Reaction Rates and Crystal Formations of Melamine-Cyanuric Acid Complexes in an. Ammoniated Environment. Rebecca Jolitz Citation Drive

Transcription

1 Reaction Rates and Crystal Formations of Melamine-Cyanuric Acid Complexes in an Ammoniated Environment Rebecca Jolitz Citation Drive Los Gatos, CA (408) Dr. James Baxter 1

2 Table of Contents List of Tables, Charts, Figures and Photos.. 2 Introduction..3 Hypothesis...6 Materials and Methods 7 Results..9 Discussion...12 Public Policy Conclusions 15 Acknowledgements 16 References..16 List of Charts: Chart 1 Chart 2 Time versus Weight Difference for Minute Trials 11 Time versus Weight Difference for Day Trials.12 List of Figures: Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Materials..7 Cyanuric acid crystals at 100x Melamine crystals at 400x..10 M-CA complex crystals at 100x Various M-CA complexes at 100x..13 M-CA complex chain at 100x...13 "Crinkled tape" M-CA complex at 100x. 14 2

3 Introduction America has a problem: our food supply is threatened with the intentional adulteration of protein products with melamine-based compounds. This threat was shown to be gravely serious worldwide in two incidents, which are known as the 2007 Pet Food Recall and the 2008 Chinese Milk Scandal. In 2007, hundreds of pets suffered or died from ingesting food contaminated with melamine and cyanuric acid (Reinberg, 2007). Kidney stones in the shape of needles clumping to form concentric rings were blocking and tearing into the kidneys of animals fed the contaminated pet food (Ciancolo et al., 2008, 732; Reimschuessel et al., 2008, 1221). Clinical and pathological studies report that one pig was discovered to have a large amount of edema of the kidneys in necropsy and developed bloody diarrhea, and in another study several cats had tubular ruptures in the kidneys due to circular wedge shaped crystals (Reimschuessel et al., 2008, 1220; Ciancolo et al., 2008, 732). A federal investigation traced the cause of the contamination to cheap wheat flour falsely mislabeled as wheat gluten sold in China, where chemical manufacturers remarked on how they often sold melamine and cyanuric acid to animal feed companies (Barboza and Barrionueva, 2007). It was ultimately determined that a Chinese company had intentionally adulterated the flour with melamine and other related compounds in an effort to artificially increase the nitrogen content. Such products would fool basic protein standards tests that count nitrogens, such as the Kjeldahl, and show a falsely high nutritional value. From this lack of nutritional value, not only were American pets poisoned, but also malnourished. After this incident, melamine and cyanuric acid detection in food became an increased priority, but was not taken seriously until 2008 when thousands of human infants were hospitalized and 6 infants died because of contaminated Chinese baby milk powder (Chang, 2008). This incident, known as the 3

4 Chinese Milk Scandal, demonstrated how dangerous this melamine and cyanuric acid adulteration of food is and indicates how much worse it could become. Melamine, or 1,3,5-triazine-2,4,6-triamine, is actually a rather non-reactive compound (Taskinen and Sherrington, 2000, 85). It is often classified as inert and baked with formaldehyde to form a resin used for kitchen utensils and other materials (known as recycled melamine in this experiment). In addition, it is generally considered only to be hazardous in high quantities. However, its high concentration of nitrogen due to its triazine ring makes it the ideal compound for mimicking proteins in the Kjeldahl test. As melamine is a byproduct of coal production, China is the world leader in melamine production, having it in serious surplus in 2006 (Wang, 2006). Within China, melamine is sold at $1.20 per protein count per ton, in contrast to protein that is sold at $6 per protein count per ton (Barboza and Barrionueva, 2007). Cyanuric acid, 1,3,5-triazine-2,4,6-triol, is also not life threatening alone (Taskinen and Sherrington, 2000, 85). As a weakly acidic analogue of melamine, it is considered to have a low acute toxicity (World Health Organization, 2008, 3). Both cyanuric acid and melamine can be easily acquired for adulteration purposes. Cyanuric acid at 99% purity is sold commercially as pool conditioner, melamine-formaldehyde resin can be ground simply from a kitchen utensil, and refined melamine can be quickly shipped from China in tons for a cheap price (approximately $100 per ton, depending on supply and demand), though the shipping cost is greater than the cost of the melamine. Within China, though, there is no issue of shipping cost. Neither melamine nor cyanuric acid alone can explain the kidney failure that caused the reported deaths in 2007 and 2008, but the infrared spectra of the needle-shaped crystals was observed to be consistent with the presence of melamine and cyanuric acid (Dobson et al., 2008, 259). Studies finally determined the cause and supported that the structures were melamine- 4

