Study On Enrichment And HPLC Determination Of Four Polycyclic Aromatic Hydrocarbon Metabolites In Urine

Polycyclic aromatic hydrocarbons are the lasting and toxic organic pollutants which distribute widely and exist stably in natural environment. This type of organic chemicals contain many different species relate to human closely and possess the properties of carcinogenesis, teratogenesis and mutagenesis which are great threatens to human health. Especially, the people working in high polycyclic aromatic hydrocarbon exposure environment suffer severe potential hazards. Protecting and promoting the vocational people’s health, preventing and controlling occupational injuries and diseases have drawn a lot of attention. By means of monitoring the polycyclic aromatic hydrocarbon metabolites in urine, the level of intoxication of the organism that contact polycyclic aromatic hydrocarbon could be obtained objectively, which further provides important basis for occupational poisoning diagnosis and treatment. As a result, it is quite necessary to develop an analytical method for the simultaneous determination of various polycyclic aromatic hydrocarbon metabolites.In this stuty, a high performance liquid chromatographic method was established for the simultaneous determination of the four polycyclic aromatic hydrocarbon metabolites in urine. The chromatographic separation and determination conditions were optimized by investigating the influence of mobile phase’s composition and ratio, flow rate, detection wavelength etc on the separation and detection of four components. The optimal conditions were:ZORBAX Eclipse XDB-C18column for separation, a system composed of methanol and water acted as the mobile phase using a gradient elution style with the flow rate was1.0mL/min; the maximum absorption wavelength for each component acted as detected wavelength which was changing with time and the injection volumn of10μL. Under the optimal chromatographic conditions, the four components could be well separated and simultaneously detected in14min, the established method was appropriate for the simultaneous separation and detection of the four components.Two pretreatment methods, i.e. dispersive liquid-liquid microextraction and bamboo charcoal solid phase extraction, were established for the simultaneous extraction and enrichment of the four components in urine. The optimal pretreatment conditions of two pretreatment methods were obtained by optimizing the experimental parameters. The optimal pretreatment conditions of dispersive liquid-liquid microextraction were:200μL carbon tetrachloride was used as the extraction solvent,0.8mL acetone as dispersive solvent, the extraction time was3min and the urine’s pH was about6.0, the dosage of NaCl was0.1g, the drying temperature was65℃. Under the optimal pretreatment conditions, the enrichment factors were between40and48. The optimal conditions for bamboo charcoal solid phase extraction was obtained, acetone-carbon tetrachloride (v:v=4:1) as extraction solvent and the extractant volume was0.5mL, the sample volume was30mL and the pH of urines was about6.0. Under the optimal pretreatment conditions, the enrichment factors were between21and41.Under the optimum conditions of DLLME-HPLC and SPE-HPLC, the working curves for the quantitative analysis of the four urine metabolites were established respectively. The correlation coefficients were ranged from0.9993～0.9999, the linear ranges were greater than or equal to103. For the DLLME-HPLC, the detection limits were0.06-0.3ng/mL (S/N=3), the average recoveries in urine samples were95.4～105%and the relative standard deviations were2.4～3.5%. For the SPE-HPLC, the detection limits were0.08～0.3ng/mL(S/N=3). The average recoveries in urine samples were95.3～107%and the relative standard deviations were2.5～5.3%.The results showed that the established DLLME-HPLC and SPE-HPLC methods for the simultaneous determination of the four polycyclic aromatic hydrocarbon metabolites in urine were simple, rapid, sensitive, accurate and environment-friendly. The analytical method can meet the requirments of biological monitoring work on the four polycyclic aromatic hydrocarbon metabolites in urine.