双酚A和烷基酚的检测与暴露评估

【作者】 肖晶；

【导师】 吴永宁； 王竹天； 邵兵；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2008， 博士

【摘要】 环境内分泌干扰物对人和动物的影响已成为国际性热门问题,尤其是环境雌激素通过与雌激素受体结合或影响细胞信号传导途径或其他方式模仿和干扰内源性雌激素作用,发挥拟雌激素样效应,对人类和动物造成很大的危害。双酚A（BPA）是制造环氧树脂、聚碳酸酯树脂、聚苯乙烯树脂的主要原料,又用于酚醛树脂、杀菌剂、抗氧化剂、聚氯乙烯、染料等的稳定剂。烷基酚壬基酚（NP）、辛基酚（OP）被广泛地用作塑料增塑剂、工业用洗涤剂、农药乳化剂、纺织行业的整理剂。人们生活中广泛用到以BPA、NP和OP为原料生产的物品,BPA、NP和OP会被迁移,污染人们的日常生活环境,尤为严重的是在食品包装中加入的BPA、NP和OP会溶入食品,而直接进入人体,危害人类健康。我国是BPA、NP和OP的使用大国,如果不严格控制,由BPA、NP和OP造成的污染问题将会非常严重。为了更好地保护我国人群健康以及国家的产业利益,本文对罐装食品中BPA、NP和OP的检测技术、尿液中的生物标志物和塑料制品的迁移等进行了较系统的研究,同时对人群摄入BPA、NP和OP的暴露进行评估。（1）罐装食品中BPA、NP和OP的检测方法建立与应用运用固相萃取、液液萃取等前处理方法,结合超高压液相色谱—质谱/质谱（UPLC—MS/MS）或高效液相色谱—荧光检测（HPLC—FLD）分析手段,建立检测罐装食品中BPA、NP和OP含量的方法。固体样品经甲醇超声提取、蒸干,溶解于碱性溶液中,酸化,用二氯甲烷萃取、浓缩。液体样品的分析采用NH₂柱浓缩、富集和净化。UPLC采用的色谱柱是粒径为1.7μm的AcquityUPLCT^TM BEH,C₁₈柱;甲醇-水作为流动相梯度洗脱,质谱仪采用三重四极杆质量分析器定性定量分析。该方法BPA、NP和OP在蔬菜、鱼和肉罐头样品中的检出限范围为0.22-0.35μg/kg、0.031-0.050μg/kg和0.08-0.12μg/kg,定量限分别为0.55-0.80μg/kg、0.10-0.94μg/kg和0.20-0.30μg/kg。BPA、NP和OP的加标回收率分别为70.0-120%、70.2-120%和71.2-133%;BPA、NP和OP的相对标准偏差分别为7.06-17.6%、4.49-18.9%和6.91-19.2%。HPLC使用的色谱柱是Waters Xterra MS（?）C18柱,采用荧光检测,甲醇-水作为流动相梯度洗脱。采用荧光检测,激发波长:225nm;发射波长:310nm。该方法BPA、NP和OP的检出限分别为0.52-0.91μg/kg、0.10-0.52μg/kg和0.14-0.53μg/kg,定量限分别为1.18-2.70μg/kg、0.28-2.15μg/kg和0.35-1.87μg/kg。BPA、NP和OP的加标回收率分别为70.1-90.2%、80.1-122%和72.1-126%;BPA、NP和OP的相对标准偏差分别为5.61-11.2%、2.35-8.88%和5.61-12.3%。本研究分别采用以上两种检测方法,对北京市售的30份食品进行了检测。两种方法均实现了BPA、NP和OP三种物质的同时测定,HPLC—FL法具有较高的灵敏度、准确度、精密度和特异性,符合食品中BPA、NP和OP的痕量分析要求,但应采用UPLC-MS/MS方法进行确证（2）人尿中BPA、NP和OP代谢产物的检测方法建立与应用双酚A、壬基酚和辛基酚在尿中的代谢产物主要是葡萄糖苷结合物和硫酸酯结合物及少量原型。本文在建立相应的检测方法的基础上,对普通人群的尿液中BPA、NP和OP含量进行检测。采用β-葡萄糖酸酐酶、硫酸酯酶水解人尿液中的结合型BPA、NP和OP,酶解后溶液采用OASIS HLB固相萃取柱浓缩、富集和净化。处理后的样品采用UPLC—MS/MS或HPLC-FL检测,建立了检测尿液中BPA、NP和OP含量的方法。UPLC—MS/MS方法BPA、NP和OP的检测限分别为0.41μg/L、0.20μg/L和0.50μg/L;定量限分别为1.02μg/L、0.85μg/L和1.28μg/L;加标回收率分别为72.3-109%、71.2-114%和76.6-116%;相对标准偏差分别为12.5-14.1%、10.0-14.0%和7.12-15.5%。HPLC-FL方法BPA、NP和OP的检测限分别为3.71μg/L、4.90μg/L和7.50μg/L;定量限分别为11.9μg/L、15.7μg/L和21.3μg/L;加标回收率分别为70.2-95.7%、73.2-108%和76.3-109%;相对标准偏差分别为8.81-10.7%、9.31-14.5%和6.65-15.5%。采用这两种检测方法,对健康人群的60份尿液进行了检测。（3）塑料制品的迁移实验研究双酚A、壬基酚和辛基酚可用于生产非离子表面活性剂、增塑剂、聚碳酸酯、环氧树脂、聚砜树脂、聚苯醚树脂、不饱和聚酯树脂等高分子材料,被广泛用于制造塑料制品、食具、涂层等。国内外有文献报道,从这类制品中溶出微量BPA、NP和OP,对人体健康及环境污染造成威胁,直接影响道人的身体健康。本文建立了塑料制品中辛基酚、壬基酚和双酚A迁移量的UPLC—MS/MS测定方法。该方法的BPA、NP和OP检出限分别为0.25-0.38μg/L、0.10-0.22μg/L和0.20-0.30μg/L;定量限为0.70-1.01μg/L、0.50-0.74μg/L和0.75-0.80μg/L。BPA、NP和OP的加标回收率分别为80.1-105%、80.8-112%和80.5-110%,相对标准偏差分别为5.02-10.9%、4.66-13.5%和4.73-10.4%。同时也建立了塑料制品中辛基酚、壬基酚和双酚A迁移量的HPLC—FL测定方法。该方法的BPA、NP和OP出限分别为1.55-2.75μg/L、1.00-1.60μg/L和1.00-1.84μg/L;定量限为4.03-5.00μg/L、2.85-3.41μg/L和2.75-4.05μg/L。