Melamine contamination

Abstract In the summer of 2008, serious illnesses and deaths of
babies in China were linked to melamine-tainted powdered
infant formula. Melamine contains several metabolites, such as
ammeline, ammelide, and cyanuric acid, and has been used for
the adulteration of foods or milk to increase their apparent
protein content. It is assumed that melamine and its metabolites
are absorbed in the gastrointestinal tract, and precipitate in the
kidney to form crystals. A new tolerable daily intake of 0.2 mg
kg 1 body weight was adapted by the World Health
- Keywords Melamine . Cyanuric acid .
Organization in 2008. This paper reviews the variety of
analytical methods that have been used for the analysis of
melamine in food. The limit of detection of these various
methods is 0.05-100 ppm. The maximum acceptable
concentration in food has been set at 50 ppb by the US FDA. A
fast and ultrasensitive procedure for screening, detection, and
characterization of melamine and its derivative compounds
needs to be established. Currently, mass- spectrometry
technologies provide an alternative to derivati- zation for
regulatory analysis of food.
High-performance liquid chromatography-tandem mass
spectrometry . Gas chromatography-tandem mass spectrometry
.
Isotope dilution

Y.-C. Tyan:S.-B. Jong

Department of Medical Imaging and Radiological Sciences, Introduction
Kaohsiung Medical University,
100 Shi-Chuan 1st Road, What is melamine?
Kaohsiung 807, Taiwan

Y.-C. Tyan:J. Shiea Melamine (2,4,6-triamino-1,3,5-triazine) is an organic

National Sun Yat-Sen University - Kaohsiung Medical University Joint compound, a base with the chemical formula C3H6N6. It was
Research Center, Kaohsiung, Taiwan
first prepared in 1834 by Liebig by heating potassium
Y.-C. Tyan (*) thiocyanate with ammonium chloride. It was subsequently
Center of Excellence for Environmental Medicine, obtained in various ways by heating guanidine carbonate,
Kaohsiung Medical University, thiourea, cyanamide, or dicyandiamide. The manufacture of
100 Shi-Chuan 1st Road,
Kaohsiung 807, Taiwan
melamine on a large scale is based on heating dicyandiamide or
e-mail: yctyan@kmu.edu.tw urea in the presence of ammonia. Melamine can be hydrolyzed
to ammeline, ammelide, and cyanuric acid. Besides, those
M.-H. Yang (*) :J. Shiea
derivatives are by-products formed during melamine synthesis.
Department of Chemistry,
National Sun Yat-Sen University, 70 Lienhai Rd,
The overall reactions and the structures of all the compounds
Kaohsiung 804, Taiwan are shown in Fig. 1.
e-mail: myang@staff.nsysu.edu.tw Melamine is a common organic compound that is often
combined with formaldehyde to be used in the manufacturing
C.-K. Wang
Department of Medicinal and Applied Chemistry, of plastics, including whiteboards, kitchenware, and commer-
Kaohsiung Medical University, cial filters. Melamine resins are known as thermoset plastic,
Kaohsiung, Taiwan because the plastic is fixed after molding. If exposed to

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2 Y.-C. Tyan et al.

