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Ecotoxicology and Human Environmental Health
Continuing Occurrence of Melamine and its Derivatives in Infant Formula and Dairy Products from the United States: Implications for Environmental Sources Hongkai Zhu, and Kurunthachalam Kannan Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00515 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018
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Continuing Occurrence of Melamine and its Derivatives in Infant Formula
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and Dairy Products from the United States: Implications for Environmental
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Sources
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Hongkai Zhu,† and Kurunthachalam Kannan*,†
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†
Wadsworth Center, New York State Department of Health, and Department of
Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA
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*Corresponding author at: Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA
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Fax: +1 518 473 2895
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E-mail:
[email protected] (K. Kannan)
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Submitted to: Environmental Science & Technology Letters
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TOC
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ABSTRACT
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A total of 52 infant formula and 42 dairy products, including milk, yogurt, cheese, and
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butter, as well as bread purchased from Albany, New York, were analyzed for
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melamine and its derivatives (cyanuric acid, ammeline, and ammelide). ∑Melamine
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(sum of melamine and its three derivatives) was found in infant formula, milk, yogurt,
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cheese, butter, and bread at median concentrations of 4.8, 7.9, 5.1, 17, 4.2, and 7.5
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ng/g, respectively. Median ∑Melamine concentrations in infant formula collected in
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2008 (9.4 ng/g) were significantly higher than those collected in 2018 (2.7 ng/g).
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Similarly, there were significant differences in ∑Melamine concentrations between
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powdered (median: 2.3 ng/g) and liquid forms (9.0 ng/g) of infant formula.
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However, no significant differences in ∑Melamine concentrations were found
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between milk based (5.0 ng/g) and soy based (2.3 ng/g) as well as between organic
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(4.4 ng/g) and non-organic (4.8 ng/g) forms of infant formula. Cyanuric acid was the
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major derivative found in all products analyzed (68–83%), followed by melamine
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(7.0–21%), ammelide (2.5–10%), and ammeline (0–6.0%). The average daily intakes 2
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of ∑Melamine by United States infants via formula and adults via dairy products were
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0.687 and 0.032 µg/kg bw/day, respectively. Our study suggests ongoing but
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low-level contamination of melamine and its derivatives in food products marketed in
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the United States.
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INTRODUCTION
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In March 2007, hundreds of dogs and cats in North America died due to acute kidney
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failure. This incident was associated with melamine adulteration in pet food
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products.1 Subsequently, more than 100 brands of dog and cat food across the United
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States (US) were recalled.2 Later, melamine, found in wheat gluten and rice protein
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concentrate exported from China to the US market, was identified as the source of
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adulteration.3 In another incident in September 2008, a high prevalence of melamine
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was discovered in infant formula in China at concentrations that ranged from 0.1 to
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2,500 mg/kg.4 Exposure to melamine-contaminated infant formula was found to be
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associated with kidney stones.5 According to a report from the Chinese Ministry of
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Health, approximately 300,000 infants were affected by melamine-contaminated
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infant formula from September to November 2008, with six confirmed cases of death
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from kidney failure.6 Melamine concentrations in the serum of affected children were
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as high as 2,563 mg/kg, with a mean concentration of 1,295 mg/kg.7
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Following these food scandals, the attention focused on melamine prompted
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worldwide monitoring of its occurrence in food items, especially milk and/or
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milk-derived ingredients.8-12 In most cases, the measured melamine concentrations
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were below the tolerance limit of 1 mg/kg in infant formula and 2.5 mg/kg in other
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milk products, as set by the US Food and Drug Administration (FDA).13 Several
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hydrolytic derivatives of melamine, such as ammeline, ammelide, and cyanuric acid, 3
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however, received little or no attention in food surveillance programs.14 Although
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these compounds are thought to have low acute toxicity, melamine in combination
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with cyanuric acid can induce severe renal damage.15,16 Therefore, a focus only on
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melamine will underestimate its risks to human health.
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Melamine and cyanuric acid have broad commercial and industrial applications.
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In the US, melamine is approved for use as a laminating agent and flame retardant and
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is found in the melamine formaldehyde resins used in the production of packaging
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materials, dishware, and kitchen utensils.17-19 Cyanuric acid is used in the production
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of plastics and disinfectants and as a disinfectant stabilizer in swimming pools.18 The
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global production of cyanuric acid in 1997 was 160,000 tons. Migration of melamine
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and its derivatives from melamine-containing tableware into food has been
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reported.20,21 These results indicate that, in addition to deliberate adulteration of foods
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(as had occurred in 2007), melamine and its derivatives can originate from approved
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uses.
