邻苯二甲酸酯及其代谢物分析方法研究及暴露评价
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摘要
邻苯二甲酸酯是一类应用广泛的环境内分泌干扰物，已在人体尿液、血液和乳汁等体液中检出。动物实验已表明邻苯二甲酸酯影响雌性动物生殖功能和胎儿发育，但尚缺乏流行病学的相关证据。目前，关于女性群体的邻苯二甲酸酯内暴露数据仍很有限。本研究建立了人体尿液和卵泡液中邻苯二甲酸酯及其代谢物的分析方法，并用于育龄妇女、不孕妇女和孕早期妇女等女性群体的邻苯二甲酸酯内暴露评价，为女性群体的邻苯二甲酸酯暴露积累了基础数据；并通过病例-对照组研究了胎停育与邻苯二甲酸酯类物质的相关性。
考察了使用塑料水杯饮水、室内降尘和室内空气等外暴露途径对邻苯二甲酸酯的污染，结果表明使用塑料水杯饮水和室内降尘带来的邻苯二甲酸酯日摄入量低于美国EPA规定的参考剂量的1%，室内空气带来的邻苯二甲酸酯日摄入量低于参考剂量的2%。与尿液代谢物估算的日摄入量比较发现，室内空气和塑料水杯饮水是邻苯二甲酸二正丁酯（di-n-butyl phthalate，DBP）的主要暴露途径；而室内空气、室内降尘和塑料水杯饮水均不是邻苯二甲酸二（2-乙基己基）酯(di-(2-ethylhexyl) phthalate， DEHP)的主要暴露途径，推测DEHP的主要暴露途径可能是食物摄入。
建立了同时测定人体尿液中邻苯二甲酸酯及其代谢物的分析方法。采用固相萃取方法对样品进行前处理，考察了萃取剂种类、萃取剂体积和萃取流速等影响固相萃取的因素，确定了最佳固相萃取条件为：用2mL乙腈、2mL乙酸乙酯和1mL乙醚-正己烷（1/19，V/V）洗脱，洗脱流速为1mL/min。采用高效液相色谱法（high performance liquid chromatography，HPLC）测定邻苯二甲酸酯及其代谢物，线性范围为20～10000μg/L，相关系数均大于0.99，最低检出限为3.8～7.0μg/L，回收率为72.2～102.3%，相对标准偏差（relative standard deviation，RSD）均小于8%。采用气相色谱-质谱法（gas chromatography-mass spectrometry，GC-MS）测定邻苯二甲酸单酯时需要进行衍生化处理，选用N，O-双三甲基硅基三氟乙酰胺（bis(trimethylsilyl)-trifuoroacetamide, BSTFA）作为衍生化试剂，考察了衍生化试剂用量、衍生化时间和温度等因素，确定衍生化条件为在样品中加入20μLBSTFA于30℃下衍生化30min。邻苯二甲酸酯及其代谢物的线性范围为5～1000μg/L，相关系数均大于0.98，最低检出限为0.3～1.1μg/L，回收率为77.9～97.7%，RSD为3.7～10.9%。比较两种分析方法，发现GC-MS法检出限较低，更适于低浓度邻苯二甲酸酯及其代谢物的分析。在尿液（GC-MS法）分析方法的基础上建立了卵泡液中邻苯二甲酸酯及其代谢物的分析方法，待测物的回收率为79.8～90.9%，RSD为6.1～11.1%，可用于卵泡液为基质的邻苯二甲酸酯暴露评价。
采用人体尿液和卵泡液中邻苯二甲酸酯及其代谢物的分析方法，对育龄妇女、不孕妇女和孕早期妇女等女性群体进行邻苯二甲酸酯内暴露评价。以50位育龄妇女为研究对象，测定尿液中邻苯二甲酸酯及其代谢物的含量。在育龄妇女尿液中检出了DEHP、邻苯二甲酸单丁酯（mono-n-butyl phthalate, MBP）、DBP、邻苯二甲酸单（2-乙基己基）酯（mono(2-ethylhexyl) phthalate, MEHP）、邻苯二甲酸单乙酯（monoethyl phthalate, MEP）、邻苯二甲酸单苄酯（monobenzyl phthalate,MBzP）和邻苯二甲酸单甲酯（monomethyl phthalate, MMP），且检出率大于75%。其中，MEP的浓度（中位值）最高，为68.4μg/L，DBP的浓度最低，为5.0μg/L。根据尿液中代谢物的含量估算日摄入量，结果表明DEHP是日摄入量最高的物质。