Fig. 1 Formation and structures of 6CO(NH2)2 C3H6N6 + 3CO2 + 6NH3

melamine and related compounds
Urea
Melamine

enough heat, melamine resins

will melt. For this reason,
melamine dishware should not
+ H2O
be exposed to high temper-
atures (around 120 °C) as in
the oven and microwave. H2N NH2 H2N
However, the plastic is able to
Melamine
withstand higher temperatures
than other plastics. Because melamine resins are a thermoset such as cyanuric acid, which might be found in the raw
plastic, they are difficult to recycle. Melamine is also used in a material. Melamine and melamine analogues (cyanuric acid,
wide range of flame-resistant materials. It protects wearers ammelide, and ammeline) are assumed to be of equal potency
from heat hazards and helps to resist the spread of fire in and will be referred to collectively in this assessment as
aircraft and buses by acting as a fire blocker. melamine compounds.
Melamine crystallizes as colorless monoclinic prisms, and It has been shown that a single oral exposure to a mixture of
its solubility in water and organic solvents is low. A saturated melamine and cyanuric acid can result in acute renal failure,
aqueous solution at 20°C contains less than 1% of melamine, which eventually caused the dog deaths. The chemical
structure of the melamine-cyanuric acid crystal is shown in
and one at 90-100°C contains less than 5% [3].
Fig. 2. Similar results were found in cats, dogs, and rats [6, 10,
Recently, it was determined that melamine may have been
11]. In 2007, the US Food and Drug Administration (FDA)
responsible for the deaths of pets after ingestion of adulterated
identified and reported melamine and cyanuric acid as
pet food. Melamine's nitrogen level is high, 66% nitrogen by
suspected contaminants in certain pet foods. This resulted in a
mass, which confers the analytical characteristics of protein
large-scale recall of these products from store shelves across
molecules. The reason for this type of adulteration is that the
North America.
test for the protein content of certain foods and food
ingredients is assayed through a nonspecific procedure, the
Melamine contamination in milk
Kjeldahl reaction [4]. The procedure quantifies the presence of
nitrogen, which is present in amino acids and therefore in
Previously, melamine was found in imported pet food in 2007
proteins, but is also present in many other nonproteinaceous
and was responsible for thousands of pet deaths in the USA.
molecules such as melamine [5]. The manufacturers of the
Today, thousands of babies in China who were given formula
milk ingredient in China, in 2008, intentionally adulterated the
contaminated with the industrial chemical melamine are
product to give spuriously high readings for protein.
seriously ill, having acute kidney failure. Allegedly, the
Thus, aside from common commercial uses, melamine
suppliers to the manufacturers added melamine to milk to
became a topic of discussion in 2007 when veterinary scientists
artificially inflate the reading of protein levels. Milk was not
determined pet food contamination of melamine was the cause
tested at that time for melamine because the regulators did not
of hundreds of pet deaths. Prior to these reports, melamine had
suspect the illegal use of this ingredient.
been regarded as nontoxic or minimally toxic. However, the
unexplained presence of melamine in pet food is most likely
the cause. The pet owners reported symptoms associated with
Toxicity of melamine
renal failure, which could be explained by the ammonia that
may result from the digestion of melamine. In addition, Melamine is not a natural product and is not approved for
previous reports indicated the combination of melamine and direct addition to food; however, melamine is approved for use
cyanuric acid formed insoluble crystals in kidneys [6]. as part of certain food-contact substances [12]. Chronic
If the melamine was low in toxicity, what caused thousands exposure to melamine may cause reproductive damage. The
of babies in China to become seriously ill? The most commonly observed effects in animal experiments of
LD50, the toxic dose, of melamine was more than 3 g kg-1 body exposure to the mixture of melamine and cyanuric acid when
weight (LD50=3,296 mg kg-1 for melamine, LD50> 7,940 mg kg- they were administered orally included body weight loss,
1
for cyanuric acid) [7-9]. The contaminants are of concern,

