The Study On The Related Substance Of Metrifonate And Its Analytical Method

[Purpose] The purpose is to develop the test method of related substances in metrifonate, establish the test conditions of HPLC, separate the related substances from trichloroethyl phosphate, and analyze the related substances separated and identify the molecular structure, and validate the HPLC analytical method, finally finish the study for the related substances in metrifonate and analytical method validation, test the related substances in metrifonate manufactured by Xinhua according to the analytical method.[Methodology] Referring to the literatures, we have developed the HPLC analytical method and selected the testing conditions, detected the corresponding compound structure m/z and its structural fragments of the major related substances through a liquid chromatograph-mass spectrometer, identified the basic structure through preparing major impurities and checking the infrared absorption spectra and ultraviolet absorption spectra.[Results] The analytical method of related substances in metrifonate has been validated, the validation includes specificity, precision, linearity and range, accuracy, limit of detection (LOD), limit of quantitation (LOQ), robustness etc. The relative standard deviation (RSD) of metrifonate peak area from system suitability test and from repeatability is0.28%, and the RSD of metrifonate peak area from intermediate precision is0.17%; There is good linear relationship when related substances are in the range of0.005mg/ml-0.5mg/ml and the correlation coefficient, R, is0.99999, the analytical method is applicable when the related substances are in the range of0.005mg/ml-0.5mg/ml. Each of recoveries is in the range of95.0%-105.0%, and all of RSDs are less than2.0%; In the robustness, metrifonate solution preserved for8hours is stable, the RSD of metrifonate peak area is less than2.0%; The specificity, LOD and LOQ for the known impurity DMN and DDVP have been conducted, the results are as follows: LOD of DMN is0.04356μg/ml, LOQ of DMN is0.2178μg/ml; LOD of DDVP is0.00572μg/ml, LOQ of DDVP is0.0286μg/ml. The validation above meets the requirements, which shows the analytical method is applicable to test the related substances in metrifonate.The correction factors of DMN and DDVP separately corresponding to metrifonate have been determined, in which, fDMN=0.54, fDDVP=0.031.