有2位被调查对象（4%）DEHP摄入量大于美国EPA规定的参考剂量(20μg/kgbw/day)。而邻苯二甲酸二乙酯（diethyl phthalate, DEP）、DBP和邻苯二甲酸丁基苄基酯（butylbenzyl phthalate, BBzP）摄入量均远低于规定值。对接受体外受精-胚胎移植的36位不孕妇女患者的卵泡液进行分析，在卵泡液中检出了DEHP、MBP、DBP、MEHP、MEP、MBzP和MMP。其中，MBP和MEHP在所有卵泡液中均检出，检出浓度（中位值）分别为12.4和6.1μg/L。MBP是浓度最高的物质，浓度最低的是DEHP，为3.6μg/L。以65名胎停育孕妇和50名健康孕妇为病例-对照组，分析了孕早期妇女尿液中邻苯二甲酸酯及其代谢物的含量，结果发现MBP、MEHP、MEP、DEHP和MMP的检出率均大于90%，检出浓度（中位值）最高的是MEP，为80.3μg/L，其次是MBP，为21.4μg/L，MBzP浓度最低，为5.2μg/L。
对胎停育和健康孕妇组（病例-对照组）进行问卷调查，采用Χ2检验和秩和检验进行单因素分析，结果显示胎停育组装修年限为4.16年，明显短于对照组（7.06年，P＜0.05）；胎停育组房间打扫次数每周≦1次的占55%，显著高于对照组（占24%，P＜0.05）；胎停育组使用保鲜膜和塑料餐具者占92%，显著高于对照组（占76%，P＜0.05）。采用Logistic逐步回归分析进行多因素分析，结果表明，房间打扫次数和装修年限是胎停育的主要影响因素。房间打扫次数越少，发生胎停育的危险性越高；装修年限越短，发生胎停育的危险性越高。比较病例-对照组尿液中的邻苯二甲酸酯及其代谢物的含量，结果表明，病例组尿液中MMP、MEP、MBP、MEHP、MBzP和DEHP的平均浓度均明显高于对照组（P<0.05）。采用列联表分析高浓度的邻苯二甲酸酯及其代谢物（高于中位值）和胎停育的相关性，其中MBP的优势比（odds ratios，OR）值高达16.1，MMP和MEP的OR值分别为7.1和5.0，MEHP、MBzP和DEHP的OR值均为2.7。结果表明高浓度的邻苯二甲酸酯更容易导致胎停育。
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.
考察了使用塑料水杯饮水、室内降尘和室内空气等外暴露途径对邻苯二甲酸酯的污染，结果表明使用塑料水杯饮水和室内降尘带来的邻苯二甲酸酯日摄入量低于美国EPA规定的参考剂量的1%，室内空气带来的邻苯二甲酸酯日摄入量低于参考剂量的2%。与尿液代谢物估算的日摄入量比较发现，室内空气和塑料水杯饮水是邻苯二甲酸二正丁酯（di-n-butyl phthalate，DBP）的主要暴露途径；而室内空气、室内降尘和塑料水杯饮水均不是邻苯二甲酸二（2-乙基己基）酯(di-(2-ethylhexyl) phthalate， DEHP)的主要暴露途径，推测DEHP的主要暴露途径可能是食物摄入。
建立了同时测定人体尿液中邻苯二甲酸酯及其代谢物的分析方法。采用固相萃取方法对样品进行前处理，考察了萃取剂种类、萃取剂体积和萃取流速等影响固相萃取的因素，确定了最佳固相萃取条件为：用2mL乙腈、2mL乙酸乙酯和1mL乙醚-正己烷（1/19，V/V）洗脱，洗脱流速为1mL/min。采用高效液相色谱法（high performance liquid chromatography，HPLC）测定邻苯二甲酸酯及其代谢物，线性范围为20～10000μg/L，相关系数均大于0.99，最低检出限为3.8～7.0μg/L，回收率为72.2～102.3%，相对标准偏差（relative standard deviation，RSD）均小于8%。采用气相色谱-质谱法（gas chromatography-mass spectrometry，GC-MS）测定邻苯二甲酸单酯时需要进行衍生化处理，选用N，O-双三甲基硅基三氟乙酰胺（bis(trimethylsilyl)-trifuoroacetamide, BSTFA）作为衍生化试剂，考察了衍生化试剂用量、衍生化时间和温度等因素，确定衍生化条件为在样品中加入20μLBSTFA于30℃下衍生化30min。