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Melamine contamination
N O
,N N„
H H
NN
O NN
N'
H H
H
,N,
NN
NN NN
Fig. 2 The chemical structure of melamine-cyanuric acid crystal
bladder and kidney stones, crystalluria, and epithelial
hyperplasia of urinary bladder [13]. However, no kidney
failure or clinical symptoms of kidney failure were observed
in current studies [14-16]. Melamine has been studied for
carcinogenicity in mice and rats for each sex by oral
administration. It produced urinary bladder and ureteral
carcinomas in male rats and urinary bladder hyperplasia in
male mice. Bladder tumors occurred only in male rats after
prolonged irritation of the epithelium by the bladder stones.
The occurrence of urinary bladder tumors in male rats
correlated strictly with calculus formation and high-dose
exposure [17]. High and continuous dietary exposure (4,500
ppm or 263 mg kg-1 body weight day-1, 2 years) to melamine in
controlled studies is associated with an increase in the
production of bladder stones and an increased incidence of
urinary bladder tumors. The two incidences are closely related
to each other [16].
It has been hypothesized that the formation of melamine
cyanurate crystals in the kidney is due to the extremely low
water solubility of this compound (2.2mg L-1) in comparison
with the parent compounds melamine (3,240 mg L-1) and
cyanuric acid (2,000 mg L-1). It is assumed that melamine and
cyanuric acid are absorbed in the gastrointestinal tract,
distributed systemically, and, for reasons that have not yet been
fully determined, precipitated in the kidneys to form
nephrotoxic melamine cyanurate crystals [18]. Another report
also characterized the toxicity potential of melamine,
cyanurate, and a combination of melamine and cyanuric acid in
cats. The study results showed that no effect on renal functions
was observed in cats fed with melamine or cyanuric acid alone.
Cats dosed with a combination were euthanized at 48 h after
3
NN /Nx/N\
H H
H -
NX N
i
i
O
i
i
NN
i
H
N
N
N^,N
O
dosing because of acute renal failure. The results demonstrate
that the combination of melamine and cyanuric acid is
responsible for acute renal failure in cats [19]. Melamine
cyanurate compounds crystallize in animal kidneys when
animals are dosed with the combination of melamine and
cyanuric acid [20].
Unlike heavy metals, such as mercury, which accumulate in
animal bodies in very low amounts, melamine does not build
up in an animal's body for a significant length of time.
Information from the US FDA suggests melamine is excreted
from an animal within 10-15 days after ingestion. In mammals,
the toxicity of melamine alone is low, with a halflife of
approximately 3-4 h [20]. In addition, the US FDA states that a
low level of melamine in food does not pose a health risk to
humans [21].
Interim safety and risk assessment
US food and drug administration
According to a report form the US FDA, dated 25 May 2007,
the base for determining melamine levels was set at 50 ppb.
The 50-ppb level represents a conservative estimate of the limit
of detection (LOD) for the methods employed, a value that will
ensure the exposure scenarios are conservative. Currently this
method is designed to only detect melamine in meat; however,
it is presumed that other melamine derivatives are present in
the tissue. For the purposes of this assessment, the
concentration of melamine assumed in the tissue (50 ppb) is
doubled to 100 ppb to account for cyanuric acid, which was not
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4 Y.-C. Tyan et al.
actually measured by the test. Doubling the measurable tissue
melamine level was based in part on the relative levels of
melamine compounds observed in the contaminated feed and
in part on the identification of the crystals in the kidneys being
a mixture of melamine and cyanuric acid [14]. In addition, it
was also indicated that the resulting tolerable daily intake
(TDI) is 0.63 mg kg-1 body weight day-1. Infants are more
sensitive than adults to exposure; thus, the US FDA has applied
an additional tenfold safety factor. This results in a TDI/10 of
0.063 mg melamine kg-1 body weight day-1. In the report by the
US FDA, melamine is not approved for direct addition to
human or animal foods and no manufacturer is allowed to
deliberately add it to any food for US consumers. The US FDA
will examine protein- containing products beyond dairy and
dairy-containing products for contamination with melamine
and melamine- related compounds [12].
European food safety authority
In the report dated 26 September 2008, the European Food
Safety Authority (EFSA) used the TDI of 0.5 mg kg-1 body
weight, which is protective for exposure over a lifetime. This
considers possible effects of exposure to melamine over a
relatively short period, and such as might occur with repeated
consumption of melamine-contaminated products. Because
high levels of melamine can primarily affect the kidneys, the
EFSA applied the TDI of 0.5 mg kg-1 body weight for
melamine in a specific case of contamination in 2007. A total
ban in the European Union of all Chinese- manufactured milk
and related products has already been established [22].
New Zealand food safety authority
The New Zealand Food Safety Authority (NZFSA) adopted a
conservative threshold of 5 ppm for most foods on 26
September 2008. This indicates that foods containing up to 5
ppm of melamine do not pose a risk to human health. However,
for infant formula, the maximum level remains as the current
detection level of 1 ppm. For dairy-based foods in their final
forms, other than infant formula, this level is 2.5 ppm. A level
of melamine above 2.5 mg kg-1 is indicative of food
adulteration. For infant formula, even at relatively low levels of
adulteration, an infant will quickly exceed the TDI for
melamine. Foods with low levels of dairy-based ingredients,
such as candies and biscuits, are likely to be infrequently
consumed in small amounts, so they are not considered to be a
high-risk food for potential dietary exposure to melamine even
if the dairy ingredient has been adulterated. Thus, the NZFSA
has adopted a higher safety factor than is normally applied, and
set the limit of 2.5 ppm before regulatory action was
considered [23].
On 5 December 2008, the World Health Organization
(WHO) adopted a new TDI of 0.2 mg kg-1 body weight. The
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new number is also lower than the TDI set by the US FDA and
the EFSA for melamine alone.
Recently, it was determined that melamine, along with
several analogues, may have been responsible for deaths after
ingestion of adulterated food. Melamine is associated with
renal toxicity owing to crystal formation and with carcinoge-
nicity secondary to urolithiasis. Urolithiasis is very common
when melamine is combined with other analogues such as
cyanuric acid [19, 24]. The current safe level, as set by the US
FDA, is 50 ppb based on the sensitivity of the analytical test
[14]. The EFSA also indicated that all products from China
containing more than 15% milk as an ingredient, or products
where the percentage of milk content cannot be established,
will be subjected to documentary, identity, and physical
checks, including laboratory analysis, to determine that any
levels of melamine present in the product do not exceed 2.5
ppm. Those products with more than 2.5 ppm will be destroyed
[25].
Detection methods for melamine
Several methods have been developed for the analysis of
melamine and related contaminates. Melamine and its
metabolites have been analyzed by cation-exchange chroma-
tography with diode-array detection and by liquid chroma-
tography with a UV or mass spectrometry (MS) detector.
These compounds were determined by derivatization before
gas chromatography (GC) or by high-performance liquid
chromatography (HPLC) with UV detection after extraction
with water, ethanol, and a solution of sodium hydroxide [26].
The presence of melamine in pet food was determined by
direct analysis in real-time MS. Liquid chromatography (LC)-
tandem MS (LC-MS/MS) was used to determine melamine as a
metabolite of the herbicide cyromazine and in porcine muscle
tissue. Cyanuric acid has also been analyzed by a variety of
methods, including derivatization and GC-MS, and HPLC with
a UV spectrophotometer based on precipitation of melamine
cyanurate [27, 28]. Other approaches, such as surface-enhanced
Raman spectroscopy (SERS), enzyme immunoassay, enzyme-
linked immunosorbent assay (ELISA), and isotope dilution
were also evaluated. Table 1 provides a compilation of
available analytical methods for the detection of melamine and
cyanuric acid in food products, based on publicly available
information. The LOD, linear ranges of the calibration curves,
and references for different analysis methods are also listed in
Table 1.
Melamine contamination 5

