Anal Bioanal Chem (2012) 402:139–162

DOI 10.1007/s00216-011-5237-3

REVIEW

Challenges and trends in the determination of selected

chemical contaminants and allergens in food
Rudolf Krska & Adam Becalski & Eric Braekevelt & Terry Koerner & Xu-Liang Cao &
Robert Dabeka & Samuel Godefroy & Ben Lau & John Moisey & Dorothea F.K. Rawn &
Peter M. Scott & Zhongwen Wang & Don Forsyth

Received: 3 June 2011 / Revised: 3 July 2011 / Accepted: 4 July 2011 / Published online: 20 July 2011
# Her Majesty the Queen in Right of Canada 2011

Abstract This article covers challenges and trends in the ants and persistent organic pollutants, and perchlorate as an
determination of some major food chemical contaminants and environmental contaminant. Ochratoxin A, fumonisins, and
allergens, which—among others—are being monitored by paralytic shellfish poisoning toxins are examples of naturally
Health Canada’s Food Directorate and for which background occurring toxins whereas sulfites, peanuts, and milk
levels in food and human exposure are being analyzed and exemplify common allergenic food additives/ingredients.
calculated. Eleven different contaminants/contaminant groups To deal with the increasing number of sample matrices
and allergens have been selected for detailed discussion in this and analytes of interest, two analytical approaches have
paper. They occur in foods as a result of: use as a food additive become increasingly prevalent. The first has been the
or ingredient; processing-induced reactions; food packaging development of rapid screening methods for a variety of
migration; deliberate adulteration; and/or presence as a analytes based on immunochemical techniques, utilizing
chemical contaminant or natural toxin in the environment. ELISA or surface plasmon resonance technology. The second
Examples include acrylamide as a food-processing-induced is the development of highly sophisticated multi-analyte
contaminant, bisphenol A as a food packaging-derived methods based on liquid chromatography coupled with
chemical, melamine and related compounds as food adulter- multiple-stage mass spectrometry for identification and
simultaneous quantification of a wide range of contaminants,
often with much less requirement for tedious cleanup
Published in the 10th Anniversary Issue. procedures. Whereas rapid screening methods enable testing
A. Becalski : E. Braekevelt : T. Koerner : X.-L. Cao : R. Dabeka : of large numbers of samples, the multi analyte mass
B. Lau : J. Moisey : D. F. K. Rawn : P. M. Scott : Z. Wang : spectrometric methods enable full quantification with confir-
D. Forsyth (*) mation of the analytes of interest. Both approaches are useful
Food Research Division, Bureau of Chemical Safety,
when gathering surveillance data to determine occurrence and
Food Directorate, Health Canada,
251 Sir Frederick Banting Driveway, AL: 2203D, background levels of both recognized and newly identified
Ottawa, ON K1A 0K9, Canada contaminants in foods in order to estimate human daily intake
e-mail: [email protected] for health risk assessment.
S. Godefroy
Food Directorate, Health Products and Food Branch, Keywords Food safety . Chemical contaminants .
Health Canada, Acrylamide . Bisphenol A . Melamine . Perchlorate .
251 Sir Frederick Banting Driveway, AL: 2203D, Sulfites . Persistent organic pollutants . Mycotoxins .
Ottawa, ON K1A 0K9, Canada
Phycotoxins . Allergens
Present Address:
R. Krska
Department for Agrobiotechnology (IFA-Tulln), Introduction
Center for Analytical Chemistry, University of Natural Resources
and Life Sciences Vienna,
Konrad Lorenz Straße 20, Food safety has become of increasing concern for consumers,
3430 Tulln, Austria governments, and producers as a result of the global
140 R. Krska et al.

marketplace where foods are produced and distributed This article covers challenges and trends in the determina-
throughout the world and also because of an increase in tion of selected food allergens and chemical contaminants,
public awareness of health and quality. Several worldwide which—among others—are being monitored by Health
incidents related to chemical contaminants in food have also Canada’s Food Directorate and for which background levels
attracted much media attention. Trace levels of chemical in food and the human exposure to these chemicals are being
contaminants in foods can originate from natural sources analyzed and calculated.
(for example mycotoxins [1, 2] and phycotoxins [3, 4]), Eleven different chemical contaminants/contaminant
environmental contamination (for example PCB [5], dioxin- groups and allergens have been selected for detailed dis-
like compounds [6], pesticide residues [7], and perchlorate cussion in this paper. They are all introduced into foods at
[8]), migration of chemicals from packaging materials some point in the food supply continuum and are listed below:
(for example phthalates and bisphenol-A) [9], veterinary
& acrylamide, as an example of a food-processing-induced
drug residues [10], by-products from food processing
contaminant
(for example acrylamide [11, 12]), and other intentional
& bisphenol A, as an example of a food packaging-derived
and unintentional adulteration (for example melamine [13] in
chemical
milk products and ethyl carbamate in wine [14]). Further-
& melamine and related compounds, as examples of food
more, instances of emerging contaminants, for example
adulteration
perfluorinated organic compounds [15] entering the food
& persistent organic pollutants and perchlorate, as examples
supply are also increasing. The occurrence of allergens in
of environmental contaminants in food
food is another important topic in food safety, resulting in the
& ochratoxin A and fumonisins, as examples of mycotoxins,
need to verify that the food labelling information on the
i.e. natural toxins produced by fungi
product is accurate and enables consumers to make informed
& paralytic shellfish poisoning toxins, cylindrospermopsin,
choices about their diet.
and β-methylamino-L-alanine, as examples of phycotox-
In Canada, Health Canada’s Food Directorate is the federal
ins, i.e. natural toxins produced by algae
health authority responsible for establishing policies, setting
& sulfites, as examples of allergenic food additives
standards, and providing advice and information on the safety
& peanuts, milk, and other allergenic food ingredients
and nutritional value of foods available for sale. An important
which exemplify the need for accurate food labelling
part of the Food Directorate’s mission is to ensure that human
exposure to chemical contaminants and residues in food,
whether from natural or man-made sources is kept to a
minimum and that it is not detrimental to Canadians’ health. Acrylamide
To support its food safety standard-setting activities and to
provide information about the background levels of these Acrylamide is currently the most actively investigated
chemicals and of possible hidden or undeclared allergens in chemical among process-induced food toxicants. The discov-
foods, it is essential for Health Canada’s Food Directorate to ery of acrylamide in heat-treated foods [16] led to many
analyse the food samples using sensitive, fast, and accurate subsequent investigations related to its formation and
analytical methods. This requirement, in conjunction with the presence in foodstuffs [17, 18]. The intensity of this research
increasing number of sample matrices and analytes of interest, is because acrylamide is a rodent carcinogen [19], a human
has led to the development of both rapid screening methods neurotoxin [20] and is classified as a probable human
for a variety of analytes, mostly based on immunochemical carcinogen [21].
techniques, and of highly sophisticated multi-analyte methods Owing to the ubiquity of asparagine and sugars—the most
based on liquid chromatography coupled with multiple-stage common reactants required for formation of acrylamide—
mass spectrometry, to enable identification and simultaneous acrylamide was detected in many heat-processed foods at
quantification of a wide range of contaminants. The Food levels ranging from sub-μg kg−1 to over 5000 μg kg−1. The
Research Division of the Bureau of Chemical Safety potential human health risk of acrylamide is still being
within Health Canada’s Food Directorate maintains an assessed and, although a maximum allowable concentration
active research program studying the distribution, sources, in foods has not been promulgated, efforts to minimize its
and potential effects of food processing on chemicals in formation are in progress, as also is continuous monitoring
food, conducts major national surveys, for example the of its concentration in the relevant food categories [22].
Canadian Total Diet Study, in support of science-based Acrylamide is a moderately reactive, very polar, α,β-
risk management approaches, when warranted, and unsaturated amide (Fig. 1). It is relatively stable at room
engages in a range of research activity related to the temperature in typical food matrices, except for roast and
determination and characterization of chemical contaminants ground coffee, in which significant degradation has been
and allergens in food. observed over time during storage [23]. Exposure of food
Challenges and trends in the determination of selected chemical contaminants and allergens in food 141

