Method Research For Simultaneous Determination Of Melamine, Cyanuric Acid And Uric Acid By Reversed Phase High Performance Liquid Chromatography

Reversed phase high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of Melamine, Cyanuric Acid and Uric Acid using a C18 column. The method has been used to detect the three compounds in blood and urinary calculi. The method is simple, accurate for the simultaneous determination of melamine, cyanuric acid and uric acid. It has high RSD and perfect coefficient of recovery.Research contents and results:(1) Selection and optimization of HPLC conditions:ion pair solution and potassium phosphate buffer are selected to be the water phase while using acetonitrile as the organic phase. Radio of the mobile phase, pH of the solution and detect wavelength are discussed to separate melamine, cyanuric acid and uric acid by C18 column. As a result, cyanuric acid can't be detected when using ion pair solution, while melamine and uric acid can be analyzed simultaneously under this condition. Melamine, cyanuric acid and uric acid are separated by C18 column using 100% potassium phosphate buffer (10mmol/L, pH=7.50) as mobile phase, and the wavelength is 200nm.(2) Application and validation of the method:①Selection and optimization of sample preparation:acetonitrile-water extraction solutions with different matching (1:1,2:1,3:1) are compared to develop a simple, cheap method for pretreatment. As a result, the 2:1 acetonitrile-water extraction solution is suitable for sample preparation with high protein precipitation and less waste.②RSD (Relative standard deviation) of the retention time and area was calculated after a repeated injection of an unknown blood sample for 6times. The RSD are between 0.48% and 1.00%; LOD (Limit of detection) and LOQ (Limit of quantitation) are calculated according S/N (Signal/Noise) 3 and 10. LOD of melamine, cyanuric acid and uric acid are 0.0045μg/mL,0.0248μg/mL and 0.0231μg/mL. LOQ of melamine, cyanuric acid and uric acid are 0.015μg/mL,0.083μg/mL and 0.078μg/mL; Recovery experiment is performed with two concentrations to add. The recoveries are between 92.59% and 107.26%.