Study On Determination Of Six Urinary Metabolites Of Aromatic Compounds By HPLC

Benzene, toluene, styrene, aniline, nitrobenzene and other aromatic compounds are common types of harmful substances, which are widely used in the fields of industrial and agricultural production and scientific research. The human body exposure to these harmful substances through inhalation or skin may cause different degrees of damage to nerve, respiration, skin, blood and other systems or tissues. Therefore, it is of great significance to monitor the above harmful substances, which are widely used in our country and easily lead to poisoning. Harmful substances are metabolized by the body and transformed into its corresponding metabolites in urine and other biological materials, which could be quantitatively detected. Such kind of biological monitoring provides an effective complement to the environmental monitoring and makes monitoring results more scientific and accurate. However, there has not been developed mature method for simultaneous determination of metabolites of a variety of harmful substances in urine of non-occupational exposed people, lacking of the background value and samples library about metabolites in urine of the normal population. As a result, it is quite necessary to develop an analytical method applied to the determination of metabolites of harmful substances.A high performance liquid chromatographic method was established for the simultaneous determination of six metabolites trans,trans-muconic acid (tt-MA), hippuric acid (HA), mandelic acid (MA), phenylglyoxy acid (PGA), p-aminophenol (PAP) and p-nitrophenol (PNP) in urine. The influence of the component and proportion of mobile phase, detection wavelength, flow rate, column temperature and injection volume on the separation and determination of six components were studied. The optimal chromatographic conditions included a Nova-Pak C18column. A system composed of methanol and water including acids (0.01%H3PO4+0.02%HAc) acted as the mobile phase using a gradient elution style. The maximum absorption wavelength for each component was used, and changed the detection wavelength along with time. The flow rate was1.0mL/min, the column temperature was 35℃and the injection volume was10μL. Under the optimal conditions, the simultaneous detection of the six components was achieved within20minutes. The linear correlation coefficients of working curves ranged from0.9993to0.9997, the linearity range was greater than or equal to103. The detection limits were0.003～0.03μg/mL (S/N=3). The average recoveries of samples were86.2%～108.1%and the relative standard deviation were2.2%～4.6%.The extraction methods of six metabolites in urine were studied systematically. The liquid-liquid extraction method was used, the optimal pretreatment conditions were determined by single factor experiments and orthogonal experimental design as follows: dichloromethane-isopropanol (v:v=7:3) as extraction solvent, sample volume was5mL, extractant volume was5mL, added0.75g NaCl, the extraction time was2min. Extracted under acidic conditions first time, then added1.5g K2HPO4into water phase, repeated the extraction once, and then merged the two extraction liquid. The extract liquor was volatilized in water bath with the volatilization temperature less than70℃, then dissolved with1.0mL mobile phase. The six components in urine were extracted simultaneously by the established pretreatment methods, while extraction efficiency was above90%.The research results show that the HPLC method, which for simultaneous determination of aromatic compounds metabolites in urine, was sensitive, rapid, lower cost, and had a good analytical performance. The analytical method could meet the needs of biological monitoring work on the metabolites of harmful substances in urine.