate). Aflatoxins account for the majority (93%) ofmycotoxins and are mainly (84%) found in nuts, of which (54%) have beenimported from Iran. Microbial contaminants include moulds, viruses andbacteria. Bacteria species were most frequently reported of which Salmonella andits subspecies were the most numerous (57% of the reports) followed by Listeriamonocytogenes (16%). It should be noted that the number of reports in the RASFFnot necessarily reflects the extent of a specific food safety problem because thenature of the RASFF system implicitly yields a multiplication of a specificfinding. Based on warnings from the EU member states authorities in othercountries will check suspicious lots which will give an additional RASFFnotification. Secondly, cost and time considerations limit the scope of survey andmonitoring programs, hence many potential food safety hazards will not bemonitored at all or at best be accidentally picked up. The application of moregeneric screening methods such as bioassays in routine monitoring programsmay circumvent this problem and increase the chance of finding new(re)emerging food safety hazards. In general, EC regulators and legislatorsrequire the availability of fit-for-purpose analysis methods having a comprehen-sive contaminant scope in order to provide the data for risk assessment, theestablishment of maximum residue limits and the development and execution ofmonitoring plans. Harmonization of validated methods and interpretation ofresults is a prerequisite in order to allow their use in data banks and to avoidinternal and external trade disputes.

Hazards reported through RASFF

July 2003 - June 2007(12,641 hazards)

Chemical

Mycotoxin

Microbiological

Others, including fraud, biological hazards, labeling, chemical hazards,quality, hygiene, defective packaging and others

Figure 2 Breakdown of food chain hazards according to Kleter et al. [3].


1.3 Issues according to the feed and food industry

According to the General Food Law [4], the producers are responsible for thesafety of food and feed they produce, storage, transport, selling (or eventuallydispose), tracking and tracing. They should not place unsafe food or feed on themarket, and should have a full traceability system for rapid withholding andrecalling in case of alerts. Last but not least, unsafe situations should beprevented using critical control points hazard analyses (HACCP) and goodmanufacturing practices. Because of this responsibility and associated demand tomaintain their core product integrity the need of the industry for rapid andinexpensive screening methods has never been greater. It is obvious thatunloading an incoming ship or truck carrying raw materials cannot wait for theresults from a full comprehensive contaminant analysis; a first screening resultshould be available in minutes, not days. As a compromise a limited number ofkey expected contaminants will be rapidly tested by, for example, dipstick-likescreening assays, and other contaminant parameters will be checked lessfrequently. Ideally a rapid screening test would allow the detection of allrelevant contaminants and still be inexpensive. But reality is far from that,putting a strong demand for international cooperation on the development ofrapid multi-detection methods for chemical contaminants in the food chain.

1.4 The consumer perception

Generally food consumers prefer high quality and intrinsically safe food for alow price, but they are also aware of food-related health and safety concerns.Following the request of the European Food Safety Authority (EFSA) and the ECDG-SANCO a special Eurobarometer was carried out in 2005 aiming at anassessment of how people in the EU perceive risk focusing particularly on food safety[5]: 42% of the EU citizens thought that their health could be damaged by thefood they eat or by other consumer goods. When they were asked to freelyassociate the food-related problems they mentioned most often food poisoning(16%), immediately followed by chemicals including pesticides and toxicsubstances (14%) and obesity (13%). However, following a reminder of possiblerisks they expressed widespread concerns having on top of their ‘‘worry-scale’’pesticide residues in fruit, vegetables or cereals and residues such as antibioticsor hormones in meat. In the mid-range environmental pollutants like mercury ordioxins were considered. Surprisingly EU citizens are less concerned about theirpersonal factors such as food hygiene and food preparation (includingpreparation induced chemical risks). Asked about regulatory consumer protec-tion 62% agreed that food safety laws are strict, despite some reservationsregarding their enforcement. Concerning information sources consumers,physicians and scientists were trusted most while economic actors such asfarmers, manufacturers and retailers are the least trusted. According to the sameEurobarometer consumers when they go for shopping indeed go for high quality(42%) and low pricing (40%). Although being based on almost 25,000 interviews(approximately 1,000 per member state) these data should be considered as an


estimation only. Classical chemical residues and contaminant issues werementioned but, for example, natural contaminants such as toxins were obviouslynot considered and/or unknown by the interviewers and the respondents.Moreover consumers might show irrational behaviour in answering questions:they tend to underestimate the safety aspect related to their own lifestyle withrespect to food hygiene during storage and cooking and to overestimate theexternal risk of chemical residues. Recently Verbeke et al. [6] discussed whyconsumers behave as they do with respect to food safety and risk information.Some irrational and inconsistent behaviours were explained based on the natureof the risk and individual psychological processes. Improvement of traceabilityand labeling, segmented communication approaches and public involvement inrisk management decision making might contribute to restore consumerconfidence and reduce the gap between risk perception of consumers andexperts.

Also the consumer expectations regarding food safety and quality require theavailability of fit-for-purpose analysis methods having a comprehensivecontaminant scope. Harmonization of methods and interpretation of results isagain a prerequisite: consumers tend to trust scientists but they will get muchmore worried when scientists provide contradictory information!

2. EMERGING CONTAMINANTS

An emerging risk is an issue that in the future may pose a risk to human health,to animals or the environment [7]. In Europe, regulation (EC) No 178/2002includes the responsibility of identification of such emerging risks [4]. Theindication of an emerging risk may relate to a significant exposure to a hazard notrecognized earlier or to a new/increased exposure to a known hazard. Somecurrent and potential future emerging risk issues are listed in Table 1.