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The threat of melamine adulteration in food items has gradually subsided, and 10
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years after China’s tainted milk-powder scandal, melamine contamination of
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foodstuffs due to environmental pathways is still not well documented. Further, the
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current status of the occurrence of melamine and its derivatives in foods is not known,
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although baseline exposure doses have been estimated for some countries.22-24
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Chronic low-level exposure to melamine and its derivatives through diet is a concern,
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owing to the renal toxicity of this class of chemicals. Dairy products are of interest
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with regard to melamine exposure due to the potential use of melamine in cattle feed
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to increase the nitrogen content and the ensuing contamination in dairy products.
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The aims of this study were to determine the occurrence of melamine and its
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derivatives, i.e., ammeline, ammelide, and cyanuric acid, in infant formula and dairy 4
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products (including milk, yogurt, cheese, and butter) and bread collected from the US
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market and to estimate the daily intake of the target chemicals in infants and adults. In
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addition, infant formula collected in 2008 was analyzed, and the data were compared
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against those collected in 2018 to elucidate temporal trends in melamine
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contamination in infant formula.
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MATERIALS AND METHODS
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Sample Collection. A total of 52 infant formula products were purchased from
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major retail stores in Albany, New York (NY), US, in 2008 (n = 26; originally
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collected for another study and were stored at -20ºC until analysis) and 2018 (n = 26).
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These products were the most popular brands commonly available in retail stores
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throughout the country. The brands of infant formula purchased in 2018 were
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estimated to account for >80% of the total sales in the US. The samples comprised
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eight commercial brands formulated for 1- to 12-month-old infants. The samples
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included liquid (n = 23), powder (n = 29), milk-based (n = 39), soy-based (n = 13),
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concentrated (n = 6), and organic (n = 10) formulas. In addition to infant formula
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samples, 42 dairy products, consisting of milk (n = 10), yogurt (n = 8), cheese (n = 8),
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and butter (n = 8) were purchased from June through July 2018 from retail stores in
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Albany, NY. Bread (n = 8) also was included due to its high consumption rate and
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potential incorporation of wheat gluten, which was shown to contain melamine in
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2007. All of these products were made in the US and packaged in plastic, glass,
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cardboard, or metal containers. All samples were popular brands, commonly
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consumed by local residents in the Northeastern US. Further details of the samples are
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provided in the Supporting Information (SI; Tables S1–S2). Infant formula and dairy
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product samples were stored at -20o C until chemical analysis. 5
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Infant formula samples were prepared, following the instructions provided on the
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labels, formula samples were mixed with high-performance liquid chromatography
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(HPLC)-grade water, following the manufacturer’s instructions, and the concentrated
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liquid formula samples were diluted 1:1 with HPLC-grade water. Ready-to-use liquid
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formula, milk, and yogurt were analyzed directly from the package, without any
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additional preparation. Cheese, butter, and bread were thoroughly homogenized prior
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to analysis.
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Chemicals. Analytical standards of melamine (99% purity), ammeline (97.9%
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purity), ammelide (98% purity), and cyanuric acid (98% purity) were purchased from
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Sigma-Aldrich (St. Louis, MO, US). Ammonium formate (99.99% purity) and
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ammonium hydroxide (28.0–30.0% NH3 basis) also were purchased from
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Sigma-Aldrich. The isotopically labeled internal standards, 15N3,13C3-melamine (15N3
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at 98% purity,
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13
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13
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MA, US). HPLC-grade acetonitrile, water, dichloromethane, reagent-grade formic
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acid, and hydrochloric acid were purchased from J. T. Baker (Phillipsburg, NJ, US).
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Oasis MAX® and Oasis MCX® solid phase extraction (SPE) cartridges (150 mg, 6 ml,
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30 µm particles) were purchased from Waters Corp. (Milford, MA, US).
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C3 at 99% purity),
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C3-ammeline (13C3 at 99% purity),
C3-ammelide (13C3 at 99% purity), and 15N3,13C3-cyanuric acid (15N3 at 98% purity, C3 at 99% purity), were purchased from Cambridge Isotope Laboratories (Andover,
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Analytical Methods. The method for the extraction of melamine and its
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derivatives in foodstuffs was similar to that described earlier, with some minor
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modifications.18,25 Briefly, infant formula samples (after appropriate preparation, as
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per instructions on the label) and dairy products were extracted with a mixture of
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acetonitrile/water (50:50, v/v), defatted with dichloromethane, and purified, using the
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mixed-mode SPE method (Oasis MAX for cyanuric acid and Oasis MCX for other 6
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analytes). The eluates from MCX and MAX cartridges were concentrated to near
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dryness under a gentle nitrogen stream, resuspended in a 200 µl mobile phase, and
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filtered through a 0.2 µm nylon syringe filter (Corning, NY, US) directly into a glass
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vial prior to HPLC- tandem mass spectrometry (MS/MS) analysis.