邻苯二甲酸酯及其代谢物的线性范围为5～1000μg/L，相关系数均大于0.98，最低检出限为0.3～1.1μg/L，回收率为77.9～97.7%，RSD为3.7～10.9%。比较两种分析方法，发现GC-MS法检出限较低，更适于低浓度邻苯二甲酸酯及其代谢物的分析。在尿液（GC-MS法）分析方法的基础上建立了卵泡液中邻苯二甲酸酯及其代谢物的分析方法，待测物的回收率为79.8～90.9%，RSD为6.1～11.1%，可用于卵泡液为基质的邻苯二甲酸酯暴露评价。
采用人体尿液和卵泡液中邻苯二甲酸酯及其代谢物的分析方法，对育龄妇女、不孕妇女和孕早期妇女等女性群体进行邻苯二甲酸酯内暴露评价。以50位育龄妇女为研究对象，测定尿液中邻苯二甲酸酯及其代谢物的含量。在育龄妇女尿液中检出了DEHP、邻苯二甲酸单丁酯（mono-n-butyl phthalate, MBP）、DBP、邻苯二甲酸单（2-乙基己基）酯（mono(2-ethylhexyl) phthalate, MEHP）、邻苯二甲酸单乙酯（monoethyl phthalate, MEP）、邻苯二甲酸单苄酯（monobenzyl phthalate,MBzP）和邻苯二甲酸单甲酯（monomethyl phthalate, MMP），且检出率大于75%。其中，MEP的浓度（中位值）最高，为68.4μg/L，DBP的浓度最低，为5.0μg/L。根据尿液中代谢物的含量估算日摄入量，结果表明DEHP是日摄入量最高的物质。有2位被调查对象（4%）DEHP摄入量大于美国EPA规定的参考剂量(20μg/kgbw/day)。而邻苯二甲酸二乙酯（diethyl phthalate, DEP）、DBP和邻苯二甲酸丁基苄基酯（butylbenzyl phthalate, BBzP）摄入量均远低于规定值。对接受体外受精-胚胎移植的36位不孕妇女患者的卵泡液进行分析，在卵泡液中检出了DEHP、MBP、DBP、MEHP、MEP、MBzP和MMP。其中，MBP和MEHP在所有卵泡液中均检出，检出浓度（中位值）分别为12.4和6.1μg/L。MBP是浓度最高的物质，浓度最低的是DEHP，为3.6μg/L。以65名胎停育孕妇和50名健康孕妇为病例-对照组，分析了孕早期妇女尿液中邻苯二甲酸酯及其代谢物的含量，结果发现MBP、MEHP、MEP、DEHP和MMP的检出率均大于90%，检出浓度（中位值）最高的是MEP，为80.3μg/L，其次是MBP，为21.4μg/L，MBzP浓度最低，为5.2μg/L。
对胎停育和健康孕妇组（病例-对照组）进行问卷调查，采用Χ2检验和秩和检验进行单因素分析，结果显示胎停育组装修年限为4.16年，明显短于对照组（7.06年，P＜0.05）；胎停育组房间打扫次数每周≦1次的占55%，显著高于对照组（占24%，P＜0.05）；胎停育组使用保鲜膜和塑料餐具者占92%，显著高于对照组（占76%，P＜0.05）。采用Logistic逐步回归分析进行多因素分析，结果表明，房间打扫次数和装修年限是胎停育的主要影响因素。房间打扫次数越少，发生胎停育的危险性越高；装修年限越短，发生胎停育的危险性越高。比较病例-对照组尿液中的邻苯二甲酸酯及其代谢物的含量，结果表明，病例组尿液中MMP、MEP、MBP、MEHP、MBzP和DEHP的平均浓度均明显高于对照组（P<0.05）。采用列联表分析高浓度的邻苯二甲酸酯及其代谢物（高于中位值）和胎停育的相关性，其中MBP的优势比（odds ratios，OR）值高达16.1，MMP和MEP的OR值分别为7.1和5.0，MEHP、MBzP和DEHP的OR值均为2.7。结果表明高浓度的邻苯二甲酸酯更容易导致胎停育。
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.
引文
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