Table 1 Some current and potential future emerging risks in the food chain

Abbreviation Description EFSA opinion

BFR Brominated flame retardants Yes

PFOS Perfluorinated organic substances Requested

ER Endocrine disruptors No

Metals Arsenic, cadmium, lead, mercury,methylmercury, organotin

Yes

Alkaloids Pyrrolizidine and ergot toxins Yes

Proteins Abuse of peptide and protein growth

promoters

No

GMO Gene modification of plants and gene doping

of animals

Yes

Nano Nanoparticles No


2.1 Brominated flame retardants

Brominated Flame Retardants (BFRs) such as polybrominated diphenylethers(PBDEs) and hexabromocyclododecane isomers (HBCD) are widespread occur-ring in the environment and belong to the group of persistent organic pollutants(POPs): BFRs show persistence in the environment like their polychlorinatedbiphenyls (PCBs) and dioxins/dibenzofurans (PCDDs/PCDFs) analogues. BFRsare suspects for various toxic effects, including endocrine disruption whichmight be enhanced following in vivo hydroxylation. The EFSA has drafted anopinion on PCBs [8] and advice on relevant compounds in the BFR group. It canbe expected that BFRs occur in the food chain accompanied by several otherPOPs in different concentrations (PCBs, PCDD/PCDF, PAH, chlorinatedpesticides).

The complex mixture thus obtained requires analysis methods having a hugeseparation and identification power. Comprehensive gas chromatographycombined with time-of-flight mass spectrometry (GC�GC/TOFMS) is anexpensive but comprehensive solution to the analysis of BFRs and relatedsubstances in the food chain [9,10]. Contrary to conventional techniques,implementation of a GC�GC/TOFMS multi-analyte/multi-class strategy infood safety control seems feasible, provided that an adequate solution is foundfor the automated data interpretation challenge related to the very large four-dimensional datasets. Apart from that, the development of bioactivity-basedscreening approaches remains challenging since they can not only provide anadditional screening tool, but even disclose the presence of unknown bioactivepollutants. The DR-CALUXs, an aryl hydrocarbon receptor-based transcriptionactivation bioassay, is being used for dioxin screening in several laboratories [11].Also a surface plasmon resonance (SPR) biosensor assay for measurement of thethyroid disrupting activity of hydroxylated halogenated aromatic pollutants hasbeen described based on binding with specific human transport proteins [12]. Areal comprehensive exposure assessment would integrate the data from theseentirely different but highly complementary approaches.

2.2 Perfluorinated organic substances

Perfluorinated compounds (PFCs) or perfluorinated organic substances (PFOS)are used in a wide variety of industrial applications [13]. As a consequence thesecompounds show a global distribution in the environment [14]. They have beendetected not only in environmental samples and fish but also in human bloodand liver, and in several wildlife species [15]. PFOS show persistence in theenvironment and some of them have been related to different carcinogenicactions, for example, perfluorooctanoic acid has been identified as a potenthepatocarcinogen in rodents [16]. Meanwhile PFOS have been recognized by theEFSA and concentration levels, contamination pathways and toxicologicalpotency should be assessed in the food chain.

So far most of the analysis methods are based on liquid chromatographycoupled to mass spectrometry or tandem mass spectrometry approaches (LC/MS,


LC/MS/MS) preceded by solid-phase extraction. A key issue is the avoidance ofcontamination during sampling, storage and analysis: PFOS are everywhereinside the laboratory and its instruments. The specific chemical and physicalproperties of PFOS hinder the development of rapid screening methods: it is forexample unlikely that antibodies can be raised successfully against the PFOSfamily, no biorecognition-based methods have been reported yet. Still manychallenges remain even for the LC/MS approach: the development of simplifiedsample preparation protocols and harmonization of methods are key issuesanyway.

2.3 Endocrine disruptors

Many contaminants occurring in the food chain can be considered as endocrinedisruptors: certain pesticides, POPs and metabolites thereof, phytoestrogens(present in fruit and vegetables and soy products), hormones like estradiolendogenously present in cow’s milk and eggs, and residues of illegally appliedhormones. Several synthetic endocrine disruptors are actually POPs and suspectfor carcinogenity; information related to phytoestrogens is rather contradictory inthis respect: both protection and promotion of cancers is claimed in literature[17]. The main classes of phytoestrogens are isoflavones, lignans, coumestans andnatural stilbenes, and show structural similarities with potent estrogens. Theirconsumption by healthy adults may be without risk but the problem might betotally different when exposure occurs at critical stages of development, i.e., atfoetal and prepubertal children. Soy-milk-based baby-food is especially relevantto check for adverse effects of phytoestrogens [18]. Cow’s milk on the other handshould be checked for both estradiol and phytoestrogens. For an adequate riskassessment it is crucial to know how much phytoestrogens (or endocrinedisruptors in general) are added to the ‘‘diet’’ of vulnerable consumers. Apartfrom endocrine disruptor analyses in the diet a clear insight into the endogenousestrogen background levels is needed. Recently, new data were presented usingsensitive gas chromatography high-resolution mass spectrometry (GC/HRMS)down to the 2 ng/L level in plasma samples [19]. It was shown that theendogenous levels in prepubertal children are much lower than previouslythought based on less specific immunoassays; as a result the diet-contribution tothe total exposure becomes much more critical and relevant. Within the scope ofthe EU project BioCop (phyto)estrogen levels have been assessed both in soy andcow’s milk products [20].