Fig. 1 Chemical structure

of acrylamide summarized current methods used for analysis of acrylamide
in coffee.
For methods employing LC–MS or LC–MS–MS cleanup
usually consists of sequential SPE using a variety of
cartridges. The combination of Oasis HLB and Bond Elut
Accucut has been used most frequently [36]; however, a
detailed study of SPE sorbents reported a superior combina-
samples to elevated temperatures within a laboratory tion consisted of Isolute Multimode and ENV+cartridges
environment should, however, be avoided and samples [29]. Figure 2 shows the ESI(+)LC–MS–MS selected
should be stored at temperatures<−15°C [24, 25]. reaction monitoring (SRM) chromatogram (also called
A plethora of analytical methods has been developed to multiple reaction monitoring (MRM) by some suppliers) at
measure acrylamide in foods over the past decade. With the a contamination level of 110 ng acrylamide g−1 corn
exception of a few methods utilizing non-MS detection, most obtained from a regular corn chip sample after extraction
methods employ isotope dilution with 13C3-acrylamide and cleanup in accordance with Ref.[30]. As quantifier
or 2H3-acrylamide with GC–MS and LC–MS or LC–MS– transition the protonated precursor ion (M+H+)→m/z 55
MS detection [26–28]. The use of a 13C3 labelled isotope is was used.
beneficial when using bromination/dehydrobromination Extraction of coffee and chocolate samples by pressur-
derivatization procedures [27], and when the deuterium ized fluid extraction using acetonitrile with concomitant in-
isotope effect [29] could affect the accuracy of the analysis situ filtration through Florisil afforded extracts that did not
(in cases when only selected column fractions are collected require any subsequent cleanup before LC–MS–MS analysis
during cleanup). [37].
Owing to the extreme solubility of acrylamide in water Chocolate, cocoa, and, especially, coffee, remain as
(~200 g/100 mL), water is the solvent most widely used for its matrices for which new and improved methods are still
initial extraction. The main disadvantage of aqueous extrac- being developed with the objective of achieving greater
tion is the formation of severe, viscous colloids with starchy robustness. It is often difficult to compare the quality of the
foods. These colloids can be completely removed by data because chromatograms of samples with the concen-
ultrafiltration with a 5-kDa molecular filter [30]; however, tration of acrylamide close to the detection limit and
care should be exercised because some batches of filters depicting data from the quantification and confirmation
were found to harbour low-ng levels of acrylamide [31]. channels are seldom available. However, comparison of
Alternative extraction solvents are methanol or n-propanol, published chromatograms indicates that the cleanup meth-
because formation of colloids is suppressed [26, 32, 33]. odology for LC–MS–MS described above (pressurized
When using alcohols, some sample matrices must be pre- fluid extraction using acetonitrile with concomitant in-situ
swelled with water to afford effective extraction. The cleanup filtration through Florisil) is superior to the GC–MS
which follows initial extraction varies with the method of method using a solid-phase dispersion/SPE step before
detection. For GC–MS with derivatization with bromine, an bromination.
additional cleanup step of brominated acrylamide on silica or GC–MS analysis of acrylamide without derivatization
Florisil is often needed, especially when detection limits might be affected by the formation of artefacts in the heated
lower than 10 μg kg−1 are required [27, 34]. An alternative GC inlet from any co-extracted acrylamide precursors. This
matrix solid-phase dispersion/SPE step before bromination may be avoided by selecting extraction solvents such as
has been used for coffee samples [35]. This last study also n-propanol or acetonitrile which do not co-extract potential

Fig. 2 Acrylamide in a regular

corn chip sample: ESI(+)
LC–MS–MS SRM chromato-
grams at a contamination level
of 110 ng acrylamide g−1 corn.
The protonated precursor ion
(M+H+)→m/z 55 was used
as quantifier transition
142 R. Krska et al.

precursors [38]. Most GC–MS methods for acrylamide are

based on derivatization of the molecule by bromination,
subsequent dehydrobromination with triethylamine (to
improve the thermal stability of the derivative), and
detection in EI mode. The most common bromination Fig. 3 Chemical structure of bisphenol A
reagent is a bromine–water solution with HBr and KBr
[28]; however, a safer KBr+KBrO3 mixture has also been monomer in the production of polycarbonate plastics and
used [39]. epoxy resins, and as an additive for elimination of surplus
Interlaboratory studies have been conducted in which the hydrochloric acid in the production of poly(vinyl chloride)
performance of a few analytical methods were compared (PVC) organosols. Polycarbonate is used in food-storage
[40, 41]. Although GC after derivatization can provide high- containers, for example water bottles and baby bottles, and
quality data down to 1–5 μg kg−1 in some laboratories [27], epoxy resins and PVC organosols are used in the internal
the broader consensus was that LC–MS–MS is generally the coating for food and beverage cans, and also in the internal
superior method [42]. The advantage of LC–MS-based coating on metal lids for foods in glass jars to protect the
methods is the determination of acrylamide without prior food from direct contact with metal. Residues of BPA in
derivatization and this could be one reason methods which polycarbonate plastic containers and coatings can migrate
use derivatization of acrylamide before LC–MS determination into foods, especially at elevated temperatures; thus,
have not been more widely accepted [43]. humans are inevitably exposed to BPA, primarily through
The state-of-the-art in the extraction and LC–MS–MS the diet. Because BPA is an endocrine disruptor that mimics
determination of acrylamide has been described by Rosen the action of the hormone oestrogen, the specific migration
et al. [29] and this methodology is able to achieve a limit of limit for BPA in food or a food simulant has been set at
quantitation (LOQ) of 5 μg kg−1. Further modifications for 0.6 μg g−1 in an EC directive in an amending document
baby foods afforded an LOQ of 0.5–2 μg kg−1 [31] This relating to plastic materials and articles intended to come
method and other, related, methods use electrospray into contact with foodstuffs [55]. The maximum acceptable
ionisation [30, 36, 44–47]. On the other hand several other dose and tolerable daily intake (TDI) for BPA were
methods use atmospheric pressure ionization [37, 48–50], established at 50 μg kg−1 body weight day−1 by the United
but it seems that neither mode of ionization is clearly States Environmental Protection Agency [56] and the
superior to the other in sensitivity or selectivity [51]. In European Food Safety Authority [57], respectively, and
recent studies, an analytical column which contains porous Health Canada established the provisional tolerable daily
graphitic carbon (Hypercarb) was used as a stationary phase intake for BPA at 25 μg kg−1 body weight day−1 [58].
[50]. This phase has a larger capacity factor for acrylamide Gas chromatography–mass spectrometry (GC–MS) and
than any other phase on the market; however, because of its high-performance liquid chromatography (HPLC) with a
unique sorption characteristics, it is prone to contamination variety of detectors are commonly used in analytical
by co-extractives, which might be present in the final methods for the determination of BPA in foods [59–67].
analytical extracts [47, 52]. It seems that cocoa and coffee Although method detection limits as low as 1 ng g−1 have
are the most difficult sample matrices for LC–MS based been reported [62, 64], methods with detection limits as
methods. Unfortunately, the exact nature of the co-extractives high as 10 ng g−1 have frequently been used in many of the
for such samples is not known, so an empirical approach for surveys to determine BPA in foods. This will overestimate
cleanup of these matrices is necessary. human exposure assessment if the detection limit (or half of
While analysis of acrylamide by traditional methods is the detection limit) is used for samples with BPA levels less
maturing, development of immunoassays for acrylamide is than the detection limits. Thus, sensitive methods with
progressing [53, 54] So far, these immunoassay-based detection limits as low as possible should be developed and
methods are still lacking ability to detect acrylamide at used for determination of BPA in food samples to assess
trace levels in a wide variety of food matrices. Other human exposure to BPA more accurately.
interesting developments include analysis of acrylamide BPA was evaluated by the Government of Canada in
and other analytes (reducing sugars and asparagine) in one 2008 and was concluded to be harmful to both human
analytical run [48]. health and the environment at current levels of exposure.
The Government of Canada has subsequently taken several
regulatory actions, including adding BPA to Schedule 1 (the
Bisphenol A List of Toxic Substances) of the Canadian Environmental
Protection Act, 1999 [68] and prohibiting the import, sale,
Bisphenol A (BPA) is the common name for 2,2-(4,4′- and advertising of polycarbonate baby bottles containing
dihydroxydiphenyl)propane (Fig. 3). It is used as a BPA under the Hazardous Products Act [69]. As part of its
Challenges and trends in the determination of selected chemical contaminants and allergens in food 143

risk-management strategy, the Government of Canada has small, polar, nitrogen-rich compounds (Fig. 5). MEL and
committed itself to monitoring levels of BPA in foods, CYA are used in the production of a variety of materials,
particularly those intended for infants [70]. To support this including plastics, adhesives, cleansers, and flame retardants;
initiative and generate exposure data for BPA in a variety of AMN and AMD are not industrially produced but are often
foods, a sensitive method based on solvent and solid-phase found as impurities in MEL and CYA feedstocks.
extraction followed by derivatization of BPA with acetic In two separate adulteration incidents, MEL and CYA
anhydride and GC–MS analysis was developed and were added to foods to boost their apparent protein content,
validated. This method was used to investigate levels of thereby increasing their market value. In 2007, vegetable
BPA in canned liquid infant formula [71], powdered infant protein containing both MEL and CYA was sold by several
formula [72], canned soft drinks [73], baby foods in glass Chinese companies. Its use in pet foods resulted in the
jars with metal lids [74], and canned food products [75]. A deaths of a number of pets in Europe and North America,
method based on headspace solid-phase microextraction because of the formation of an MEL–CYA complex that
and GC–MS analysis has also been developed [76] and precipitates in the kidneys and induces renal failure [80]. In
used to investigate migration of BPA from polycarbonate 2008, thousands of Chinese infants became ill after
baby and water bottles under severe conditions [77] and drinking MEL-adulterated milk, with six confirmed deaths
BPA levels in bottled water products [78]. [81]. These incidents prompted many countries to examine
Since BPA in foods is mainly a result of migration from can the extent of contamination in their own food supply, to
coatings, BPA is rarely measured in non-canned foods. Thus, establish limits for these chemicals in foods, and to recall
the contribution from the non-canned foods to the overall products which violated these limits.
dietary intake of BPA is unknown. There is also a lack of Methods for the analysis of MEL and related compounds
information on the effect of food processing, preparation, and in foods for human consumption have been adapted from
cooking procedures on BPA levels in the final cooked foods. those used for animal feed [82]; there are also methods for
Because polycarbonate tools and containers and containers analysis of the pesticide cyromazine, which degrades to
with epoxy coatings may be used during food preparation for MEL [83] and methods used to evaluate the migration of
cooking, BPA could be introduced into the final cooked foods, MEL from melamine–formaldehyde plastic food-contact
because of migration from polycarbonate and coatings. To materials [84]. Typically, homogenized food samples are
address these issues, Health Canada is currently investigating extracted with a polar solvent, for example water, acetoni-
the presence of BPA in the composite food samples from the trile–water [82, 85] or methanol–water [86]. Aqueous
Canadian total diet study. Health Canada is also investigating extraction has been performed under acidic [87, 88] and
the presence of BPA in human milk samples as part of the basic [89, 90] conditions, which are thought to dissolve or
MIREC study (Maternal–Infant Research on Environmental inhibit the formation of the MEL–CYA complex. However,
Chemicals) to update the exposure assessment for infants. basic extraction and storage conditions, and other practices
Because of the wide varieties and complex matrices of intended to prevent MEL–CYA complex formation (for
food samples, interferences are sometime unavoidable example keeping MEL and CYA extracts separate) do not
during analysis. For example, substantial interference with seem to be necessary unless a threshold concentration is
the quantification ion m/z 213 and the qualification ion m/z exceeded [80, 91]. Others have reported that basic
228 from sample matrices were observed during analysis conditions promote hydrolysis of MEL into AMD and
of all beer samples for BPA [79]. Figure 4 shows typical CYA [92].
GC–MS ion chromatograms obtained from a beer sample After extraction, samples are centrifuged and/or filtered,
with interferences and from a canned soup sample without then diluted for analysis. Methods requiring high sensitivity
interferences. However, if MS was not used as the detection usually incorporate further cleanup by solid phase extraction
method, false results for BPA would have been reported for (SPE). Mixed-mode reversed-phase and/or ion-exchange
all beer samples. Thus, MS detection and subsequent sorbents have frequently been used: cation-exchange phases
identification by use of ion ratios are essential in the analysis are used to isolate MEL [82, 88] whereas CYA is isolated
of foods for various contaminants to avoid reporting of false with anion-exchange sorbents [86]. AMN and AMD are
positive results. partially retained by both anion and cation-exchange
sorbents [85]. Graphitic carbon has also been used for
sample cleanup [87].
Melamine and the related triazines ammeline, ammelide Because MEL and its analogues are highly polar, they
and cyanuric acid are usually chromatographed using normal-phase LC. Some
groups have used ion-pair reagents to increase retention on
Melamine (MEL) and the related triazines ammeline reversed-phase columns [93], but most recent methods
(AMN), ammelide (AMD), and cyanuric acid (CYA) are use hydrophilic interaction liquid chromatography (HILIC)
144 R. Krska et al.

Fig. 4 GC–MS ion chromato- Abundance

grams: (A) canned beer sample Ion 213.00 (212.70 to 213.70): 2009APR30A17.D\ data.ms
(interferences with ions m/z 213 6000 Ion 228.00 (227.70 to 228.70):
Ion 270.00 (269.70 to 270.70):
2009APR30A17.D\ data.ms
2009APR30A17.D\ data.ms
and 228), (B) canned soup Ion 312.00 (311.70 to 312.70): 2009APR30A17.D\ data.ms
5500 Ion 224.00 (223.70 to 224.70): 2009APR30A17.D\ data.ms
sample (no interferences).
Bisphenol A: m/z 213, 228, 5000
270, 312 (in decreasing order);
bisphenol A-d14: m/z 224 4500

4000 A
3500

3000

2500

2000

1500

1000

500

0
Time--> 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00

Abundance
Ion 213.00 (212.70 to 213.70): 2009MAY15A16.D\ data.ms
Ion 228.00 (227.70 to 228.70): 2009MAY15A16.D\ data.ms
20000 Ion 270.00 (269.70 to 270.70): 2009MAY15A16.D\ data.ms
Ion 312.00 (311.70 to 312.70): 2009MAY15A16.D\ data.ms
Ion 224.00 (223.70 to 224.70): 2009MAY15A16.D\ data.ms
19000
18000 Bisphenol A
17000
Bisphenol A-
16000 d14
15000
14000
13000 B
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Time--> 12.80 12.90 13.00 13.10 13.20 13.30 13.40 13.50 13.60

[86, 91]. Gas chromatography (GC) has also been used samples and validate for each sample matrix. For LC–MS
[94], but derivatization of the analytes is required. (−MS), MEL is analyzed in positive-ionization mode and
The analyte is usually detected by single-stage or tandem CYA in negative-ionization mode: AMN and AMD have
mass spectrometry (MS). UV-based detection methods have been analyzed in either mode [85, 90]. If there is sufficient
also been used [89], but because these are less selective chromatographic separation between compounds analyzed
than MS methods, greater care must be taken to cleanup in positive mode and those analyzed in negative mode, the
Fig. 5 Chemical structures
of melamine, ammeline,
ammelide, and cyanuric acid
Challenges and trends in the determination of selected chemical contaminants and allergens in food 145

MS polarity can be switched during the run and all determine the protein content of food, rather than using
compounds can be analyzed in a single run. Figure 6 shows nitrogen as a proxy [98]. This approach should be extended
ESI(+)LC–MS–MS chromatograms obtained from the quan- to other nutritional assays, particularly for foods for which
tification SRM transitions for an infant formula sample the result of the assay has a direct result on the commodity
fortified at 0.05 mg kg−1 (CYA) and 0.01 mg kg−1 (MEL, price. Such testing would discourage the intentional addition
AMN and AMD). CYA was analyzed in negative mode, of other novel adulterants, chosen specifically to evade the
AMN and AMD in positive mode. food control system [99].
For GC–MS(−MS), derivatized MEL analytes are analyzed
in positive electron-impact mode [94]. 13C and 15N-labelled
MEL, AMN, AMD and CYA have recently become Persistent Organic Pollutants (POPs)
commercially available for use as internal standards, to
correct for the matrix effects that are frequently observed The general category of chemicals classified as persistent
during food analysis [87]. organic pollutants (POPs) are halogen-substituted organic
MEL can also be determined using a variety of rapid compounds that have similar behaviour when released into the
methods, including enzyme-linked immunosorbent assay [95] environment. POPs are known to persist in the environment,
and non-chromatographic mass spectrometric methods are subject to long-range transport and deposition, and are
[96, 97]. Although currently limited to MEL, these techni- ubiquitous throughout the environment [100]. These com-
ques require little or no sample preparation, and many are of pounds also are lipophilic and accumulate in the lipid tissues
equivalent sensitivity to LC–MS–MS methods. of living organisms causing levels to biomagnify through the
Further research is required to resolve conflicting informa- food chain [101, 102].
tion on the behaviour of some analytes, including conditions Owing to the toxicity of POPs to humans and wildlife,
required for MEL–CYA complex formation, and transforma- international action was initiated via the Stockholm Convention
tion and/or hydrolysis of MEL into AMN, AMD, and CYA. on POPs to regulate their release into the environment. The
These questions have important toxicological and analytical compounds listed by the Convention may be identified for
implications. elimination of their production, restriction of their production
Although MEL is of low toxicity, the adulteration incidents and use, or for reduction of unintentional release. Originally, the
in China have prompted many countries to re-evaluate the Stockholm Convention identified twelve compounds or groups
safety of their food supply. The intentional nature, economic of compounds which included organochlorine insecticides,
motivation, and technical sophistication of the contamination, industrial chemicals, and industrial by-products (Table 1) [103].
combined with a global food ingredient network, highlighted Since the original POPs were identified, amendments to the
significant weaknesses in the food-control system. The World list have occurred.
Health Organization has recommended the development and The toxicological effects associated with exposure to
use of rapid, inexpensive, and reliable methods that directly POPs include alteration of endocrine function, effects on

Fig. 6 ESI–LC–MS–MS
chromatograms of the quantita-
tion SRM transitions for an
infant formula sample fortified
at 0.05 mg kg−1 (CYA) and
0.01 mg kg−1 (MEL, AMN,
and AMD). CYA was analyzed in
negative mode, AMN and AMD
in positive mode
146 R. Krska et al.

the reproductive and immune systems, on liver function, and chemicals measured in The Canadian Total Diet Study
on neurobehavioral development and carcinogenesis [104– (TDS), a dietary surveillance program for chemical contam-
106]. In some cases, the POPs identified are classes of inants that has been conducted by the Food Directorate for
compounds (e.g., polychlorinated dibenzo-p-dioxins/ diben- over 30 years. Results from the TDS, covering five cities over
zofurans (PCDD/Fs), and polychlorinated biphenyls (PCBs)) the period 1992 to 2008 are available online. Levels of these
which have many individual structural analogues within each environmental contaminants in food are generally low (pg g−1
class. The individual analogues or congeners may cause to ng g−1 fresh weight) compared with other chemicals in
similar toxic responses, although they have different potency, foods that are present because of direct addition, contact with
as in the case of 2,3,7,8 tetrachlorodibenzodioxin (TCDD) food during processing, or are produced during food
which is the most toxic PCDD/F. Because of this different processing.
toxicity, toxic equivalency factors (TEFs) relative to TCDD Although concentrations of POPs are not high, their
have been developed for the other congeners [107, 108]. The presence in food and dietary supplements can result in court
combination of TEF values and concentration data are challenges, as has been observed recently in the US [110]. POP
generally used to establish the toxic equivalent (TEQ) levels in fish are frequently higher than observed in other
concentration which are used for risk characterization and food types [111, 112]. PCDD/F levels in foods are generally
risk management activity [107, 108]. low and may be reported on a TEQ basis (pg TEQ g−1
These lipophilic compounds (see chemical structures in sample fresh weight) whereas other POPs (e.g. organochlo-
Fig. 7) are present in foods of animal origin and it is generally rine insecticides, PCBs, PBDEs) are generally observed at
accepted that diet is the main route of exposure of humans to higher concentrations (ng g−1 fresh weight) [112–115].
these compounds. Food safety authorities, therefore, have The determination of POPs generally involves multi-
been required to develop methods for their detection and residue methods requiring extensive sample preparation
quantification in foods [109]. POPs are amongst the before analysis. The methodology applied to both extrac-

Table 1 Persistent organic pollutants (POPs) as identified by the Stockholm Convention (Stockholm Convention Secretariat, 2010)

Compound (or class) Type of chemical Listed

Aldrin Pesticide Original POP

Chlordane Pesticide Original POP
Dichlorodiphenyltrichloroethane (DDT) Pesticide Original POP
Dieldrin Pesticide Original POP
Endrin Pesticide Original POP
Heptachlor Pesticide Original POP
Hexachlorobenzene Pesticide Original POP
Mirex Pesticide Original POP
Toxaphene Pesticide Original POP
Chlordecone Pesticide Added in 2009
α-Hexachlorocyclohexane Pesticide/by-product Added in 2009
β-Hexachlorocyclohexane Pesticide/by-product Added in 2009
Lindane (γ-hexachlorocyclohexane) Pesticide Added in 2009
Pentachlorobenzene Pesticide/industrial chemical/by-product Added in 2009
Hexachlorobenzene Industrial chemical/by-product Original POP
Polychlorinated biphenyls (PCBs) [209 congeners] Industrial chemical/by-product Original POP
Hexabromobiphenyl Industrial chemical Added in 2009
Hexabromodiphenyl ether Industrial chemical Added in 2009
Heptabromodiphenyl ether Industrial chemical Added in 2009
Perfluorooctane sulfonic acid and its salts Industrial chemical Added in 2009
Perfluorooctane sulfonyl fluoride Industrial chemical Added in 2009
Tetrabromodiphenyl ether Industrial chemical Added in 2009
Pentabromodiphenyl ether Industrial chemical Added in 2009
Polychlorinated dibenzo-p-dioxins and Industrial chemical Original POP
polychlorinated dibenzofurans (PCDD/Fs) [75 congeners]
Challenges and trends in the determination of selected chemical contaminants and allergens in food 147