Table 1 Summary of the detection methods used for melamine and cyanuric acid

Method Category LOD Linear range Screening/confirmation Advantage Reference

1 1
UPLC-ESI-MS/MS Feeds 10 igkg -
10-5,000 igkg -
Both High sensitivity [29]
LC-MS/MS Swine and poultry tissues 50 ppb Both High sensitivity [30]
HPLC-MS/MS Plant-origin protein powers 0.5 mg kg 1 -
Both High sensitivity [31]
HPLC-MS/MS Kidney tissue 50 ng mL 1 - 50-1,000 ng mL 1 -
Both High sensitivity [27]
DART-MS Pet food 100 ppm Both High throughput [32]
HPLC-DAD Dog food 0.1 igmL 1 - 0.1-200 igmL 1 -
Screening High throughput [42]
HPLC-DAD Plant-origin protein 10 mg kg 1 -
Screening High throughput [31]
HPLC-UV detection Cereal flours 5 i gg 1
-
Screening High throughput [28]
GC-MS Protein-based foods 0.1 igmL 1 -
0.1-1.0 igmL 1 -
Both High sensitivity [33]
Isotope dilution LC-MS Pet food 2 ppm 2-21 ppm Confirmation High sensitivity [6]
[15N6]melamine
Isotope dilution LC-MS Meat and pet food 10 igkg 1 -
10-100 igkg 1
-
Confirmation High sensitivity [39]
[13C3]melamine
SERS Aqueous solutions 33 ppb Screening High throughput [41]
ELISA Dog food 9 ng mL 1 -
10-250 ng mL 1 -
Screening Low cost per assay [42]
EIA Pet food 0.02 igmL 1 -
0.02-0.5 igmL 1 -
Screening Low cost per assay [43]