Several bioactivity-based approaches are feasible for the screening ofendocrine disruptors in the food chain. Apart from the SPR biosensor assaybased on binding with specific human transport proteins already mentioned [12],robust transcription activation bioassays are available for estrogens [21]. Theperformance of the latest generation based on recombinant yeast cells fulfilled allvalidation and ISO 17025 accreditation requirements and the results obtainedcompared very well with GC/MS data [22]. Also a highly challengingtranscriptomics approach is being explored within BioCop [20]. An MCF7 cellline is exposed to sample extracts. Next the messenger RNA (mRNA) is extracted


from the cells and the cDNA is hybridized on a microarray carrying 47,000human transcripts. Up and down regulation of specific transcripts was observedwhich will allow the design of dedicated microarrays having a limited number oftranscripts for endocrine disruptor fingerprinting. A major challenge will be toensure compatibility of real sample extracts with the cells, overall robustness andvalidation issues.

2.4 Metal speciation

The total content of the classical heavy metals, lead, cadmium and mercury infoodstuffs and animal feeding stuff, is regulated by EU maximum levels.However, for some trace elements their speciation is very important in terms offood and feed safety and information on the total content only does not giveadequate information for correct toxicological risk assessment. For example, inthe case of arsenic, the inorganic forms are the most toxic while for mercury,methylmercury is the most toxic form. Seafood is the major dietary source forarsenic and mercury in the European population [23]. For speciation analysis oftrace elements the use of either LC or GC with inductively coupled plasma MS(GC- and LC/ICPMS) is currently the state-of-the-art [24,25]. These techniqueshave been known for a couple of decades but their use is not routine yet,probably due to rather expensive instrumentation, the need for skilled personneland the lack of standardization, thus clearly defining some of the urgentchallenges. Another issue is the lack of rapid and simple field sensors forspeciation analysis which address at least partly the toxicity of the specific metalforms. In the past, different microbial biosensors were developed and evaluatedin aqueous model systems [26,27]. These transcription activation biosensorscontain a reporter gene under metal responsive element(s). Once the cell and theresponsive element detect a metal/metalloid the responsive element on the DNAwill switch on the transcriptional and translational machinery producing fireflyluciferase leading to light emission directly responding to the concentration.These metal biosensors determine intrinsically the bioavailable fraction ofthe metal, giving an estimation of the toxic potential and complementing thechemical methods where total concentration of the metals is determined. Theability of this type of biosensors to work in real food and feed extracts is a majorchallenge that still needs to be explored. Some people are hesitating to work withsuch genetically modified bacterial cell biosensors. To overcome these hesitationsluminescent cells immobilised onto a fibre dipstick might be applied to metal andmetal speciation analysis [28].

2.5 Alkaloid biotoxins

The EFSA is very interested in alkaloid biotoxins and has requested scientificopinions for ergot alkaloids (EA) in food, and for tropane and pyrrolizidinealkaloids (TA and PA) in feed; an opinion on EA in feed has been completedshowing that there is a lack of data on EA patterns in feed materials and on toxiceffects [29]. In particular ergotamine and ergocristine are of concern. PA are


widespread and can be found in many plant genera and therefore also in feed,food and herbs, jacobine and lycopsamine being the most abundant. TA, such asatropine and scopolamine, are mainly found in feed as contaminants from Daturaspecies. Plants producing TA have expanded dramatically in parts of Europe andproblems are emerging. Data on the sensitivity of animal species towards thealkaloids are incomplete and do not allow the establishment of tolerance levelsfor individual alkaloids and mixtures thereof; nevertheless the data available sofar indicate that adverse effects may occur in animals. The very limited and oftenincomplete data on tissue distribution and residual concentrations in edibletissues, milk and eggs do not allow an estimation of carry-over rates to food forhuman consumption. Data on human exposure and sensitivity towards thealkaloids are very incomplete and do not allow the establishment of tolerancelevels for individual alkaloids and mixtures thereof.

No harmonized methods are available yet, although different suggestionsranging from ELISA to thin layer chromatography (TLC) and LC/MS/MS can befound in literature [30]. A general issue in the determination of these alkaloids isa lack of reliable analytical standards, (certified) reference materials, proficiencyschemes and a lack of harmonized regulation.

2.6 Peptide and protein growth promoters

The use of growth promoters in food producing animals has been banned inmany countries since 1988 [31]. Thanks to harmonization efforts most of the EUmember states are capable of detecting steroids and b-agonists at the requiredlevel, although large differences in specific analyte coverage still exist. Hormonecriminality is believed to be linked with sports doping and to occur viainternational networks. As in sports doping it can be predicted that the abuse willshift from classical growth promoters such as steroids and b-agonists to peptidesand proteins when the veterinary control of the former becomes more effective.Bovine and porcine somatotropin (bST and pST), the equivalents of humangrowth hormone, are 22 kDa proteins and commercially available as recombinantpreparations. They are important endocrine factors influencing metabolic andsomatogenic processes including growth, immune function, reproduction andlactation. Their species specificity implies low toxicity in humans, apart frompotential rare allergic reactions. Major concerns are the observed increased levelsof insulin-like growth factor (IGF-1) in milk [32], which are connected to theincidence of cancer [33]. Detection via instrumental LC/MS/MS approaches isfeasible [34]. Recently a rapid SPR biosensor immunoassay has been proposed todetect somatotropins in illegal preparations [35]. However, routine sampling andanalysis of somatotropins and associated proteins in blood, milk and otherrelevant matrices is still a major challenge due to the complexity of theendogenous regulation (Figure 3), protein binding and the pulsatile secretion bythe pituitary gland. As a result the blood levels show a high intra- and inter-individual variability.

In view of this it is rather unlikely that measurement of a single parameter canprovide a validated tool for the enforcement of the ban on somatotropin and


related growth promoters. A huge challenge is envisaged that an assembly ofparameters has to be measured followed by appropriate statistics in order tovalidate its diagnostic value versus untreated control populations.