BPA、NP和OP的加标回收率分别为80.9-107%、80.4-108%和80.4-107%,相对标准偏差分别为4.24-11.5%、4.93-12.4%和3.64-9.88%。用以上方法分别考察了不同条件下的迁移情况,包括不同浸泡液:蒸馏水、3%醋酸、4%醋酸、15%乙醇、20%乙醇、65%乙醇、正己烷;不同温度:室温、水浴40℃、60℃、80℃、100℃;不同浸泡时间:12h、24h和48h。结果表明,当模拟物为65%乙醇,60℃水浴浸泡3小时时,检测到BPA、NP和OP迁移量最大。因此,选用模拟物65%乙醇,60℃沸水浴浸泡3小时为迁移检测条件。（2）BPA、NP和OP的测定及其暴露评估30份罐装食品（10份蔬菜罐头、10份鱼罐头、10份肉罐头）中11份检出BPA,其含量水平为0.180-12.3μg/kg;27份检出了NP,其含量水平为1.35-100μg/kg;6份检出了OP,其含量水平为0.05-23.5μg/kg。此结果表明,壬基酚NP广泛存在于罐装食品中。在被检的60人份尿样中,8份检出BPA,占13.3%,暴露水平为0.515-49.5ng/ml;29份检出NP,占48.3%,暴露水平为2.32-28.5 ng/ml;17份检出OP,占28.3%,暴露水平为0.720-15.2ng/ml。女性检出率高于男性检出率,但不同年龄组间检出率差别不大。被检的塑料饭盒、奶瓶、水杯、油壶等15种塑料制品中,13份检出BPA,其迁移量为10.3-1004ng/L;14份检出NP,其迁移量为103-9875ng/L;5份检出。应考虑累积的环境暴露是伤害我们健康的OP,其迁移量为1.02-12.1ng/L。此结果表明,塑料制品存在双酚A、壬基酚和辛基酚的迁移。迁移条件（温度、溶剂和贮存时间）不同,迁移量也不同。虽然在众多样品中检出了双酚A、壬基酚和辛基酚,暴露水平远低于文献报道的毒性阀值。但是,由于双酚A、壬基酚和辛基酚广泛存在于各种环境介质中,累积的环境暴露对我们的健康仍然存在较大的风险。更多还原

【Abstract】 The influence of environment endocrine（内分泌）interference to the person and the animal was becoming an international popular problem.Particularly,the environment estrogen（雌激素）initiated and interfered endogenesis estrogen by combining with estrogenic receptor or influencing the way of cell signal conduction or the other ways,which has esteogen-like action,brings hazard to the mankind and the animal.BPA was key raw material of making epoxide resin、polycarbonate、polystyrene resin,which was used to the stabilizing of agent phenol formaldehyde resin、bactericidal agen、antioxidanK polyvinyl chloride、dye.The alkylphenol NP、OP was widely used to elasticizer、technica grade abstergent、pesticide emulsifies、trimming Iin the spinning and weaving,ect.People usually use some articles,whose raw material was BPA、NP and OP, BPA、NP and OP were often migrated and contaminate living environment .Especially,BPAn、NP and OP were migrated to food when food packing has BPA、NP and OP,enter a human body directly,endanger mankind’s health.Our country is important country using BPA、NP and OP,if we didn’t control strictly,the pollution problem of the BPA,NP and OP would be very serious.For the sake of the protection our country people health and the industry benefits of the nations, we carried on detection technique、organism marker and migration of plastic products of BPA、NP and OP,and make a comprehensive comment to the BPA,NP and OP in the meantime,mainly include several aspects as follows:（1）The detection technique of the BPA,NP and OP in the canned foodIn this study,the comprehensive analytical methods based on Ultra-high Pressure Liquid Chromatography-mass/mass（UPLC-MS/MS）or high performance