5 cyanuric acid complexes (M-CA complexes) that caused the blockage in the renal tubules (Dobson et al., 2008, 261; Reimschuessel et al., 2008, 1225). M-CA complexes are formed when melamine and cyanuric acid spontaneously hydrogen bond with each other, creating insoluble complexes that can hydrogen bond even more to form larger structures (Reimschuessel, 2008, 1225, Seto & Whitesides, 1992, 906). Three important questions critical to this issue that should be answered are 1) how much melamine and cyanuric acid are necessary to form complexes in the environment of the kidney, 2) how long does it take these M-CA complexes to grow, and 3) what kind of melamine was used as the adulterant? The amounts necessary to form observed quantities of M-CA complexes are important to understand the extent of the food adulteration and quantities of adulterant in individual foods. Because Chinese chemical manufacturers have already casually mentioned selling melamine and cyanuric acid for the purpose of adulterating foodstuffs, it is unfortunately likely that there could be many more unknown sources of contamination in the food chain that cause M-CA complex accumulation over time in the kidneys (e.g. wheat flour, milk powder are already documented sources of contamination). Methods such as GC-MS (gas chromatography mass spectrometry), LC-MS (liquid chromatography mass spectrometry), and ZIC-HILIC (zwitterionic hydrophilic interaction chromatography tandem mass spectrometry) have been developed to more accurately sense the levels of melamine and cyanuric acid in specific foods (Food and Drug Administration, 2007; Varelis & Jesekelis, 2008, 1209; Heller & Nochetto, 2008, 3624). However, by the time we know what foods to test with these fancy new instruments, many innocents are already irreversibly damaged from the poisoning. Knowing the quantities of kidney precipitate and thus the quantities of adulterant would aid in detecting adulterated foodstuffs since it is easier to deal with food adulterants in large amounts rather than 5

6 as trace contaminants. So far, there has been no consideration for the effect of time on complex formation though it is critical to understanding how much contaminant is in the American food supply. As far as the American public knows, we could have been poisoned for a long time or a short time, in possibly large or small amounts we simply do not know. Knowing the kind of melamine used for adulteration is important because detection methodologies would differ for pure melamine and recycled melamine. Hypothesis: A much more accurate quantification of melamine in the American food supply could be better ascertained from amounts of crystals formed by known amounts of reacted melamine and cyanuric acid as well as rates of formation than from testing random specific foods. Using such an approximation, it is possible to estimate how much melamine and cyanuric acid could have reacted in the kidneys to form complexes of the weights observed in pets and infants. From there, an assay could be developed to determine the likely level of melamine adulteration independent of food sample analysis given approximations of percent of melamine absorbance by the human body. In addition, the kind of melamine used in adulteration is most likely recycled because melamine resin is much more commercially common in finished products. I hypothesize that if M-CA complexes are precipitated from reacting known amounts of melamine and cyanuric acid, M-CA complexes precipitated will form in solution over a timeframe of minutes with a linear relationship between time and product formation and be more likely to form using recycled rather than pure melamine. If the conditions of this hypothesis are met, the contamination level and time of complex formation can be quantified. 6