2.7 Genetically modified organisms (GMO)

Within the framework of safety assessment of food produced by means ofmodern genetic engineering the comparative analysis of the genetic modified(GM) crop with its unmodified genetic counterpart is an important part [36]. Twoapproaches are followed to detect intentional or unintentional alteration of thechemical composition of the GMO, i.e., a targeted approach (i.e., investigatingdefined constituents) and an untargeted approach using profiling technologies toanalyse differences in RNA, proteins and metabolites [37]. Targeted analysis ofsingle compounds with focus on important nutrients and critical toxicants hasbeen successfully applied to demonstrate the safety of GM foods. Some criticismshave been expressed on the targeted approach as being biased and focused onknown compounds and expected results [38]. This may become increasingly aproblem in the second generation of GM crops where the introduction ofcompletely new biosynthetic pathways or modifications in key enzymes in theprimary or secondary structure may result in unexpected changes. Non-targetedapproaches using profiling technologies based on DNA (e.g., microarraymethods), proteins (two-dimensional gel electrophoresis followed by MSand/or metabolites (using e.g., nuclear magnetic resonance (NMR), GC/MS and LC/MS) are nowadays explored for their potential within the food safety assessmentof GM food crops.

Figure 3 Endogenous regulation along the somatotropin (growth hormone) axis.


In the EU the release of GMO and GMO-derived ingredients into theenvironment, and the marketing of GMO-derived food and animal feed isregulated within various specific directives and regulations. The EU regulations1829/2003 and 1830/2003 concern the premarket safety assessment and thetraceability and labeling of food and feed products derived from authorizedGMOs establish that food or feed materials containing GMO-derived ingredientsabove the set threshold of 0.9% must be labeled as such. Implementation andenforcement of this regulation is generally performed by PCR-based methodsusing GMO-specific DNA probes. These methods must meet a number of agreedquality requirements. On the other hand, the presence of unauthorized GMO isnot allowed at all in food and feed. In these cases, qualitative methods, such asDNA microarray methods may in the near future become very useful since thesemethods allow the detection of many different GMOs and GMO elements in oneanalysis, albeit their quantitative performance is limited [39]. Major challenges inGMO detection are quantitative aspects, validation and harmonization. Knowl-edge about the measurement uncertainty at the level of 0.9% is crucial already,and once a minimum required performance level (MRPL) has been assigned tothe ban on unauthorized GMO that challenge will become immense.

2.8 Nanoparticles

Application of nanoparticles — particles with one or more dimensions at thenanoscale — in food, medicine and agricultural products is booming and manynanobased products are already on the market [40]. Inherent to their size andsurface-to-volume ratio nanoparticles often show a high chemical reactivity. Thequantum-size and Coulomb-charging effects of nanoparticles may yield particleswith exceptionally electric conductivity or resistance, high capacity for storing ortransferring heat or changed solubility properties. Within food technologynanoparticles are used in food conservation, dietary supplements, food additives,packaging materials, functional foods and intelligent food. Some typicalexamples of application in the food area can be found: bakeries in WesternAustralia have incorporated nanocapsules containing omega-3 fatty acids inbread, the capsules will open only in the acidic environment of the stomach; thecompany NutraLeaset utilizes micelles with a diameter of 30 nm to deliverlycopenes, beta-carotene, lutein, phytosterols, CoQ10 and omega-3 fatty acids;Unilever is developing low fat ice creams by decreasing the size of the emulsionparticles and The Oilfresh Corporation marketed a new nanoceramic productthat prevents the oxidation and agglomeration of fats in deep fryers. It isenvisaged that within agricultural practices nanoparticles can be used forprecision farming, meaning that autonomous nanosensors are applied for real-time monitoring and early warning of plant health issues, and for controlledrelease of pesticides and herbicides encapsulated in nanomicelles. Some of thesedevelopments already come into reality are: Syngenta is using nanoemulsions inits pesticide products and also marketing the microencapsulated productGutbusters that releases its content in the alkaline environment of the insectstomach; and a growth-promoting product PrimoMaxxs is used to strengthen


the physical structure of turfgrass. The unique properties of nanoparticles makethem attractive in the applications mentioned previously but also impose newunforeseen risks, hence making an evaluation of the appropriateness of thecurrent risk assessment protocols and methods necessary. The appropriateness ofrisk assessment methodologies currently in place to deal with the new propertiesof the nanoparticles is being addressed by different authorities. Current methodsfor the identification of nanoparticle hazards are probably adequate but themethods for characterization of the hazard (i.e., establishment of toxicologicaldose-response relationships) and subsequent exposure assessment need to beadapted. Improvement of current assessment methodology should consider thefollowing aspects: (i) physical parameters such as particle size, size distribution,surface charge, (ii) agglomeration and disagglomeration properties in differentenvironments, (iii) impurities within, and adsorbed species onto the surface and(iv) biological processes involving nanoparticles, including translocation, cellularuptake and toxicological mechanisms.

The analytical challenge for the required monitoring of nanoparticles in thefood chain is immense. A vast array of analytical techniques is typically used forthe characterization of the physical properties of manufactured nanoparticles: themean size and its distribution is measured by techniques like photon correlationspectroscopy (PCS), laser diffractometry (LD), light scattering (LS), differentialmobility analysis, TOFMS and microscopy, while electrophoresis is typically usedto determine the particle charge density [41,42]. The crystalline structure of ananosuspension can be assessed by differential scanning calorimetry (DSC) [41],polarized optical microscopy and scanning electron microscopy [43]. Electronspectroscopy for chemical analysis (ESCA), X-ray photoelectron spectroscopy(XPS), secondary ions mass spectroscopy (SIMS) and matrix assisted laserdesorption ionization MALDI TOFMS are used for surface structure andchemical composition analysis of nanoparticles [42]. It should be stressed thatso far the key issue of isolation and sample preparation of nanoparticles fromfood or feed samples has been hardly addressed. Definitely it will be verydifficult to maintain the integrity of the nanoparticle during sample preparationand the subsequent analysis. Apart from that, many of the characterization toolsemployed and cited previously will not be easily implemented within a routinefood control environment.