The units of the limit of detection (LOD) and the linear range were obtained from the references.
UPLC ultraperformance liquid chromatography, HPLC high-performance liquid chromatography, LC liquid chromatography, ESI electrospray ionization, MS
mass spectrometry, DART direct analysis in real time, DAD diode-array detection, GC gas chromatography, SERS surface- enhanced Raman spectroscopy,
ELISA enzyme-linked immunosorbent assay, EIA enzyme immunoassay

The most common methods for melamine detection utilized
were by GC-MS or HPLC combined with UV or MS detectors.
The LOD and linear ranges of the calibration
curves of these methods were 0.1-0.01 ppm and 0.015 ppm,
respectively. The US FDA laboratories have used analytical
tools such as GC-MS and LC-MS to identify melamine as a
contaminant in foods [27-33].
In selected ion monitoring mode, the GC-MS/MS method
can obtain a limit of quantification (LOQ) of about 100 igg in
melamine. Using selected reaction monitoring of GC-MS/ MS,
this method can reach a lower LOQ of 10 ig g-1. The minimum
reporting level for the GC-MS/MS procedure to screen for the
presence of melamine and related compounds is defined as 10
ig g-1. The GC-MS/MS procedure has been evaluated with
protein materials, pet foods, and animal feeds, and provided
semiquantitative analysis of all four target compounds in one
sample preparation [34].
A method for the determination of melamine residue in food
was developed using LC-MS/MS. For sample extraction
procedures, a mixed-mode strong cation exchange with solid
phase extraction cartridge was used to clean up sample extracts
and remove interfering compounds in dirty matrices such as
seafood and meat. Melamine is a polar molecule, which is a
good candidate for aqueous normal phase (ANP) LC. In ANP
LC, a polar hydrophilic analyte partitions between a relatively
polar stationary phase and a relatively nonpolar mobile phase.
ANP LC is commonly referred to as hydrophilic interaction
chromatography, or HILIC, but the term HILIC implies a
mechanism that is one of several that may be operative under
ANP conditions. Detecting the melamine content with the
HILIC mode results in better retention of this polar molecule
than reversed-phase chromatography. Both compounds,
melamine and cyanuric acid, were analyzed in one run by
utilizing the chromatographic system, based on a zwitterionic
HILIC LC column [35-37]. In its combination with
electrospray ionization MS/MS, the negative ion (cyanuric
acid) and positive ion (melamine) modes were detected. The
methods are described in laboratory information bulletin 4421,
“Determination of melamine and cyanuric acid residues in
infant formula using LC-MS/MS,” and bulletin 4422, “Interim
method for determination of melamine and cyanuric acid
residues in foods using LC-MS/MS.” With use of this
procedure, the amounts of melamine and cyanuric acid
determined from sample extracts from fortified infant formula
were from 0.25 to 5 ig g . The LOQ and the limit of
confirmation were 0.25 ig g 1 for both analytes in dry infant
formula [38].
However, these analyses required extensive sample prep-
aration because the foods were complicated matrices composed
of many compounds. To enhance the sensitivity and accuracy,
the isotope dilution method was the most advantageous [6, 39].
New methods for the routine determination of melamine in
foods have been proposed. Several kinds of isotope-labeled
melamine (Fig. 3) are used for isotope dilution: [ 15N6]melamine
[6], [13C3]melamine [39], [15N3] melamine [10, 36], [13C3,
amino-15N3]melamine, and [13C3, 15N3]cyanuric acid [40]. By
utilization of the isotope dilution method, the matrix effect in
different samples could be prevented. The technique of stable

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6 Y.-C. Tyan et al.
isotope dilution LC-MS/ MS used in previous studies
demonstrates the application of