7 Methods and Materials: All laboratory reactions took place in a fume hood. Laboratory safety precautions were exercised (lab coat, goggles, gloves.) All solutions were not heated beyond 30 degrees Celsius. Melamine is notoriously difficult to dissolve in water. Acetonitrile is the ideal solvent used in GC-MS and LC-MS to dissolve melamine and cyanuric acid, but cannot dissolve M-CA complexes (Filigenzi, 2008, 7597). Diethylamine is typically added in solution along with acetonitrile to prevent M-CA complex precipitation. These possibilities were considered, but denied as acetonitrile can cause blood toxicity with the lack of a vapor respirator and diethylamine also dissolves M-CA complexes (MSDS(b), 2008, 5). Anhydrous ammonia was also recorded as a good solvent, but was denied due to its corrosivity and a lack of an adequate respirator (Sato, 1967, 1547; MSDS, 2005, 2) Ammonium Figure 1 - Materials hydroxide was eventually chosen as the solvent to use because of its availability. The choice of ammonium hydroxide also can simulate how the reaction takes place in the human body. Ammonium hydroxide in the human body is both secreted by the kidneys to neutralize acidic substances and a waste product of cells in the intestines and liver (Tanner, 1984, 95; Zanghi, Mendoza, Knop & Miller, 2002, 135). Pure melamine powder was purchased from a chemical manufacturer. Recycled melamine powder was obtained through filing a melamine ladle of an undefined concentration of melamine resin. Pure cyanuric acid was obtained in the form of a 4 lb. container of pool conditioner containing 99% cyanuric acid (therefore classified as pure) from Leslie s Swimming pool supply. Ammonium hydroxide solution came from the Ohlone College Chemistry 7

8 Department. Equipment and lab access was provided by the Ohlone College Biology Department (see Figure 1). Whatman qualitative filter paper with a retention factor of over 1 micrometer was used to catch filtrate. A Corning hot plate/stirrer was used to dissolve the chemicals in question, set to 5 on the stirring dial and to 4 on the heating dial. A weighing scale was used to determine weights and amounts of solute to dissolve. Sample Preparation and Solubility Tests: Each reactant (pure melamine, recycled melamine, and cyanuric acid) needed to be dissolved in ammonium hydroxide before it could be reacted. To make each reactant soluble, a certain amount of reactant would be stirred into a known amount of ammonium hydroxide to form a 0.01 mol/l solution (for cyanuric acid, grams were poured into 179 ml; for pure and recycled melamine, grams were poured into ml). If the reactant did not visibly dissolve, it would be heated to 30 degrees Celsius and magnetically stirred via the Corning Stirrer. If the reactant had reached 30 degrees Celsius and still didn t visibly dissolve, the mixture would be filtered through a preweighed filter paper in a 700mL Buchner funnel. Because the last step does not form a 0.01 mol/l solution, the molarity was calculated by dividing the weight of reactant in solution (initial amount of melamine in solution subtracted by the difference in filter paper weight) by the initial volume of ammonium hydroxide. The final step was also used to calculate solubility limits of reactants through increasing the concentration to 0.1 mol/l. If one compound ran out, the process that was the best for dissolving the compound was repeated to make more. 8