3. MASKED CONTAMINANTS

A masked contaminant is defined as a contaminant present in such a form that itwill escape from rapid screening or instrumental analysis and remainundetected. A contaminant might be conjugated with carbohydrates, sulfates,amino acids, fatty acids or bound to proteins or nucleic acids in the sample ofinterest. Rapid screening tests such as immuno or receptor assays are based onmolecular recognition and will fail recognition when the binding sites of themolecule become less accessible due to modification or steric hindranceelsewhere in the molecule. Chromatographic and mass spectrometric methods


will fail as well due to retention time shifts and changes in m/z. A typicalexample is the occurrence of glycosylated Fusarium mycotoxins in wheat andmaize, as recently shown for deoxynivalenol (DON) and zearalenone byBerthiller et al. [44,45]. In addition to that it was shown that the ratio betweenfree and glycosylated mycotoxins can change during fermentation processes, forexample, in beer breweries [46]. It can be assumed that all biotoxins arevulnerable to some degree of conjugation and escape at least partly fromdetection, unless appropriate modifications to sample preparation schemes arebeing implemented. Risk assessments and intake data of biotoxins should bereconsidered taking this phenomenon into account, but first of all appropriateanalytical methods should become available.

Another example is residues of the nitrofuran antibiotics, the use of which hasbeen prohibited within the EU because of their potentially harmful effects onhuman health. Former analysis of the parent drugs turned out to be completelyinappropriate since they do not persist in edible tissues due to rapidmetabolization. The nitrofuran metabolites on the other hand form persistingprotein-bound residues which will remain undetected. Conneely et al. [47]described a method for the determination of the total content of nitrofuranmetabolites in tissues incorporating an acidic hydrolysis combined with anitrobenzaldehyde derivatization step. The method was validated for porcineliver at the 5-ng/g level. Subsequent application to poultry had a global impact:high percentages of non-compliant samples were detected and an MRPL wasassessed by the EC for harmonization of residue control in importing andexporting countries.

4. UNKNOWN BIOACTIVE CONTAMINANTS

Unknown bioactive contaminants might emerge in the food chain due to climatechanges, illegal production methods such as the application of designer steroidsand b-agonists in cattle fattening or even because of an act of terrorism.Irrespective of the origin, they will remain undetected and escape from control aslong as contaminant monitoring is restricted to a defined list of target substances.At least three different approaches can be distinguished for the detection andmass spectrometric identification of unknown contaminants, a bioactivity-directed, a metabolomics-like and an in silico prediction approach.

4.1 Bioactivity-directed identification of emerging unknowncontaminants

Ideally, the sample should be extracted and purified in such a way to maintain allrelevant contaminant classes of interest, the first challenge being clearly in thefield of generic sample preparation development. The sample extract is analysedfor bioactivity using a suitable assay (whole cell-, receptor-, immuno-,microbiological inhibition assay, etc.). When the sample has been screenedsuspect in one of these assays and the cause cannot be identified using the


prescribed confirmatory analysis method targeted for the presence of knowncontaminants, then possibly an emerging unknown contaminant is present in thesample. Next the unknown can be identified using the generic experimental set-up shown in Figure 4. In short, the suspect sample is fractionated by gradient LCand narrow fractions are collected in duplicate using a parallel 96-well plate set-up. One plate is re-analysed for bioactivity using the original bioassay yieldinga bioactivity chromatogram or ‘‘biogram’’. The duplicate well number(s) ofthe suspect positions in the biogram are subjected to full-scan LC/TOFMS orGC/TOFMS techniques in order to perform chemical identification of the bio-active unknown, preferably using accurate mass measurement and isotope fittingwhich allows elemental composition assessment of the molecular ion and itscollision-induced dissociation fragments. This approach has been demonstratedfor cell-, immuno- and SPR biosensor assays and applied successfully to theidentification of b-agonists in feed, hormones in urine and antibiotics in chickenmuscle [48–50]. A key issue in this approach is the biocompatibility between theLC fraction and the subsequent bioactivity measurement. In this respect we havegood experience with wells filled with a mixture of 2 mL dimethylsulfoxide(DMSO) and 50 mL water as a keeper solvent prior to the fractionation. The

Sample pretreatmentand clean-up

Gradient Liquid Chromatography

Bioassay plate

Collection plate

Optional on-line(Q)TOF MS(/MS)

Identification off-line

UPLC-(Q)TOFMS(/MS)GC-TOFMS

Bioactivity screening

Flowsplit

Figure 4 Generic set-up for bioactivity-directed identification of emerging unknown

contaminants.


water–acetonitrile eluate from the LC gradient yields a large number of filledwells which are simply evaporated overnight in a fume hood, leaving thesubstances of interest in a tiny volume of water/DMSO. Of course, an LCfractionation is also directly compatible with atmospheric pressure ionizationMS. That option requires only one well plate for fraction collection andbioactivity assessment and the suspect well number is then simply correlatedwith the retention time in the parallel MS system [51].

4.2 Metabolomics approach to the identification of emerging unknowncontaminants

Another sophisticated means of identification of unknown contaminants is basedon a metabolomics-like approach. Again the sample should be extracted andpurified in such a way to maintain all relevant contaminant classes of interest.Next the sample, still being a highly complex mixture, is analysed by a high-resolution chromatographic technique such as ultra performance liquidchromatography (UPLC), GC or even comprehensive GC�GC, combined witha sensitive full-scan MS technique such as TOF, ion trap or FT Orbitrap. The datafrom the sample replicates are aligned in the retention time domain andcompared with the data from a set of reference sample replicates. Finally uni- ormulti-variate statistics are applied in order to assess the significant differencesbetween the suspect sample and the regular reference situation. By usingappropriate data analysis software contaminants could be retrieved automati-cally from an oily preparation, drinking water and grass samples [52].

