Study On The Synthesis, Sepreation And Analytical Methods Of Organophosphate Ester Metabolites

Organophosphate(OPEs) compounds were widely used in many products for high flame retardant property and strong plasticity, such as varnish, polyurethane foam, upholstery and textiles etc. Usually, OPEs was filled into materials by physical way, so it will be worn and released to environmental medium in the process of production, transportation and service. Currently, OPEs were detected in various environmental media including air, groundwater, sediment, organisms and humans. Confirmed through in vivo and in vitro experiments, OPEs can produce a metabolic transformation in human body, metabolism is the main factor to determine the toxic and biomagnification of pollutants in life, so the research on the metabolism of OPEs in human body has become particularly important. However, the domestic and international research was mainly focus on the synthesis of new type of OPEs, separation and analysis methods and ecological toxicity of OPEs. Only Birgit Karin Schindler’s research group from Germany studied limited several metabolites of OPEs in human urine. The lack of OPEs metabolites samples was a key factor in restricting the further research. The commonly used preparation method of OPEs metabolites was mainly via hydrolysis of prototype compounds, the reaction degree was difficult to control and the purity of the product was not high enough. In view of this situation, this paper aimed to synthesize a series of OPEs metabolites and study their separation and analysis methods. The main contents are as follows:1. Synthetizing OPEs metabolites. Phosphorus oxychloride and hydroxyl compounds(ethylene glycol butyl ether, ethylene chlorohydrin, 2-Propylene Chlorohydrin, phenol,p-cresol, m-cresol) was as raw materials, di-phosphate ester compound was synthetized through controlling the reaction temperature and the equivalent. Then via hydrolysis reaction, the chlorine atoms were converted into hydroxyl groups to obtain final six kinds of OPEs metabolites. The influences of synthetic temperature, concentration and reaction time on the yields were studied through the response surface method(RSM). Design of Expert software analyzed the optimum conditions of hydrolysis reaction. 1HNMR and EIMS were employed to study the structure and molecular weight of six kinds of OPEs metabolites. It provided strong evidence for successful synthesis of a series of metabolites of OPEs sample.2. Liquid chromatography tandem mass spectrometry(LC-MS/MS) method for detection of OPEs metabolites in human urine. The LC-MS/MS analysis method for OPEs metabolites was optimized by adjusting the instrument parameters. The qualitative and quantitative of 6 OPEs metabolites ion were determined by collision and forming dissociation fragments(CID). The quantitative ion of DBEP, BCEP, BCPP, DPh P, Dp CP, and Dm CP were 299, 222, 246, 251, 279 and 279, respectively. During sample pretreatment, the different effects of purification and extraction between solid phase extraction extraction(SPE) and liquid-liquid extraction(LLE) of target compounds were compared through the standard addition experiment. The results showed that the recovery rate for controlled standard samples was above 80%, but there are large differences between water standard samples and urine standard samples. The optimized LLE method was established for satisfying the dual requirements of high stability and high recovery rate. The recovery rate for controlled standard samples, water standard samples and urine standard samples were reached more than 80%, 90%-125% and 95-180%, respectively. Although the method would lead to higher recovery rate of urine standard samples because of matrix effects, the separation and analysis method of organophosphate metabolites in human urine were determined.3. Gas chromatography-mass spectrometry(GC-MS) method for detection of OPEs metabolites in human urine. Based on the limitations of LC-MS/MS, The target compounds were derivatized by 2,3,4,5,6-Pentafluorobenzyl bromide and gas chromatography-mass spectrometry(GC-MS) was established for detection of OPEs metabolites in urine. The influences of different solvents, catalysts, derivatization reaction time and reaction temperature on derivative effect were studied. During adding standard samples experiments, when the water standard, urine standard samples samples at a concentration of 300-900 mg /kg, the values of RSD were less than 6% while recovery rates were more than 80%. The effectiveness of the proposed method was verified.