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Melamine, cyanuric acid, ammeline, and ammelide were analyzed, using a
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Shimadzu LC-30 AD Series HPLC system (Shimadzu Corporation, Kyoto, Japan),
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connected to an API 5500 triple-quadrupole mass spectrometer (Applied Biosystems,
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Foster City, CA, US). Chromatographic separations were performed, using a Kinetex
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hydrophilic-lipophilic interaction liquid chromatography (HILIC) column (100 mm ×
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2.1 mm, 2.6 µm particle size; Phenomenex, Torrance, CA, USA), serially connected
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to a Betasil C18 guard column (20 mm × 2.1 mm, 5 µm particle size; Thermo
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Scientific, Waltham, MA, US) with acetonitrile (A) and 5 mM ammonium formate
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buffer (pH = 4.0; B) as mobile phases. Cyanuric acid and ammelide were analyzed,
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using multiple reaction monitoring (MRM) negative ion electrospray mode (ES-),
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whereas MRM positive ion electrospray mode (ES+) was used in the detection of
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melamine and ammeline. Details of the extraction and instrumental analysis are
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presented in the SI; chromatograms of a standard and an infant formula sample are
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shown in Figure S1.
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Quality Assurance (QA)/Quality Control (QC). To minimize background levels
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of contamination, disposable polypropylene tubes and pipettes were rinsed with
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methanol prior to use. The HPLC syringe was washed twice before and after any
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injection with a mixture of acetonitrile/water (50:50, v/v). No target analytes were
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found above the limits of quantification (LOQs) in procedural blanks, which were
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analyzed with every batch of 10 samples. Analytes were quantified, using a ten-point
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calibration curve, ranging in concentrations from 0.05 to 200 ng/ml. The regression 7
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coefficient of the calibration curve was >0.996. The recoveries of target compounds in
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fortified blanks (at three different levels of 10, 50, and 100 ng/g) ranged from 75% to
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106%
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15
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into samples, determined by comparison of peak areas between pre-spiked and
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post-spiked samples, were 91 ± 6.6%, 81 ± 11%, 78 ± 14%, and 85 ± 7.2%,
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respectively. The LOQs were 0.042 ng/g for melamine, 0.072 ng/g for ammeline,
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0.058 ng/g for ammelide, and 0.063 ng/g for cyanuric acid, which were determined
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from the lowest acceptable calibration standard (signal-to-noise ratio of 10), a
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nominal sample weight of 2.0 g, and the sample concentration factor of 2.
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with a
relative
standard
deviation
of
Group III (0.47–1.1 µg/kg
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bw/day) > Group I (0.22–0.52 µg/kg bw/day). These results indicate that liquid
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formula contributes higher exposure to melamine and its derivatives than does
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powdered formula. In 2008, the EDIs of melamine via infant formula was reported at
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0.57 to 2.4 µg/kg bw/day in Canada,9 and these values were similar to those
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calculated in our study for US infants in 2008 (0.47–3.2 µg/kg bw/day). In general,
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the exposure doses of melamine for a US infant (0.52–1.3 µg/kg bw/day) fell within
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the reported baseline range at 0.54–1.6 µg/kg bw/day.23 The EDIs of ΣMelamine for
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1- to 3-month-old infants were higher than those for other age groups, which could be
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attributed to the highest intake rates of infant formula and lower body weight for this
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age group.
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Among the five types of dairy products analyzed, the EDI of ∑Melamine by US
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adults varied by an order of magnitude. The highest exposure dose was through milk 13
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consumption (average: 0.020 µg/kg bw/day), followed by cheese (0.005 µg/kg
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bw/day), bread (0.004 µg/kg bw/day), yogurt (0.001 µg/kg bw/day), and butter (0.001
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µg/kg bw/day). The average exposure dose of ∑Melamine by US adults via dairy
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products was 0.032 µg/kg bw/day, which was one order of magnitude lower than the
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EDI calculated for infants via infant formula (average: 0.687 µg/kg bw/day; for infant
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formula purchased in 2018). On the basis of melamine concentrations reported for
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urine from the general US population,38 an estimated total daily intake of 0.050 µg/kg
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bw/day was calculated. The EDI through dairy product consumption was 60% of the
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total exposure dose for the US adults. In addition to dairy products, several other
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sources of melamine exposures exist for adults. The contribution of various food
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categories to human melamine exposure was reported by the European Food Safety
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Authority.39 Among 15 food categories, milk and dairy-based products (average EDI:
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0.25 µg/kg bw/day); vegetables, nuts, and pulses (0.24 µg/kg bw/day); cereals and
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cereal products (0.23 µg/kg bw/day), tap water (0.21 µg/kg bw/day); juices and drinks
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(0.17 µg/kg bw/day); and meat and meat products (0.13 µg/kg bw/day) account for
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three-quarters of the total dietary exposure to melamine in adults from 19 European
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countries. Other potential sources of melamine exposure, including the use of
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trichloromelamine as a disinfectant in food processing (7 µg/kg bw/day), migration
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from melamine-containing food contact materials (plastics: 13 µg/kg bw/day;
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adhesives:
