Study On The Analytical Method And Exposure Of Phthalates And Their Metabolites

Phthalates are environmental endocrine disruptors those are widely used inindustrial and commercial applications. They have been found in body fluids such ashuman urine, serum, breast milk, and so on. Animal experiments have shown thatphthalates have adverse effects on the female reproductive system and fetaldevelopment. However, it is lack of epidemiological data. Reports about phthalatesexposure of women are rather limited. We developed the methods for determination ofphthalates and their metabolites in human urine and follicular fluid, and applied todetect phthalates and their metabolites in reproductive age women, infertile women,and early pregnant women. It accumulated data for phthalate exposure in women. Wealso studied the relationship between phthalate exposure and early embryonic death.Phthalate intakes from drinking water in plastic container, inhalation of indoorair and ingestion of indoor dust were estimated. Phthalate intakes through drinkingwater and indoor dust were less than1%according to the reference dose(RfD)established by USEPA. Phthalate intake from indoor air was less than2%accordingto RfD. Compared to the daily intake estimated from urinary metabolites, inhalationof indoor air and drinking water were the main routes of di-n-butyl phthalate(DBP)exposure. But drinking water, inhalation of indoor air and ingestion of indoor dustwere not the main routes for di-(2-ethylhexyl) phthalate(DEHP) exposure. It ispossible that food is the main route for DEHP intake.Analytical method was developed for the simultaneous determination ofphthalates and their metabolites in human urine. Sample pretreatment was carried outby solid phase extraction. The conditions of pretreatment such as eluent, elutionvolume and elution velocity were optimized. The optimized conditions were2mL ofacetonitrile,2mL of ethyl acetate and1mL of ethyl ester-n-hexane(1/19,V/V)in theflow rate of1mL/min. Under the optimized conditions, phthalates and theirmetabolites were determined by high performance liquid chromatography(HPLC).Good linear relationships were obtained in the range of20to10000μg/L with thecorrelation coefficients above0.99. The limits of detection (LOD) ranged from3.8to7.0μg/L. The recoveries varied between72.2and102.3%. Precision measured by therelative standard deviation (RSD) was lower than8%. It is necessary to derivatizatephthalate monoesters when they are measured by gas chromatography-massspectrometry(GC-MS). Bis(trimethylsilyl)-trifuoroacetamide (BSTFA) was chosen asderivative reagent. The amount of BSTFA, derivatization temperature and time wereinvestigated. Phthalate monoesters could be completely derivatizated with20μL ofBSTFA at30℃for30min. The linear range was from5to1000μg/Lwith correlation coefficients greater than0.98. The LOD of phthalates and their metabolites werebetween0.3and1.1μg/L. For all analytes, the mean recoveries ranged from77.9to97.7%,RSD was between3.7and10.9%. Compared to HPLC, GC-MS with lowerLOD was more suitable for the determination of low levels of analytes. On the baseof the method of GC-MS, we developed an analytical method to determine phthalatesand their metabolites in human follicular fluid. Recoveries were in the range of79.8to90.9%. RSD was between6.1and11.1%. It was applied to estimate exposure tophthalates by human follicular fluid.The methods for the determination of phthalates and their metabolites in humanurine and follicular fluid were applied to estimate exposure to phthalates inreproductive age women, infertile women, and early pregnant women. Thefrequencies of detection for DEHP, mono-n-butyl phthalate (MBP), DBP,mono(2-ethylhexyl) phthalate(MEHP), monoethyl phthalate(MEP), monobenzylphthalate (MBzP), and monomethyl phthalate(MMP) were more than75%in50urinesamples from reproductive age women. MEP with the highest concentration was68.4μg/L(median). DBP with the lowest concentration was5.0μg/L(median). Thetotal daily intake of DEHP estimated from DEHP metabolites was the maximumphthalate intake. DEHP intakes of two investigators were higher than theRfD(20μg/kg bw/day). Daily intakes of diethyl phthalate(DEP), DBP and butylbenzylphthalate(BBzP) were much lower than the RfD. Phthalates and their metaboliteswere determined in follicular fluids collected from36infertile women accepted invitro fertilization and embryo transfer. DEHP, MBP, DBP, MEHP, MEP, MBzP, andMMP were found in follicular fluids. MBP and MEHP were detectable in all samples.MBP had the highest concentration of12.4μg/L (median). The median concentrationfor MEHP was6.1μg/L. DEHP with the lowest concentration was3.6μg/L(median).Phthalates and their metabolites were also determined in urine samples from65pregnant women who had early embryonic death and50controls who had embryonicsurvival. The frequencies of detection for MBP, MEHP, MEP, DEHP and MMP inpregnant women were more than90%. The highest concentration was measured forMEP (median80.3μg/L). The following was MBP with the level of21.4μg/L. Thelowest concentration was measured for MBzP (median5.2μg/L).Questionnaires were finished in case-control groups. The single factor analysisshowed that the time after decoration in case group was4.16years, which wassignificantly shorter than those of7.06years in controls(P<0.05). House cleaningfrequency≦1/week in case group was55%, which was significantly higher thanthose of24%in controls (P<0.05). The percentage of using fresh-keeping film andplastic containers in case group was92%, which was significantly higher than thoseof76%in controls(P<0.05). The results of multivariate logistic regression showed that house cleaning frequency and the time after decoration were the risk factors ofearly embryonic death. The fewer house cleaning frequency, the higher risk of earlyembryonic death. And the shorter time after decoration, the higher risk of earlyembryonic death. The mean concentrations of MMP, MEP, MBP, MEHP, MBzP andDEHP in case group were higher compared to the controls (P<0.05). Crosstabs wasused to analyze the relationship between high concentrations of phthalates and theirmetabolites (above the median) and early embryonic death. The greatest oddsratios(OR) was observed for MBP(OR=16.1), followed by MMP(OR=7.1),MEP(OR=5.0), MEHP, MBzP and DEHP(OR=2.7). It suggested that highconcentrations of phthalates easily leaded to early embryonic death.