liquid chromatography fluorescence detection（HPLC-FLD）with solid phase extraction,Liquid liquid Extraction before have been established for determination of BPA,NP and OP in food samplesBPA,NP and OP was extracted with methanol,the residue was dissolved in alkaline solution,acidized,extracted by dichloroacetamide from the foods and the extract was concentrated.Clean-up of samples was performed on an NH₂ column solid extraction cartridges.The UPLC separation was performed on a Acquity UPLC^TM BEH,C₁8 column（particle diameter 1.7μm） with a gradient elution system of methanol-water as the mobile phase.triple-quadrupole mass spectrometry analyzer was performed for the qualitative and quantitative analyse of LC-MS/MS system.The limit of detection of BPA、NP and OP was 0.22-0.35μg/kg、0.031-0.05Oμg/kg and 0.08-0.12μg/kg;The limit of quantification of BPA,、NP and OP was0.55-0.80μg/kg、0.10-0.94μg/kg and 0.20-0.30μg/kg;The Recoveries of BPA,NP and OP were 70.0-123%,70.2-125%and 71.2-133%;The precision was 7.06-17.6%,3.98-18.9%and 6.91-19.2%,respectively.The HPLC separation was performed on Waters XTerra^? MS C₁8 column with a gradient elution system of methanol-water as the mobile phase and fluorescence detector with the excitation and emission wavelength at 225 nm and 310nm respectively were used for the determination..The limit of detection of BPA、NP and OP was 0.52-0.91μg/kg、0.10-0.52μg/kg and 0.14-0.53μg/kg;The limit of quantification of BPA、NP and OP was 1.18-2.70μg/kg、0.28-2.15μg/kg and 0.35-1.87μg/kg;The Recoveries of BPA、NP and OP were 70.1-90.2%、80.1-122%and 72.1-126%;The precision was 5.61-11.2%、2.35-8.88%and 5.61-12.3%,respectively.The two methods both may detect the BPA,NP and OP at the time,The results showed that the method is with high sensitivity、accuracy、precision、specificity.the method is suitable for the determination of BPA、NP and OP in can food.30 food samples from Beijing’s markets were determined by using the improved methods,the method is simple,the result is accurate、credibility.（2）The study of biomarker-human urine The BPA,NP and OP is esteogen-like "endocrine chemical materials".the general people contacted with the BPA,NP and OP through using plastics packing material、the can food、abstergent,ect.The metabolic product in urine is mainly glucoside conjugate and sulphate conjugate.In this study,we detected BPA,NP and OP in the urine of health crowd.The urine samples was de-conjugated by addingβ-glucuronidase and sulfatase,after the enzymatic treatment,the samples was subjected to the OASIS HLB column solid extraction cartridges to Clean-up