Byungchul Kim

Date of Award


Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)


Food and Nutrition Sciences


Rodney J. Bushway

Second Committee Member

L. Brian Perkins

Third Committee Member

Alfred A. Bushway


Melamine and cyanuric acid are the compounds that caused the global incidences of kidney related disease to pets and infants in North America and China during the last two years. After a concerted research effort by U.S. laboratories, it was discovered that they were intentionally added in raw ingredients to pet foods or milk to increase nitrogen levels without providing protein. Melamine and cyanuric acid can easily combine by hydrogen bonding and produce a melamine-cyanurate complex that directly caused renal failure to pets and infants who consumed the tainted pet food or milk. Many analytical methods for the determination of melamine and cyanuric acid have been developed. In terms of sensitivity and validity, not all of the methods are useful. In the research described in this thesis, various analytical methods and techniques were explored to find better and easier methods for melamine and cyanuric acid analysis. In the first chapter, methods of enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS-MS) were studied for melamine analysis. The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 µg/mL, respectively. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.997 and 0.989. The r2 values for UPLC-MS-MS with HPLC-DAD and with EIA were 0.957 and 0.949, respectively. The commercial melamine EIA kit used in this study proved to be a rapid and inexpensive alternative to the HPLC-DAD method to quantify melamine in pet foods. In the second chapter, pressurized liquid extraction (PLE) was evaluated and compared with typical extraction methods such as polytron and sonication. Recoveries obtained by the PLE method were significantly higher (P=0.05) than those of sonication and polytron methods for dry pet food samples. For the analysis of adulterated pet foods, PLE resulted in the highest melamine content followed by sonication and polytron. PLE provided the best extraction efficiency compared to sonication and polytron. In the third chapter, liquid chromatography with tandem mass spectrometry (LC-MS-MS) was used for determination of melamine and cyanuric acid using an alkaline pH aqueous extraction solvent (0.1 N ammonium hydroxide). When melamine cyanurate complex was fortified, alkaline extraction solvent yielded better recoveries than the acetonitrile/water (50/50) mixture, which is the most commonly used solvent for extracting melamine and cyanuric acid from various sample matrices. Since similar results were obtained from the adulterated pet food samples, it is assumed tht the adulterated samples were contaminated with melamine cyanurate complex rather than free melamine and cyanuric acid. Therefore, the method developed in this study is effective for the accurate determination of melamine and cyanuric acid.