9 Rate Reactions and Crystal Formation: Cyanuric acid solution and melamine solution were smeared together on a slide, allowed to dry, and observed for crystal formation. If no crystal formation was observed, the experiment wasn t continued with that melamine mixture. To determine a correlation between time in minutes and precipitate formation, 8mL of melamine solution and 8mL of cyanuric acid solution were immediately mixed and allowed to react for 2, 5, 6, 10, 15, or 20 minutes. The resulting solution was then filtered through a preweighed piece of filter paper in a Buchner funnel. The filter paper was then removed, allowed to dry, and then weighed. The experimental control repeated the procedure with 16mL of ammonium hydroxide. To determine a correlation between time over days and precipitate formation, mL of each reactant were poured together into an Erlenmeyer flask and sealed for 1, 2, 3 or 7 days. Then 10-20mL of the solution were pipetted onto a preweighed piece of filter paper in a Buchner funnel. The filter paper was then removed, allowed to dry, and then weighed. Results were interpreted in grams of weight difference divided by milliliters of reacted solution transferred. All photographs of crystal forms were taken through afocal projection, where I set the camera to maximum magnification as I adjusted the focus of the microscope for a sharp photograph. Results Sample Preparation and Solubility Determinations: Recycled melamine had significant difficulty dissolving at 30 degrees Celsius while being stirred, as there was no visible dissolution. The solubility limit of recycled melamine was determined to be ± mol/l. Both cyanuric acid and pure melamine visibly 9

10 Rebecca Jolitz dissolved after stirring, as no solid particles were visible when the mixtures were heated at 30 degrees Celsius while being vigorously stirred. In later experiments, the solubility limit of pure melamine was determined to be ± mol/l in ammonium hydroxide. Constant and Rate reactions: There was no M-CA complex precipitation in the reacted solution of recycled melamine and cyanuric acid when observed under a microscope at 400x. Recycled melamine was discarded and further data on recycled melamine was not collected. M-CA complex Figure 2 - Cyanuric acid crystals at 100x precipitation was observed under a microscope for reacted solutions of pure melamine and cyanuric acid. Melamine samples took the form of large, granular particles (see Figure 2) and cyanuric acid samples took the form of needles (see Figure 3). M-CA complexes appeared as small, rectangular boxes (see Figure 4) that in solution tended to form chains. There were also large rectangular crystals that formed at the edges of the area stained with the M-CA mixture. Due to a lab clean-up error, bacteria were observed to be growing in one M-CA complex sample. No crystal precipitate or change of color was visible with the naked eye over minutes. Powder-like precipitate was Figure 3 - Melamine crystals at 400x 10

11 Rebecca Jolitz observed in the reactions over days, though. Chains of precipitate also formed on the surface of the solution, but were easily disrupted by turbulence. Figure 4 - M-CA complex crystals at 100x Comparison Graph of Minutes of Reaction versus Weight Difference for Trial 1, Trial 2, and a Control Trial 1 Trial 2 Control Weight Difference (grams/milliliter) Time (minutes) Chart 1- Time versus Weight Difference of Minute Trials

12 Comparison Graph of Days of Reaction versus Weight Difference for Trial 1, Trial 2, and a Control Weight Difference in Filter (grams/milliliter) Trial 1 8 Trial 2 Control Trial 3 Time (days) Chart 2 - Time versus Weight Difference of Day Trials Discussion Recycled melamine didn t dissolve at 30 degrees Celsius or form a crystalline complex with cyanuric acid. For safety s sake, the recycled melamine was only dissolved at a temperature of 30 degrees Celsius; so recycled melamine may have dissolved at a slightly higher temperature. But if recycled melamine was adulterating the food supply rather than pure melamine, the kidney stones might have not formed and caused the recent deaths concerning contamination. Given that the adulteration process has been discovered only in China and China has a surplus of pure melamine on the world market, food manufacturers could have bought it much cheaper than labor for grating recycled melamine as in this experiment. It is a strong possibility that pure rather than recycled melamine was used to adulterate food cheaply. 12

13 Rebecca Jolitz The appearance of the M-CA crystals in this experiment is not consistent with the M-CA complexes described in the histological analyses of the pet kidney precipitates, but similar (Reimschuessel, 2008, 1221, see Figure 5). Conditions in the kidneys may have promoted linking of the M-CA complexes in concentric arrangements. Residual undissolved Figure 5 - Various M-CA complexes at 100x cyanuric acid may have formed the needles that pierced the renal tubules. As most crystals form in the distal tubules rather the proximal tubules (Brown, 2007,526), it could have been that the melamine and cyanuric acid were not dissolved till they reached the proximal tubules. Melamine and cyanuric acid would then be dissolved by ammonium hydroxide ions at body temperature (37 degrees Celsius), and began to build complexes. However, the complexes probably built up slowly and only started forming major kidney stones Figure 6 - M-CA complex chain at 100x in the distal tubules and collecting ducts. The crystals in the kidneys and crystals in this experiment also differ in color. The brown appearance of the kidney precipitate could have been caused by staining of the crystals with materials passing through the kidneys, such as blood where complexes have cut into the tubular walls. 13

14 Rebecca Jolitz According to Zerkowski, Seto and Whitesides, melamine and cyanuric acid can hydrogen bond and form three main types of M-CA complex, dubbed rosette, crinkled tape, and linear tape (1992, 5473). These complexes would be formed from smaller 1:1 M-CA complex units (Seto and Whitesides, 1992, 906). It could be that only one type of larger complex forms in the kidneys because it would be much stronger than the other Figure 7 - "Crinkled tape" M-CA complex at 100x M-CA complexes that could form. It is also possible that the box-like units observed under the microscope link together to form these different structures. The chain like structure observed in Figure 6 and macroscopically was probably an example of a weaker structure, since it broke apart easily under turbulence. The structure in Figure 7 could be an example of a more durable form, or maybe the crinkled tape forms observed in the Seto and Whitesides papers. Perhaps M-CA complex buildup in the kidneys takes just the time for stronger crystals to aggregate around a center, while other crystal formation is washed out and broken. Public Policy The results of this research have several international and national public policy implications. Several factors led to the major 2008 Chinese Milk Scandal, including and especially cheap melamine production as a byproduct of coal mining. Countries that have large coal mining industries face similar issues of food adulteration as China. Despite the development of new tests to detect melamine, third-world countries would tend to stay with the ineffective and cheap Kjehldahl tests for nitrogen because newer testing equipment is significantly more 14

15 expensive to implement and maintain with integrity. Those countries would be at substantial risk from people intending to make an extra profit through adulterating food with melamine. Since America s most common form of melamine is recycled rather than pure, the economic incentive is not as likely to impact peoples health to the degree seen in China because it was determined that recycled melamine did not dissolve or form dangerous complexes. Malnourishment would still result, though, because of the nutritional displacement in bulk. Because of this, possible recycled melamine adulteration should not be ignored as a threat within America. America is also in danger due to cheaply imported food components from countries with a high degree of coal mining, such as melamine-adulterated wheat flour in the 2007 Pet Food Recall. Although America can afford newly developed expensive tests for melamine, they are minimally effective due to the huge volume of imported products, rendering small sample techniques difficult. Because of the ease through which an adulterated product can be relabeled, repackaged, and remixed through multiple transit points to obscure the country and company of origin, every nation is at risk. As such, reliance on scientific methods and reactive procedures such as recalls may not be enough. A proactive approach to public policy such as regulatory, legal, and trade agreements are critical to preventing widespread tragedies in the future. Conclusions The hypothesis was incorrect. Rate data charting product formation in minutes displayed conflicting trends (see Chart 1). This was most likely due to contamination in several samples because of an unclean tray. Rate data charting product formation over days displays increasing trends that gradually plateau after 3 days. In trial 2 after the amount of M-CA complex plateaus, M-CA complexes form at a rate of grams per hour for a 150mL mixture. Recycled 15