Successful application of this approach requires the availability of a clean,stable and highly reproducible chromatography system, including reproduci-bility in solvent and column impurities. Moreover, the reference situation iscrucial, requiring a more or less reproducible sample matrix background. Forhomogeneous samples such as drinking water this can be relatively easilyachieved but an adequate reference for inherently inhomogeneous sampleshaving a fluctuating composition such as feed will be very difficult to obtain. Lastbut not least, intelligent data analysis software is required which canautomatically correct for small changes in retention time and/or mass accuracyand is capable of keeping the underlying raw data accessible for retrospectiveanalysis, reprocessing and evaluation in its original software format. Note that aGC�GC/TOFMS analysis creates a challenging four-dimensional dataset.Ignoring all these factors will yield many irrelevant data from system impurities,bulk composition changes, etc., and probably not identify the unknown emergingcontaminant.

4.3 In silico prediction approach to the identification of unknowns

Starting from a chemical core structure of a specific contaminant or contaminantclass and having knowledge about reactivity at specific atom positions in thatcore structure, one can predict in silico the chemical structure of unknowncontaminants, metabolites and emerging risks. On the basis of such a structure


physicochemical parameters such as solubility, polarity, acidity can be estimatedas well, thus providing a basis for the prediction of chromatographic and massspectrometric behaviour of the unknown. Finally, GC- or LC/MS techniques canbe used to perform a targeted search on the presence of the predicted unknownin samples of interest. Recently we demonstrated the feasibility of this approachby a UPLC/TOFMS search for in silico predicted metabolites and designermodifications of glucocorticosteroids in urine [53]. Even the potential modifica-tions can be automated: we modified a commercially available software packagefor metabolite finding in such a way that all kinds of chemical modifications wereautomatically searched for in the TOFMS dataset yielding a listing of both knowncompounds already present in a user library and unexpected options caused by acombination of different reactions relative to the core structure, all supported byelemental compositions thanks to the accurate mass measurement.

5. EMERGING TECHNOLOGIES

5.1 Omics

Genomics, proteomics and metabolomics (‘‘omics’’) are extremely valuable toolsin studying biological processes, in bio- and disease marker discovery, in drugdiscovery, in nutrition and toxicology. Generally, cells, plants or animals areexposed and divided into a treated and an untreated group. Following theexperiment both groups are analysed at the mRNA, the protein or the metabolitelevel using DNA microarrays, 2D-gel electrophoresis plus MALDI/TOFMS orLC/MS, NMR plus GC- or LC/MS, respectively. Next the differential regulationof thousands of targets is assessed using appropriate statistics. Usually bothexperimental groups are well-defined and identical, except for the treatment, andthe dose of exposure is relatively high as compared to levels normallyencountered in food contaminant and residue analysis. On the other hand,biomarkers thus obtained might be used for the development of dedicatedscreening assays based on PCR, tailored DNA microarrays, receptor orimmunoassays. Assuming that such screening assays will be developed at leasttwo scenarios can be distinguished: (i) exposure of a standard cell system ororganism to the food sample extract of interest followed by isolation of mRNA,proteins or metabolites from the cells and analysis using the developed dedicatedscreening assay and (ii) isolation of mRNA, proteins or metabolites directly frombiofluids in the food sample, usually restricted to farm animals and crops. Amajor challenge would be to obtain, purify and maintain the integrity of arepresentative isolate. But most important of all will be the validation of thebiomarker targets versus their natural background variability in food, feed andbiofluid matrices: real-life is quite different from standard cells or organisms! Theon-going European project on new technologies to screen multiple chemicalcontaminants in food, acronym BioCop, has taken this challenge and is studyingboth transcriptomics and proteomics for chemical food contaminant analysis [20].An MCF-7 standard cell line is used and exposed with food sample extracts for


phytoestrogen and mycotoxin analysis, next the extracted mRNA from the cells isanalysed by tailored DNA microarrays. In the proteomics topic of BioCop amultiplex SPR biosensor immunoassay is being developed to analyse proteinbiomarkers in blood of bovines for the screening of steroid abuse in cattlefattening.

5.2 Bioassays

The acceptance and application of whole-cell bioassays in routine chemical foodcontaminant analysis is progressing slowly but steadily. Typical examples are theDR-CALUXs bioassay and hormone reporter gene bioassay. The principle ofsuch assays is illustrated in Figure 5: Genetically modified cells are exposed to thesample extract containing the contaminant(s). The cells are modified in such away that they express the receptor protein of interest, in this example the arylhydrocarbon (Ah) receptor which binds to dioxins and PCBs. Upon bindingthe receptor–ligand complex travels into the cell nucleus and binds onto themodified DNA at specific responsive elements which act as a switch for theactivation of a gene encoding for the production of a marker protein such asluciferase or green fluorescent protein (GFP); as a result the cells will generatelight upon exposure, even at trace levels [54]. Cell assays can be performed inparallel in 96-well plates and require hardly any reagents. Only the suspectsamples will require confirmatory analysis using expensive instrumental analysismethods such as GC/HRMS in the case of dioxins. A key issue in the applicationto food and feed samples is the stability of the cells. In general, mammalian cellsseem to be more vulnerable to cell toxicity caused by matrix components andrequire a more stringent sample clean-up in contaminant and residue analysis.Yeast cells on the other hand are inherently more robust, allowing application to

Figure 5 Schematic representation of the mechanism of the DR-CALUXs. Reproduced and

kindly provided by T.F.H. Bovee [54].


residue analysis of estrogens in feed and urine samples following a simple solid-phase extraction step [54].

5.3 (Bio)nanotechnology

The nanosciences intend to deliver radical new products and processes atnanoscale dimensions by manipulation of surfaces and macromolecularassemblies. In 2005, Bruce et al. [55] reviewed the role of nanotechnology infood analysis but no real-life application of (bio)nanotechnology in chemical foodcontaminant analysis was presented. Since then the situation is not so muchdifferent. However, it should be noted that any SPR biosensor instrument alreadyused in chemical food contaminant analysis is actually an example ofbionanotechnology since the biointeraction is measured within a distance of150 nanometre of the functionalized surface and the flow cells of the microfluidicsystem are typically in the order of some tens of nanolitres. For example, byapplying SPR, Farre et al. [56] showed part-per-trillion sensitivity for thepesticide atrazine in natural water samples and Haasnoot et al. [57] showed theSPR determination of sulfonamide antibiotics in broiler serum and plasma as aprediction tool for residue levels in edible tissues. Another option is the use oflocalized surface plasmons (LSPs) sustained by functionalized silver or goldnanoparticles: a highly specific, label-less immunosensor has been constructedfor the residue analysis of the anabolic steroid stanozolol down to the nanomolarrange [58]. The nanoscale format of SPR can also be coupled successfully tonanoLC/TOFMS as demonstrated for the residue analysis of the antibioticenrofloxacin in chicken muscle [59]. Gold nanoparticles can also be applied toenhance the performance of immunochromatographic strip tests (‘‘dipsticks’’) asshown by Tanaka et al. [60]. A special application of nanoparticles in thedetection of chemical food contaminants will be the use of quantum dots (QDs).QDs are semiconducting crystals of a few nanometres and have uniquephotophysical properties such as size-tunable luminescence spectra, highquantum yields, broad absorption and narrow emission wavelengths. As aresult they will be increasingly used as fluorescent labels in, for example,immunoassays [61], also in chemical food contaminant and residue analysis.Apart from detection, nanomaterials might be used in sample preparation aswell. Zhao et al. [62] used carbon nanotubes as a solid-phase extraction adsorbentfor the GC/MS/MS analysis of barbiturate drug residues in pork at sub-ppblevels.

5.4 Ambient mass spectrometry

In 2004 a novel ambient ionization technology, called Desorption ElectroSprayIonization (DESI), was proposed by the Cooks group at Purdue University[63,64]. For the first time, surfaces can be probed and analysed by MS under realnative ambient conditions, without any sample preparation or matrix addition.The typical lay-out of a DESI source is shown in Figure 6. In short, apneumatically assisted electrospray is used to produce charged microdroplets


and gas-phase solvent ions that are directed onto the sample at a surface. Theionization process closely resembles conventional ESI-MS; however, the sampleis not present in the solvent nor ionized during the electrospray process, and istherefore less vulnerable to ionization suppression caused by the presence ofsalts and other interfering matrix components. Instead, ionization of the samplemolecules takes place at or above the surface as a result of proton transfer,electron transfer, or ion transfer by gas-phase ions [65]. Reagents can be addedto the electrospray solvent in order to tune the selectivity and the sensitivity ofthe DESI process [66]. That versatility is a great advantage over the directanalysis in real time (DART) ionization alternative in which helium or nitrogenis used as a reaction gas [67]. Depending on the capabilities of the MS, targeted(selected ion mode), untargeted (full-scan mode) or accurate mass analysis ispossible, yielding for example, new biomarkers from differential metabolomicswith the aid of principal component analysis (PCA) [68]. The ion transfer line tothe atmospheric inlet of the MS can be extended and integrated with theelectrospray nozzle, thus creating a probe for in situ sensing of any native or cutsurface. Widely variable substrates such as paper, metal, Teflon, human skin,animal organs such as pancreas, liver and brain, plant parts such as stems,seeds, flowers and roots, glass, leather, bricks, polymers (Nylon, latex, PDMS)and silica gel have been sampled successfully. Measurements take on average 3 sbut high-throughput analysis (3 samples/s) with a moving belt has also beendescribed [69]. Automated chemical imaging of surfaces is possible. Dependingon the dimensions of the electrospray tip and the distance to the surface, spotsizes will vary from 2� 2 mm down to 50� 50 mm. In terms of analytes, thetechnique is more versatile than standard ESI-MS, as also highly non-polarmolecules such as carotenoids, steroids, terpenes and even volatiles can beanalysed. The sensitivity of the technique lies in the low picogram or high

Sample

inlet

ofMS

electrospray

moving sample stage in air

desorbed ions

Solvent

HV

N2

Figure 6 Schematic view of a DESI source.


femtogram range. DESI-MS is also believed to be quantitative but we have somereservations in that respect. So far, the applicability of DESI technology has beendemonstrated in forensics (detection of explosives, of drug residues onbanknotes, Ecstasy), pharmaceutical [69–71], plant sciences (in vivo analysis ofplant alkaloids) [72], and clinical analysis, diagnosis of cancerous tissue [73] andmetabolomics [68].

The option of direct surface analysis of plant alkaloids, pesticide residuesand natural toxins will be very attractive for chemical food contaminant analysis.For complex, mixtures the combination of TLC and DESI can be considered, asrecently demonstrated for alkaloid dietary supplements [74].

6. VALIDATION AND QA/QC CHALLENGES

The validation requirements for contaminants and residue analysis in the foodchain are laid down in different international regulations. Some contaminantplus sample matrix groups are using the concept of normalized andharmonized reference methods, in other situations the requirements are limitedto method validation and data interpretation issues. In any case, the number ofavailable certified reference materials and interlaboratory studies is far too low,mainly due to financial limitations and other priorities. Two issues areparticularly challenging: (i) the validation of biochemical or biological rapidscreening methods and (ii) the validation of generic comprehensive instru-mental methods.

6.1 Biochemical or biological rapid screening assays

Validations for classical instrumental chemical analysis methods are relativelystraightforward, but the introduction of (multiplex) biochemical or biologicalrapid screening assays requires a new fit-for-purpose approach. Typically thedata output is generating qualitative or semi-quantitative results, while the scopeof bioactive analytes covered might be rather diverse in terms of physicochemicalparameters. As a pragmatic solution we applied the following validationprocedure to the validation of a whole-cell estrogen bioassay: first, five differentbioactive estrogen representatives were selected ranging from polar to apolarchemistries assuming that both known and unknown estrogens would havephysicochemical characteristics within that range. Then, representative samplematrices, in this case calf urine and three different types of feed matrix, andacceptable spiking levels were defined based on current legislation and/orexpected sensitivity of the bioassay for that particular compound. For eachsample matrix both unspiked and samples spiked with the representativeindividual estrogen were analysed. By doing so the unspiked samples werealways screened compliant while the spiked samples were always screenedsuspect non-compliant at the levels chosen, allowing validation as a quali-tative screening method and assessment of CCa and CCb values according to2002/657/EC; even ISO 17025 accreditations could be obtained [75,76]. Moreover,


as shown in Figure 7 routine operation of the validated bioassay showed that thenon-compliant control sample was always screened above the CCa decision limitand declared non-compliant while the compliant blank was only screened twotimes false non-compliant over a two-year period, despite the inherent signalvariability of such a bioassay [22]. In other words, even a biological assay canprovide consistent results as an on/off screening tool in residue and contaminantcontrol.

6.2 Comprehensive instrumental analysis methods

Thanks to the introduction of highly sensitive ion trap and TOF mass analysersin GC/MS and LC/MS full-scan positive and negative ion data of the residuesand contaminants in a particular sample of interest can be easily obtained,provided that the analytes are effectively extracted and survived the clean-upstep, the injection, chromatographic separation and ionization. Then the sky isthe limit and hundreds of residues and contaminants can be searched for. QA/QC considerations will require the co-analysis of control samples, and thenparticularly the preparation of a non-compliant control sample having a knownconcentration of all analytes of interest will be a huge task. The co-analysis ofsuch a control sample will be crucial for the assessment of false compliantresults, especially because in the end automated raw data processing andevaluation will be the only option to handle the screening for hundreds ofresidues and contaminants. It should be noted that state-of-the-art raw dataprocessing and evaluation software will function quite well in academicstandards but most often fail in real samples at residue or contaminant level.

-200

0

200

400

600

800

1000

# control samples

resp

on

se u

nit

schem + 1 ng/ml 17bE2

urine + 1 ng/ml 17bE2

blank chem

blank urine

CCα

July 2003 May 2005

Figure 7 Long-term performance of a validated estrogen bioassay for blank and estradiol-

containing control samples [22].


Considering validation of the total analysis including data processing it mightbe questioned whether the immense efforts of a full validation for all thesehundreds of analytes in all sample matrices of interest is really required. Inanalogy with Section 6.1 a set of a limited number of representatives might bechosen for an initial in-house validation covering a range of physicochemicalanalyte characteristics and/or contaminant (sub)groups. Ideally, the choice ofthese representatives should be validated by performing at least a within-dayreplicate analysis experiment of blanks and control samples containing allanalytes of interest. Additional contaminants of interest might be added to thecontrol sample during routine operation thus providing a continuous extensionof the scope of the method.

7. CONCLUSIONS AND FUTURE TRENDS

The current use of routine monitoring systems for a limited number of chemicalfood contaminants and residues in food industry and food control laboratoriesdoes not mean in any way that the challenges we are facing are less, they simplyevolved under the influence of globalization, new food technologies, climatechange, etc. Not surprisingly different stakeholders have different wishes forimprovements in chemical food contaminant and residue analysis: in industrypriority will be at the implementation of rapid and inexpensive methods having ascope for those substances which are of paramount importance to their specificcore product integrity. Recognized challenges are further increase of speed andreduction of costs, and extension of the scope. Despite their primaryresponsibility for food safety as laid down in the General Food Law, it cannotbe reasonably expected from the food industry that they take care of anyemerging known or unknown risk, which might pop up somewhere in the foodchain. On the other hand, some of the emerging issues raised in this chapter willcause a problem sooner or later, requiring the availability of analysis methodshaving a comprehensive scope in order to provide an early warning to the foodsafety authorities and generate the data for risk assessment. We believe thatbioactivity-based screening methods and full-scan accurate mass spectrometricidentification methods are highly complementary technologies and both essentialin this respect. It is recognized that still major efforts should be put to genericsample preparation and automated data processing and evaluation before suchcomprehensive tools can be routinely applied in a real-life environment. CurrentEU monitoring plans are restricted to a limited list of target contaminants andresidues in a limited number of samples. Also current validation requirementsare typically developed for target analysis. The advocated more comprehensivescope of analysis does not fit easily into such policies. It is recommended that atleast a part of the associated efforts and resources is moved towards the actualapplication of comprehensive methods for known and unknown emerging foodcontaminants.


ACKNOWLEDGEMENTS

Partners in EU-framework projects SAFEFOODS, BioCop (www.biocop.org) and CONffIDENCE(www.confidence.org), and colleagues at RIKILT-Institute of Food Safety are acknowledged for theirstimulating contributions.

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