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Melamine contamination
NH2
■X
NN
JL J' N
(b)
2 H2 N 15
NH2
NN
N 15NH
H.'5r-r
2
(d)
OH
13
'
15N 15N
A HPLC-UV method), GC-MS or GC-MS/MS (US FDA
15
N laboratory information bulletin 4423), and LC-MS/MS (US
HO OH
(e)
Fig. 3 Structures of isotope-labeled melamine and cyanuric acid: a
[15N6]melamine, b [13C3]melamine, c [15N3]melamine, d [13C3, amino-
15
N3]melamine, and e [13C3, 15N3]cyanuric acid
these isotope-labeled compounds to the quantitative analysis of
melamine and cyanuric acid in pet foods [6, 39]. The LOQs for
isotope dilution LC-MS/MS of melamine and
cyanuric acid were 25 and 50 pg kg-1, respectively. With use of
this method, any matrix effects or volume changes can be
corrected [40].
Another new approach to measure melamine and cyanuric
acid is SERS coupled with gold nanosubstrates. SERS is a
branch of Raman spectrosocopy that measures molecular
virations by light scattering. The LOD of SERS for melamine
was determined to be 2.6x10 7mol L (about 33 ppb) calculated
from the relationship between the Raman intensity of the most
prominent peak at around 676 cm-1 and log values of melamine
concentrations. During the measurement, the incident laser
light is inelastically scattered from a sample and the frequency
or wavelength of this light shifts in a manner characteristic of
molecular vibrations. This method can quickly detect and
characterize a small amount of melamine and its derivative
compounds in aqueous solutions [41].
Owing to the melamine contamination in milk and foods,
commercial ELISA test kits were developed for the quantita-
tive analyses as a potential solution to meet the needs ofhigh-
throughput screening of samples. For the ELISA test kit, the
melamine from samples and melamine horseradish peroxidase
conjugate were bound to melamine antibody. After washing, a
clear substrate was added to the wells and any bound enzyme
Conclusions
7
conjugate resulted in a blue color. The color was compared
with the calibrators and the melamine concentration was
derived. Each ELISA plate can analyze around 80 samples. It
has been a reliable, sensitive, and fast method for screening
NH2 melamine [42, 43]. The results from ELISA could include the
effects of possible cross-reactions with related compounds. If
melamine is not detectable, there is no need for further
analysis. Other tests may have to be run if it is detected to meet
regulatory requirements and to ensure that a cross-reacting
2
compound did not cause the response.
These methods have good repeatability and high sensitivity,
and can be applied for the determination of melamine residue
in foods. Several methods for analyzing melamine and co-
contaminants were checked by the US FDA and WHO. Since
the outbreak of melamine contamination, the US FDA has
published six methods for melamine analysis utilizing HPLC
with UV detection (US FDA Forensic Chemistry Center
FDA laboratory information bulletins 4396, 4421, and 4422).
However, melamine is a chemical commonly used in the
manufacture of durable plastics and foam products; but it
should not be present in foods or milk. It is important to
analyze melamine in foods to prevent the violative residues
from entering the human food supply.
A study performed by the US FDA has described the risk to
human health associated with melamine and its analogues.
Melamine has also been shown to be carcinogenic for male
rats. There are no established regulatory limits for melamine
and related triazines in any type of food. None of them should
be present in foods because they are toxic at high-dose
exposures. However, the presence of this nitrogen-rich
chemical in food commodities, such as milk, wheat gluten, and
rice protein, artificially skews the result of protein analysis and
causes the food to be tested as a more protein-rich product.
When melamine is in combination with another triazine,
cyanuric acid, it can result in the formation of insoluble
melamine cyanurate crystals in the kidneys, causing renal
failure. A standardized method for fast determination of
melamine residue in foods should be established. It is
important to monitor all kinds of foods to prevent melamine
contamination.
Acknowledgements We are thankful to S. Sheldon (ASCP) of the Edmond
Medical Center Laboratory (USA) for fruitful discussions. This work was
supported by research grants Q097004 from the Kaohsiung Medical
University Research Foundation and NSC96- 2321-B-037-006, NSC97-
2320-B-037-012-MY3 from the National Science Council, Taiwan,
Republic of China.
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