BLBS102-c03 BLBS102-Simpson March 21, 2012 11:56 Trim: 276mm X 219mm Printer Name: Yet to Come
48 Part 1: Principles/Food Analysisgas chromatography, immunoassay, and mass spectrometry. The
drug tends to be conjugated in biological systems, and Bogusz
et al. (2004) recently developed an assay to distinguish between
the free and conjugated forms. They usedβ-glucuronidase in
the extraction process to hydrolyze the conjugated to the free
form, enabling selective analysis of the drug in honey, shrimps,
and chicken.
Fluoroquinolones, antimicrobial agents used for treatment and
disease prevention in food-producing animals and sometimes
used as feed additives to build up animal body mass, is another
group of drugs that has come under scrutiny. This is due to their
misuse, potentially leaving residues in meats, which could lead
to drug resistance and allergic hypersensitivity (Martinez et al.
2006). Therefore, maximum residue limits have been instituted
in the European Union, and a number of ELISA methods have
been developed for their detection in milk, chicken, pork, eggs,
and other foods (Coillie et al. 2004, Huet et al. 2006, Lu et al.
2006, Scortichini et al. 2009, Sheng et al. 2009). Euro Diagnos-
tica B.V. (Arnhem, Netherlands) has a commercial kit currently
available for analysis of quinolones.
Melamine is a triazine compound that contains 67% nitrogen
and is used in the production of plastics, construction materials,
and furniture. This compound was brought to public attention in
the fall of 2008 following growing incidents of infants coming
down with kidney stones in China as a result of adulteration of
infant formula with the compound. Similar melamine adulter-
ation in an effort to raise the apparent protein content of pet foods
resulted in several pet deaths. These incidents were presumably
possible due to protein analysis of foods being routinely con-
ducted with the Kjeldahl method, which is nonspecific and does
not distinguish between protein and nonprotein nitrogen. Sev-
eral alternative methods for detection of melamine and related
compounds (cyromazine, ammelide, cyanuric acid, and amme-
line), including ELISA, have been developed and implemented
(Tittlemier 2010) to ensure food safety. Romer Labs and Beacon
Analytical Systems have commercially available ELISA test kits
for melamine analysis in a broad range of foods.
Biotechnology, and particularly genetic modification, is un-
doubtedly a key factor for addressing the challenge of boosting
food supplies to meet the demands of an exploding global pop-
ulation. This is clearly evident by the increase in the global area
cultivated for biotech crops from 1.7 million hectares in 1996 to
125 million hectares in 2008 with the major food crops being
soybean, canola, and maize (Lee et al. 2009). However, there is
a deep sense of cynicism over the potential health and environ-
mental impact of GMO-based foods. There have been calls for
controls on GMO, and strict regulations on traceability and la-
beling of GMO and foods or feed made from GMO technology
have been established in many countries (Council Regulation
2000, Lee et al. 2009). Hence, a number of detection and quan-
tification methods have been developed based on analysis of two
main components characteristic to GMO: (i) a specific transgenic
DNA and (ii) a specific novel protein expressed by the GMO.
The basic principle underlying these methods is the use of the
polymerase chain reaction (PCR) to establish the presence of
the most common recombinant elements in the vast majority
of genetically modified crops (Vollenhofer et al. 1999, Miche-lini et al. 2008, Lee et al. 2009). These are the 35S promoter and
nopalin synthetase (NOS) terminator, both of which are tran-
scription sequences (Barbau-Piednoir et al. 2010). Variations of
this method have been used to detect and quantify GMO in soy,
maize, and other food crops (Matsuoka et al. 2001, James et al.
2003, Meric et al. 2004, Yang et al. 2006, 2007, Wu et al. 2007,
Lee et al. 2009, Costa et al. 2010). In one of these methods, a
disposable genosensor for detecting the NOS terminator (Meric
et al. 2004) was used. It involved immobilization of a synthetic
single stranded 25-base oligonucleotide that is complimentary to
the NOS terminator to a screen-printed carbon electrode. In the
presence of exogenous genetic materials, DNA polymerase cat-
alyzes the PCR to synthesize the complimentary base sequence.
Detection of hybridization is achieved by monitoring the reduc-
tion signal from the high-affinity binding of methylene blue to
guanine.
In the alternative method based on detection of expressed
proteins by the genetically modified organisms, PCR-ELISA
has been the preferred approach and has been used for detect-
ing and quantifying various GMO materials. This is demon-
strated by the use of a sandwich ELISA for the detection of the
Cry1Ab endotoxin characteristic of some transgenic maize. A
Cry1Ab protein-specific antibody was first immobilized on a mi-
crotiter plate to capture the Cry1Ab toxin in the sample. This was
followed by a second anti-Cry1Ab antibody and a horseradish
peroxidase-labeled antibody for detection of the toxin using a
chemiluminescent probe (Roda et al. 2006). A similar sandwich
ELISA has been developed for the detection of phosphinotricin-
N-acetyltransferase, which is encoded by a gene in genetically
modified pepper (Shim et al. 2007). Using two commercially
available ELISA kits, Whitaker et al. (2001) detected and quan-
tified the distribution and variability of the Cry9C insecticidal
protein produced by the genetically modified corn (StarLink)
in a bulk of corn flour and meal. There are a number of other
reports of PCR-ELISA application for GMO analysis in soy and
maize products (Landgraf et al. 1991, Brunnert et al. 2001, Petit
et al. 2003).Food ToxinsFood toxicity is of high importance due to their potential im-
pact on safety and risk to the environment. These toxins may
be naturally present (endogenously), process induced, or from
the environment. Food toxins such as phytotoxins (terpenoids,
glycoalkaloids, phytosterols, flavonoids, lignans) and mycotox-
ins (aflatoxins, fumonisins, ochratoxins, citrinin, mycophenolic
acid, etc.) are all naturally occurring and their toxic effects have
been well noted. This section discusses the enzymatic methods
for analyses of these and other toxins.Endogenous ToxinsMycotoxins are toxic secondary metabolites produced by vari-
ous fungi (Aspergillus, Fusarium, Penicillium,etc.) and excreted
in their substrates. Aflatoxins first came to the attention of re-
searchers following the outbreak of the so-called Turkey X dis-
ease among turkeys in the 1960s and is by far the most widely