16 melamine was significantly less likely to produce M-CA complexes. Future work would involve examining the mechanisms for melamine and cyanuric acid absorption in the body. The rates of formation determined in this experiment would be significantly impacted by the time melamine and cyanuric acid persists in the bloodstream, how much is absorbed, and the effects of other body secretions on complex formation. Such a study would need to be at a much larger scale as well as requiring specialists, significant lab resource, and time to explore these effects. Acknowledgements: I d like to thank Professor Baxter of the Ohlone College Biology Department for supervising the research and mentoring me. I d also like to thank Dr. Grant and Dr. Ganguly of the Ohlone College Chemistry Department for their advice throughout the project. References: Barboza, D. & Barrionueva, A. (2007, Apr 30). Filler in Animal Feed Is Open Secret in China. New York Times. Brown, C.A., Jeong, K., Poppenga, R.H., Puschner, B., Miller, D.M., Ellis, A.E., et al. (2007). Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and Journal of Veterinary Diagnostic Investigation. 19, Chang, Anita (2008, Dec 30). China dairy boss delayed reporting issues. New York Times. Filigenzi, M.S., Puschner, B., Aston, L.S., & Poppenga, R.H. (2008). Diagnostic determination of melamine and related compounds in kidney tissue by liquid chromatography/tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 56, Cianciolo, R.E., Bischoff, K., Ebel, J.G., Van Winkle, T.J., Goldstein, R.E., & Serfilippi, L.M. (2008). Clinicopathologic, histologic, and toxicologic findings in 70 cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid. 233(5), Dobson, R.L.M., Motlagh, S., Quijano, M., Cambron, R.T., Baker, T.R., Pullen, A.M, et al.. (2008). Identification and characterization of toxicity of contaminant in pet food leading to an outbreak of renal toxicity in cats and dogs. 106(1), Food and Drug Administration, (2007, May 22). GC-MS screen for the presence of melamine, ammeline, ammelide, and cyanuric acid. Retrieved January 9, 2009, from U.S. Food and Drug Administration Web site: 16

17 Heller, D.N., & Nochetto, C.B. (2008). Simultaneous determination and confirmation of melamine and cyanuric acid in animal feed by zwitterionic hydrophillic interaction chromatography and tandem mass spectrometry. Rapid Communications in Mass Spectrometry. 22, MSDS. (2008). Melamine MSDS. MSDS. (2008). Acetonitrile MSDS. MSDS. (2005). Ammonia MSDS. Reimschuessel, R., Gieseker, C.M., Miller, R.A., Ward, J., Boehmer, J., Rummel, N, et al.. (2008). Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs. 69(9), Reinberg, S. (May 4, 2007). Pet food recall widens again on new threat. Washington Post. Sato, K. (1967).New method for the study of melamine resin (New solvents for melamine resin). Bulletin of the Chemical Society of Japan. 40(6), Seto, C.T. & Whitesides, G.M. (1993) Molecular self-assembly through hydrogen bonding supramolecular aggregates based on the cyanuric acid-melamine lattice. Journal of the American Chemical Society. 115, Taskinen, K.A. & Sherrington, D.C. (2000). Self-assembly in synthetic macromolecular systems via multiple hydrogen bonding interactions. Chemical Society Reviews. 30, Tanner, G.A. (1984).Renal regulation of acid-base balance: ammonia excretion. The Physiologist. 27(2), Varelis, P., & Jesekelis, R. (2008). Preparation of [13C3]-melamine and [13C3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry. Food Additives and Contaminants. 25 (10), Wang, Ruilin. (2006, Jan 6). Melamine capacity is serious surplus. China Chemical Reporter. World Health Organization (2008). Melamine and cyanuric acid: Toxicity, preliminary risk assessment and guidance on levels in food. Retrieved January 9, 2009, from World Health Organization Web site: Zanghi, J.A., Mendoza, T.P., Knop, R.H., & Miller, W.M. (2004). Elevated pco2 inhibits the polysialylation of the neural cell adhesion molecule in CHO MT2-1-8 cell cultures. New 17

18 developments and new applications in animal cell technology (pp ). Netherlands: Springer. Zerkowski, J.A., Seto, C.T. & Whitesides, G.M. (1992). Solid-state structures of "rosette" and "crinkled tape" motifs derived from the cyanuric acid-melamine lattice. Journal of the American Chemical Society. 114,