O
congener-specific determination. The dioxin-responsive
Polychlorinated dibenzo-p-dioxins
(PCDDs) chemical-activated luciferase expression assay (DR-CALUX)
O
is used when total dioxin-like compound levels are required
Clx Cly
x+y=1–8 [128].
O
As the number of POPs continues to increase, the
Polychlorinated dibenzofurans requirement for methods of analysis for these compounds
(PCDFs)
will continue to grow. Brominated flame retardants (e.g.,
Clx x+y=1–8 Cly hexabromocyclododecane; HBCD) and organochlorine insec-
ticides still in use (e.g., endosulfan) have been of recent
Polychlorinated biphenyls interest.
(PCBs)
Clx Cly
x + y = 1 - 10
Perchlorate
O Polybrominated diphenyl ethers
(PBDEs) Perchlorate is an environmental contaminant that arises as a
Brx
x + y = 1 - 10
Bry
result of anthropogenic processes and from natural sources.
Perchlorate can originate from the use of perchlorate salts in
Fig. 7 The structures of polychlorinated dibenzo-p-dioxins (PCDDs), military and industrial products, for example solid rocket
polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls fuels, munitions, explosives and fireworks, road flares, air bag
(PCBs), and polybrominated diphenyl ethers (PBDEs)
inflation systems, some fertilizers, and places near potash
deposits [129–132]. In the human body, the perchlorate anion
tion and cleanup for removal of co-extracted compounds is can inhibit uptake of iodide, an essential trace element, by
critical. Analyte extraction generally involves homogeniza- the thyroid gland at the sodium iodide symporter (NIS) [133,
tion with organic solvent [111, 116, 117]. Soxhlet extraction 134]. Sustained inhibition of iodide uptake can impair
is also used for isolation of POPs from foods [114, 118]. thyroid function by reducing the amount of iodide stored in
Cleanup techniques applied to POPs may include acid the thyroid and, consequently, that which is available for
digestion or gel-permeation chromatography to remove production of the thyroid hormones triodithyronine (T3) and
large-molecular-weight co-extractives (e.g., lipid)s, followed thyroxine (T4), leading to hypothyroidism [135]. Perchlorate
by column chromatography using a variety of sorbents exposure is an especial risk to pregnant women and their
including Florisil, silica gel, alumina, and activated carbon foetuses, neonates, and people with iodine deficiency or with
[111, 119, 120]. thyroid dysfunction.
With the numerous steps required for cleanup of sample Perchlorate contamination emerged as a public health
extracts, the potential for analyte loss is greater than for concern in the late 1990s This initiated the conduct of
methods requiring no cleanup. The application of stable scientific studies and guideline development related to
isotopes (e.g., 13C) as surrogate standards enables analysts perchlorate in drinking water, plants, food, and other
to correct for losses during sample preparation and is contamination sources in the United States and around the
routinely used [121–123]. world. A number of recent studies have shown that plant
The analytical methods employed for some of the POPs species are able to absorb perchlorate from soil and
include GC coupled to specific detectors (e.g., electron-capture irrigation water, and elevated levels of perchlorate have
and flame-photometric) [124, 125], although GC–MS is most been found in food crops, including lettuce [136], fish
frequently applied to the determination of POPs in foods [137], seaweed [138, 139], and some beverages [140].
[114, 119, 121, 126]. Because of the great number of POPs of Studies from different regions of the United States [141],
interest and the low detection limits required (for, e.g., Chile [142], Japan [143], and China [144] have also
PCDD/Fs), their measurement requires the application of reported detectable levels of perchlorate in milk samples.
high-resolution mass spectrometry rather than low-resolution In 2005, the United States Food and Drug Administration
GC–MS. Examples of GC–MS chromatograms obtained (US FDA) launched a study to determine perchlorate levels
from measurement of PCB, PCDD/F/coplanar PCB, and in total diet study (TDS) samples, and a mean value of
PBDE in egg yolk extracts is given in Fig. 8. In contrast, the 5.8 μg L−1 perchlorate was reported in 125 dairy milk
more recently identified POPs (e.g., perfluorinated com- samples [145]. The recent discovery of perchlorate in infant
pounds) are analysed by use of LC–MS [127]. formula samples by the United States Centers for Disease
There has recently been increased interest in developing Control and Prevention (US CDC) gave rise to intense
rapid methods for screening of contaminants in food, public concern regarding infant exposure to perchlorate
although quantitative analytical methods are required for [146].
148 R. Krska et al.

100

47
PCB

HCB

p,p’-DDE
%

28

118
49
18

0
77 28.00
100

Coplanar PCB, PCDD/F

Response

1,2,3,4,6,7,8 HpCDD
1,2,3,4,6,7,8 HpCDF
%

126

OCDD
OCDF
169
81

0
28.00
99

100

PBDE
%

47

100

153
154

0
17.00
Time (min)

Fig. 8 GC–HRMS chromatograms obtained from measurement of PCB, PCDD/F/coplanar PCB and PBDE in egg yolk extracts

A variety of analytical methods has been developed for fruits and vegetables, dairy milk, and human milk. Table 2
determination of perchlorate. Ion chromatography (IC) shows the mean and range values for 150 fruit and vegetable
coupled with conductivity cell detection (CD) is the most samples collected in Ottawa, Canada [153].
common approach [147], and it is the first method approved As a continuous study of perchlorate surveillance, an
by the Environmental Protection Agency (EPA) for deter- improved ID IC–MS–MS method enabling accurate identifi-
mination of perchlorate in drinking water [148]. To cation and quantification of perchlorate in infant formula was
determine trace levels of perchlorate in complex matrices, developed and 39 infant formula samples collected in Ottawa,
IC was coupled with tandem mass spectrometry (MS–MS), Canada were analyzed by this method.
taking advantage of the high selectivity and sensitivity of Future work is planned to determine the concentration of
mass spectrometry operated in SRM mode. IC–MS–MS perchlorate and iodide in Canadian human milk in order to
was demonstrated to yield limits of detection (LOD) of better assess dietary risk. Additionally, direct sampling of
5–25 ng L−1 when determining perchlorate in a variety of foods, for example infant foods, specific to high risk groups, is
matrices, including water, urine, amniotic fluid, wine, and also planned.
food [140, 149–151]. Figure 9 shows a typical SRM mass
chromatogram obtained from perchlorate analysis by ID
IC–MS–MS. Sulfites
In Canada, preliminary analysis of ground water and
surface water samples revealed non-detectable or very low Sulfites are sulfiting reagents that include SO2, sulfite,
concentrations of perchlorate [152], indicating that perchlo- sodium and potassium salts of metabisulfite, and bisulfite.
rate contamination of drinking water supplies is not expected They are widely used additives in the food industry and
to be a significant issue. Nevertheless, exposure to high have several technological functions, including as an
concentrations of perchlorate from sources such as food has antioxidant, bleaching agent, flour-treatment agent, and
the potential to cause adverse health effects. In 2005, Health preservative [154]. Sulfites have attracted increasing atten-
Canada began to investigate the occurrence of perchlorate in tion recently because of their allergy-like effect on
Challenges and trends in the determination of selected chemical contaminants and allergens in food 149

Fig. 9 Typical SRM mass 100

MRM of 4 channels [35Cl16O4]-to[35Cl16O4-16O]-
chromatograms from perchlorate
98.9>82.9
analysis by ID IC–MS–MS

0
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00

100
MRM of4 channels
100.9>84.9 [37Cl16O4]-to[37ClO4-16O]-

0
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00

% 100
MRM of4 channels
106.9>88.9 [35Cl18O4]-to[35Cl18O4-18O]-

0
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00

100
MRM of4 channels
108.9>90.9 [37Cl18O4]-to[37Cl18O4-18O]-

0
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00
Time(min)

hypersensitive individuals. The symptoms include respiratory (expressed as sulfur dioxide equivalent) must be declared on
sensitivity, triggering asthma, severe skin irritant, vitamin B the label and also prohibits the use of sulfites in any product
and E deficiencies, bronchospasm, wheezing, chest tightness, to be served or sold raw to consumers [156]. Although when
flushing, hypotension, etc [155]. added as a food additive, the presence of sulfite has long been
When sulfites are present at levels above 10 mg L−1 but are required to be declared on the ingredient list of most
not indicated on the label, this may pose a potentially serious processed foods, Health Canada has recently proposed that
health risk to sulfite-sensitive consumers. As a result, the when sulfites are present in pre-packaged foods at levels
international regulations on residual sulfite levels in food in exceeding 10 ppm they must also be declared on the labelling,
Europe and the US specify sulfites at concentrations >10 ppm to increase the level of protection and product choice for

Table 2 Mean and range

of perchlorate concentration Food product Sample number (n) Mean±SD (μg kg−1) Range (μg kg−1)
in fruit and vegetables
Apple 24 0.48±0.60 N.D.-1.60
Cantaloupe 18 56.1±151 N.D.-536
Carrot 12 1.31±1.26 N.D.-3.08
Grapes 12 27.7±23.0 1.96-62.1
Honeydew 6 0.30±0.33 N.D.-0.66
Lettuce 18 11.5±14.2 1.27-46.7
Orange 12 0.50±0.82 N.D.-2.89
Potato 24 N.D. N.D.
Spinach 6 133±24.9 99.4-175
Tomato 18 0.30±0.47 N.D.-1.38
150 R. Krska et al.

sulfite-sensitive individuals [157]. However, sulfites occurring be achieved by use of the isotope dilution (ID) techniques,
naturally in foods would be exempt from the label declaration. which was demonstrated by incorporating ID into GC–
A variety of methods are available to quantify sulfites in ICP–MS systems in which detection limits in the range of
foods. The most widely used is the optimized Monier– 0.5–10 ng g−1 sulfur species could be achieved [171]. The
Williams (M–W) method [158]. The method converts free use of matrix-independent detection and calibration methods
and reproducible amounts of bound oxosulfur(IV) anions in could also possibly simplify quantification of sulfur species
foods to sulfur dioxide, which is distilled and oxidized to and improve the accuracy of results for natural sulfur-
sulfuric acid in a hydrogen peroxide trap. The acid is then containing foods, for example onions, garlic [172], broccoli,
determined by titration. Alternative methods include enzy- and soy products [173].
matic [159], LC [160], differential pulse polarographic
[161], capillary electrophoresis [162], and flow-injection
techniques [163, 164]. The discrepancies in sulfite content Mycotoxins: ochratoxin A and fumonisins
amongst methods, and the potential for natural sulfur-
containing compounds (besides sulfur dioxide) to contribute Recent advances in the determination of mycotoxins in
to overestimation with the M–W assay, bring into question the foods have been well reviewed by Shephard et al. [174],
actual sulfite content of some samples. Therefore, a more who covered aflatoxins, Alternaria toxins, cyclopiazonic
accurate method that is able to differentiate between added acid, fumonisins, ochratoxins, patulin, trichothecenes, and
sulfite and matrix-derived sulfite must be developed, and zearalenone. A recent review by Maragos and Busman of
accurate information on sulfur speciation at low detection methods for analysis of mycotoxins [175] stresses the need
limits is becoming an important component of health risk for methods with increased throughput. Ochratoxin A and
assessment. fumonisins are mycotoxins of concern selected below for
Challenges in developing a sensitive and specific sulfite further discussion.
detection method are twofold: first, sensitive monitoring of Ochratoxin A (OTA, L-phenylalanylcarbonyl-5-chloro-8-
low-level sulfite is difficult owing to the effect of complex hydroxy-3,4-dihydro-3-R-methylisocoumarin; Fig. 11) is a
matrix and low signal response of sulfite in previously used mycotoxin formed by some species of Aspergillus and
techniques. Second, it is difficult to analyze the mixtures of Penicillium [176, 177]. It is carcinogenic, nephrotoxic,
sulfur species, because they are unstable and participate in teratogenic, immunotoxic, and hepatotoxic [178] and has
complex equilibria with other sulfur compounds formed by been classified by the International Agency for Research on
their auto-redox reactions [165]. Cancer (IARC) as possibly carcinogenic to humans (group
Ion chromatography (IC) has been the favoured separation 2B) [179]. OTA is found in grain and many other kinds of
technique for sulfur species, with electrochemical detection foodstuffs, notably including wine, beer, spices, dried vine
and spectrophotometric detection being widely used [166– fruits, botanicals, liquorice, coffee, and cocoa.
168]. The inductively coupled plasma mass spectrometer New developments in analytical methods for OTA have
(ICP–MS) is a more robust detector with very high been summarized by Shephard et al. [174]). LC with
selectivity and sensitivity. Health Canada’s Food Research fluorescence detection (FLD) continues to be important. A
Division has developed an IC–ICP–MS method for determi- simple, sensitive, and reproducible LC–FLD method for
nation of sulfur species in order to achieve a substantial determination of OTA in wine was developed by Tafuri et al.
reduction of the detection limit of sulfite, and species- [180]. It features direct injection of the wine on to a 5-μm
specific detection [169]. An ICP–MS has been used as an monolithic C18 column, with no need for extraction or
element-specific detector for sulfide, sulfite, sulfate, and cleanup of the wine. A new hollow-fibre liquid micro-
thiosulfate after their separation on a Dionex IonPac AS17 extraction method for determination of ochratoxin A (and T-2
column. The 32S species were detected as SO at m/z 48 with toxin) in wine and beer has been reported; extraction was
the ICP–MS tuned to high oxide ion production. The followed by UHPLC–MS–MS [181]. LC–MS–MS is now
stability of sulfite, metabisulfite, and bisulfite in different increasingly being used, particularly in multimycotoxin
stabilizers was also studied. Preliminary studies on wine methods, which usually include OTA. Other types of method
and preserved vegetable samples gave satisfactory results. for OTA include use of biosensors, aptamers, solid-phase
Figure 10 shows a typical chromatogram obtained from a microextraction (SPME), immunochromatographic strips, and
standard solution containing four sulfur species and spiked molecularly imprinted polymer (MIP) solid-phase extraction.
and unspiked pickled vegetable samples. Fumonisins produced by Fusarium verticillioides (formerly
Future studies will focus on extension of the IC–ICP– known as F. moniliforme) which caused leucoencephaloma-
MS method to a greater variety of food matrices including lacia in horses were discovered in South Africa in 1988.
wine or other alcoholic beverages labelled with “no sulfites Fumonisin B1 (FB1) is hepatocarcinogenic in rodents and a
added” [170]. In addition, matrix-independent accuracy can kidney carcinogen in rats [182, 183]. It is classified as a
Challenges and trends in the determination of selected chemical contaminants and allergens in food 151

S2-
x105 A
1
2- S2O32-
0.5 SO32- SO4

5.0 10.0 15.0

RT(min)

SO42-
x104 B
2
Counts

1
SO32-

0
5.0 10.0 15.0
RT(min)

SO42-
x104 SO32- C
2

0
5.0 10.0 15.0
RT(min)

Fig. 10 (A) standard solution containing S2−, SO22−, SO42−, and S2O32−, (B) pickled vegetable sample, (C) pickled vegetable sample spiked
with SO32−

possible human carcinogen (IARC Group 2B). The structures oxalylfumaric acid), the FD series with fewer carbon atoms
of FB1 and two related metabolites are shown in Fig. 12. in the backbone and several others, including FB5 and
Several other Fusarium spp., Alternaria alternata f. sp. FBK1 [186]. FB6 is a new fumonisin found in cultures of
lycopersici and, most recently, Aspergillus niger [184] are Aspergillus niger, which is mainly a producer of FB2 and
also known producers of fumonisins. FB1 is the most FB4 [185]. The sensitivity of LC–MS–MS for fumonisin
commonly found fumonisin, mainly in corn (maize) and determination is illustrated by the mean FB1 contamination
corn foods, and also in beer, rice, sorghum, triticale, cowpea level of 0.26 μg L−1 found in commercial milk samples in
seeds, beans (navy, mung, adzuki), milk, and asparagus. Italy [187]. Also, with the wide range of different
Now fumonisins have been found in additional grain foods/ fumonisins and food matrices of concern, it is apparent
feed, including barley, wheat, millet, and oats, and in other that LC–MS–MS is now the procedure of choice for their
types of food, including garlic and onion powder, turmeric, analysis [174, 188, 189]; fumonisins are frequently included
tea, coffee beans, medicinal plants, soy products, peanuts, in multimycotoxin determination by this procedure.
figs, buckwheat, and wine. The widespread occurrence of
FB2 in the last of these [185] will probably prove a topic of
interest comparable with that of ochratoxin A in wine. Also,
A. niger contaminates many foodstuffs, for example cereals,
peanuts, coffee beans, and grapes.
Newly discovered fumonisins that have recently been
detected by LC–MS–MS in F. verticillioides cultures are the
FBX series comprising 12 compounds with the tricarbal-
lylic acid moiety replaced by other carboxylic acids
(for example cis-aconitic acid, oxalylsuccinic acid, and Fig. 11 Chemical structure of ochratoxin A
152 R. Krska et al.

Fig. 12 The structures of FB1

and two related metabolites

Other recent analytical procedures are lateral flow tests— Paralytic shellfish poisoning toxins (PSTs), for example
membrane-based colloidal gold immunoassays for rapid saxitoxins, gonyautoxins, and C-toxins (Fig. 13), are
detection of fumonisins B1, B2, and B3. Lateral flow tests produced by marine dinoflagellates, e.g. Alexandrium,
are applicable to analysis of corn, rice, and other commodities Gymnodinium, and Pyrodinium. They have now been
[190, 191] and are available commercially. MIP solid-phase found in freshwater cyanobacteria, e.g. Cylindrospermopsis
extraction has been applied to pepper, rice, and corn flakes raciborski, Anabaena spp., and Aphanizomenon spp.
[192] and LC–fluorescence using post-column derivatization Official control of paralytic shellfish toxins (PSTs) remains
with o-phthaldialdehyde (OPA) and N,N-dimethyl-2-mercap- a subject of major concern to regulators [200]; there is
toethylamine [193] has also been used. debate over whether the mouse bioassay, LC with pre-
“Hidden (masked) fumonisins” are of current interest column oxidation (Fig. 14), or LC with post-column
because they are not detected by the usual analytical methods. oxidation should be used for shellfish analysis. LC–MS
The issue of terminology has been discussed by Dall’Asta based methods can also be used and may eventually
et al. [194]. One type is non-covalently bound and is an supersede the oxidation and/or fluorescence methods
associative interaction of fumonisins with matrix constitu- [201]. Other approaches are being studied; two of these are
ents. These hidden fumonisins can affect determination of based on the sodium channel, which is the target of the
free fumonisins when immunoaffinity columns are used for toxins, and another is use of antibodies [28]. Sensitive
cleanup [195]. The term “bound fumonisins” applies to methods incorporating solid phase microextraction (SPME)
fumonisins covalently bound to proteins (PBFB), starch, and have been used for analysis of drinking water [202].
sugars after heat processing. Total bound fumonisin FB1 Cylindrospermopsin (CYN) (Fig. 15) is one of three
(TBFB1) is determined indirectly either by subtracting free classes of cyanotoxins on the US Environmental Protection
FB1 and HFB1 found before hydrolysis from HFB1 found Agency Contaminant Candidate List III and, together with
after hydrolysis or by washing away free fumonisins with microcystins and anatoxin-a, may require regulation in
extraction solvent before the hydrolysis. There can be more drinking water [203]. A “guideline value” of 1 μg L−1 has
bound fumonisins than free FB1 in heat processed naturally been proposed [204]. CYN has been found to be genotoxic
contaminated foods [196, 197]. In vitro digestion has been
used to estimate the bioaccessibility of TBFB1 from naturally R4
contaminated solvent-washed corn flakes as 37–64% [198].
So bound fumonisins should be regarded when evaluating H
R1 H
exposure to fumonisins in a risk assessment. N
N
NH
Phycotoxins: paralytic shellfish poisoning toxins
N NH
(PSTs), cylindrospermopsin (CYN) NH
and β-methylamino-L-alanine (BMAA)
OH

Updates on progress in the validation of analytical methods R2 OH

for marine and freshwater algal toxins are provided R3
annually by James Hungerford [199]. Three of the many
known phycotoxins are briefly discussed below. Fig. 13 Basic structure of PSP toxins
Challenges and trends in the determination of selected chemical contaminants and allergens in food 153

Fig. 14 Chromatogram obtained LU

from naturally contaminated lob-
ster tomalley sample containing 12
GTX 2,3 (224 μg/100g) and STX GTX2,3
(40 μg/100g). Determination by 10
LC–FL after pre-column peroxide
oxidation 8

6 STX

0
0 2 4 6 8 10 12
min

in in-vitro systems and carcinogenic in mice [204]. Human protein-bound) in cycad seeds, flying foxes, brain tissue
poisoning (gastroenteritis) has occurred [204]. CYN is a of Alzheimer patients, and Chamorros in Guam who died of
metabolite of several other freshwater cyanobacteria be- amyotropic lateral sclerosis (ALS)/parkinsonism–dementia
longing to the genera Anabaena, Umezakia, Raphidiopsis, complex (PDC) [212]. BMAA is neurotoxic—it injures
and Aphanizomenon [204] and, more recently, Lyngbya neurons in mice and induces motor system disease in
[205]. CYN, originally regarded as a subtropical problem, monkeys. BMAA can be determined by LC of the
is now occurring more frequently in European countries. fluorescent derivative formed with 9-fluorenylmethyl chlor-
Recent applications of LC–MS and LC–MS–MS for oformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccini-
determination of CYN in lake or river water from, for midyl carbamate (AQC) or by LC–MS–MS [214]. Analysis
example, the USA, Mexico, the Czech Republic, Italy, of cyanobacteria and other biological samples for BMAA is
Germany, and Poland have been published [37, 203, 206– complicated because it often occurs partly as a protein-
210]. Oehrle et al. [203] noted that single-ion monitoring bound form, so the sample must be hydrolysed to give
would have resulted in a false positive for an Ohio River additional free BMAA [212]. Recently, an LC–FLD method
sample compared with tandem MS. LC–MS–MS was also has been developed which is applicable to the analysis of
recently used to find CYN in freshwater snails consumed blue green algae (BGA) food supplements, freshwater fish,
by humans in Mexico [207]. An enzyme-linked immuno- and bottled water [215]. BMAA has been found in a
sorbent assay (ELISA) is now commercially available Peruvian food, Nostoc commune [216], and recently in
[206]; however, overestimation compared with LC–MS Nostoc flagelliforme used as the food item fa cai [217];
was observed. BMAA was not detected in a sample of genuine fa cai
β-Methylamino-L-alanine (BMAA) (Fig. 16) is pro- sold in Canada but the isomeric 2,4-diaminobutyric acid
duced by some marine cyanobacteria (e.g. Trichodesmium) (DAB) was found; this is also a neurotoxin known to occur
and by many other cyanobacteria, including Nostoc root
symbionts [211, 212]. BMAA was detected in a seawater
sample from a Trichodesmium bloom near Hawaii and was Fig. 16 Chemical structure
recently found in shrimps and other aquatic animals in of β-methylamino-L-alanine O OH
Florida waters [213]. It has also been found (mainly (BMAA)
C
OH
_ CH NH2
O3 SO
O

CH2
N NH HN NH

H3C + NH
NH O

Fig. 15 Chemical structure of cylindrospermopsin

CH3
154 R. Krska et al.

in numerous cyanobacterial samples, as determined by tial of peanuts [223]. A recent study of egg proteins has
LC–MS–MS [218, 219]. shown there is a similar decrease in the ability of the
ELISA to detect the proteins of interest in salad dressing
[224]. This matrix is likely to have a high vinegar
Allergens component and it has been shown that vinegar also reduces
the allergenicity of eggs [225]. In the latter investigation it
For specific individuals with food allergies or sensitivity to seemed that egg protein detection correlates with egg
gluten, consuming foods containing an allergen may be of protein allergenicity when exposed to vinegar, in contrast
concern. Recent estimates of numbers suffering from food with the results obtained after roasting peanuts. Unfortu-
allergies are approximately 4% for adults and 6% for nately, the correlation between allergenicity and ELISA
children and have been increasing [220]. Some of these response is difficult to determine beforehand and informa-
allergies will be outgrown, but the most severe typically tion on food matrix and processing effects is needed for
remain throughout adulthood. Individuals with celiac confidence in a result. The development of incurred
disease number around 1% of the population, but this is materials that have a known amount of allergen that has
thought to be a conservative estimate as many individuals undergone industrial processing steps would be invaluable
go undiagnosed [221]. Most allergenic reactions occur to in validation of a method.
one of the nine priority allergens in Canada—peanuts, tree The development of reference materials, for example
nuts, milk, egg, fish and/or crustaceans, sesame, soy, wheat, incurred materials or any material used to evaluate the
and sulfites. In both types of food sensitivity the only solution effect of processing and food matrix on the response from
is complete avoidance of the food to reduce the risk of a an ELISA, will depend on the availability of material that
reaction. Unfortunately, cross-contamination of other foods has been fully characterized. The continuing lack of these
with allergens during processing remains a possibility and thus materials for the development and validation of ELISA
ongoing surveillance of products to verify labelling accuracy is methods is partly because of the difficulty in deciding what
a necessity. Analytical methods are needed to determine the will make a good reference material for a given allergen or
validity of allergen label statements and assess the risk to the commodity. The lack of recognized reference materials
food-sensitive consumer. Immunological and DNA-based means that most kit manufacturers supply a calibration
assays are the most developed methods for detection and standard developed for their own particular assay, which is
quantification of food allergens. However, the preferred likely to be different from the calibration standard from
method is to detect the presence of the protein as it the direct another ELISA manufacturer. These differences in protein
cause of the allergic reaction. As a result, DNA-based methods content in the calibration standards sometimes result in
have limited use. Immunological methods, for example ELISA different final results; this is very evident from interpreta-
and lateral flow devices (dipsticks) have become important tion of results from ELISA assays that determine gluten
tools for detection and quantification of food allergens. content. Most commercial ELISA methods for gluten rely
Commercial assays are available from a variety of companies on two different antibodies, the R5 antibody which reacts to
for most of the priority food allergens. These methods have a five-amino-acid sequence found in the gliadin fraction of
improved since their introduction in the late 1990s; however, wheat [226] and the omega-gliadin antibody which reacts to
there are still some limitations in their application including the the omega-gliadin fraction of wheat [227]. The R5-ELISA
lack of reference materials, knowledge of processing and is calibrated by use of a purified gliadin material based on
matrix effects, and a harmonized validation procedure specific multiple wheat cultivars while the omega-gliadin assay is
to protein quantification in food matrices. calibrated by use of a wheat material from NIST [228]. The
Processing effects and food matrices can have a R5-ELISA also responds to gluten from barley and rye
significant affect on the ability of an ELISA to detect the because these proteins also contain the five amino acid
proteins of interest. In a recent study it was shown that peptide sequence, but the omega-gliadin assay has low
roasting peanuts resulted in a significant decrease in the cross-reactivity (5%) towards barley hordeins and typically
detection of proteins and the ability to determine peanut results in different estimates of gluten content if the source
content. In addition, a prolonged baking time (26 min is barley. Interestingly, the R5-ELISA is likely to overesti-
compared with the standard 16 min) resulted in reduced mate the gluten content of barley because it relies on a 1:1
detection by a factor of 5–10 [222]. This decrease in the prolamin-to-glutenin ratio to calculate total gluten, which is
ability of the ELISA to detect the proteins of interest could not valid for other grain [229].
be because of changes in the solubility of the proteins, The difficulties associated with the analysis of gluten
changes in protein structure, or changes in the epitopes. and those associated with some processed proteins when
Interestingly, heat treatment by roasting of peanuts has the using ELISA methods emphasize that immunological
opposite effect and actually increases the allergenic poten- assays are not perfect. Both false-positive and false-
Challenges and trends in the determination of selected chemical contaminants and allergens in food 155

negative results can occur, which reveals the need for a arginine residues that are not adjacent to proline [236], an
conformational method of analysis. Mass spectrometric assumption that has been challenged [237]. This digestion
analysis of proteins does not have these specific drawbacks must be efficient and reproducible for reliable qualitative
and if the proteins are soluble and can be digested, typically and quantitative analysis of proteins. Like ELISA methods,
by trypsin, positive identification can be achieved, typically mass spectrometry will benefit from the use of reference
with only one peptide. Analysis of proteins in biological materials, incurred materials, and method-validation proce-
systems has developed rapidly since the discovery that dures. One difficulty in the validation of these methods is
these large molecules can be introduced into the gas phase the choice of peptides and proteins to target. For a given
and measured by mass spectrometry [230]. In study of food allergenic food there are many targets that could be used for
allergens a significant body of work has been conducted on qualitative and quantitative analysis, but these targets can
peanut proteins. Early work using mass spectrometry was vary within cultivars and thus whole food reference
performed either to characterize all of the proteins present materials will be vital [238, 239]. Mass spectrometric
in a sample [231] or those determined, in other inves- methods for detection, confirmation, and quantification of
tigations, to be allergenic [232]. Initial work using mass food allergens have not been developed to the extent of
spectrometry as a tool to investigate the presence of commercial ELISA methods, but these types of validation
allergens in different food matrices was confirmatory in studies are starting to appear in the literature [240]. The
nature, with specific peptides being used to confirm the promise of mass spectrometric methods of analysis,
presence of allergenic proteins in the samples. One of these whether confirmatory or quantitative, could alleviate some
early studies relied on finding peptides specific to the of the problems associated with ELISA methods, including
allergenic peanut protein (Ara h1) and then using a processing effects, but a procedure for harmonized multi-lab
combination of LC and tandem mass spectrometry to investigations will be essential to the production of results
confirm the presence of peanut allergenic proteins [233]. of which we can be confident, as they will be for ELISA
In another study LC coupled to high-resolution time-of- methods [241].
flight tandem mass spectrometry was used to gather
information on all of the peptides in the digested sample,
and then determining if any food allergen proteins were Summary and outlook
present by use of a database search [234]. In the first
example, knowledge of specific protein markers is needed The contaminants and allergens discussed above have a
to develop multiple reaction monitoring (MRM) methods. wide range of chemical and physical properties, ranging
These methods have been used extensively for analysis of from lipophilic to hydrophilic, from volatile to non-volatile,
small molecules because they are very sensitive, and and from small molecules, for example chemical contam-
quantitative results can be obtained by use of isotope- inants, to large allergenic proteins. In addition, many of
dilution studies. These authors were able to confirm the these analytes have unknown toxicological or allergenic
presence of Ara h1 protein spiked at a level of 10 ppm into effects in humans and, therefore, maximum allowable levels
an ice cream matrix and in a subsequent study were able to set by government regulatory agencies are also driven by
confirm the level of the same protein at a level of 2 ppm in the achievable limits of detection. Therefore, matrix-
dark chocolate [235]. A difficulty in using one or two independent methods and low quantification limits are still
peptides for confirmatory or quantitative analysis is that if required to gather surveillance data about the occurrence
these peptides were altered in some way by processing they and background levels of both recognized and newly
would become invisible to the method and a false negative identified contaminants in foods in order to estimate human
result would be obtained. In the latter example, in which all daily intake for risk assessment. Clearly, the choice of
the digested peptides are monitored, processing would not appropriate extraction and cleanup methods using modern
be such a problem because there would probably be SPE techniques in combination with chromatographic
additional peptides from the allergenic food that were not methods, usually with MS detection, are crucial for success.
altered and could be used for identification. However, this There is still a need for appropriate reference materials,
method would be very dependant on the level of other particularly for allergens.
proteins in solution and higher-protein-content samples
would be likely to mask the peptide signals from the
allergenic food, producing a false-negative result. In either Acknowledgements This article was written as a result of a one-year
type of analysis the experimental workflow requires tenure of the first author at the Food Research Division of Health
Canada’s Bureau of Chemical Safety. In this respect, the first author
enzymatic digestion of the proteins into smaller tryptic
would like to express his great gratitude to Dr Samuel Godefroy, John
fragments before analysis. These digestions are performed Salminen, Barbara Lee, and all members of the FRD for their great
using trypsin, which cleaves the protein at lysine and support and enthusiasm.
156 R. Krska et al.

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