and concentrate.A comprehensive analytical method based on Ultra-high Pressure Liquid Chromatography-mass/mass（UPLC-MS/MS）or high performance liquid chromatography fluorescence detection（HPLC-FLD） has been established for the determination of BPA,NP and OP in urine.UPLC-MS/MS:The limit of detection of BPA、OP and NP was 0.41、0.20 and 0.50μg/L;The limit of quantification of BPA、OP and NP was 1.02、0.85 and 1.28μg/L;The Recoveries of BPA、OP and NP were 72.3-109%、71.2-114%、76.6-116%;The precision was 12.5-14.1%、10.0-14.0%、7.12-15.5%,respectively.HPLC-FL:The limit of detection of BPA、OP and NP was 3.71、4.90 and 7.50μg/L;The limit of quantification of BPA、OP and NP was 11.9、15.7 and 21.3μg/L;The Recoveries of BPA、OP and NP were 70.2-95.7%、73.2-108%and 76.3-109%;The precision was 8.81-10.7%、9.31-14.5%and 6.65-15.5% respectively.60 urine samples were determined by using these two methods,the method is sensitivity,selectivity and good reproducibility.（3） The migration experiment research of the plastics product BPA、OP and NP were one kind of environment estrogen,which were used to make non-ion surface activity agent、elasticizer、polycarbonate、epoxy resin、polysulfone resin、polyphenyl Ether resin、selectron,ect.They were generally used to make plastic products、tableware、coating,ect.According to documents,many plastic products may dissolute a little BPA、OP and NP,brought threat to the human body health and pollute the environment,influence directly the person’s healthy.This study investigated migration circumstance,respectively,under different test conditions:the different simulant:Distilled water、3%acetic acids、4%acetic、 acids、15%alcohol、20%alcohol、65%alcohol、hexane;the different temperature: 25°C、40°C、60°C、80°C、100°C;The Different soaks time:12h、24h and 48h.The result show,when the simulant is 65%alcohol、at 60°C、soak for 3 hours,the amount of migration is greatest.A UPLC-MS/MS method was developed for quantifying levels of BPA、OP and NP in plastic products.The limit of detection of BPA、OP and NP was 0.25-0.38μg/L、0.10-0.22μg/Land 0.20-0.30μg/L;The limit of quantification of BPA、OP and NP was 0.70-1.01μg/L.0.50-0.74μg/L and 0.75-0.80μg/L;The Recoveries of BPA、OP and NP were 80.1-105%、80.8-112%and 80.5-110%.The precision was 5.02-10.9%.4.66-13.5%and 4.73-10.4%respectively.A HPLC-FL method was developed for detecting levels of BPA、OP and NP in plastic products.The limit of detection of BPA、OP and NP was 1.55-2.75μg/L、1.00-1.60μg/Land 1.00-1.84μg/L;The 1imit of quantification of BPA、OP and NP was 4.03-5.00μg/L、2.85-3.41μg/L and 2.75-4.05μg/L;The Recoveries of BPA、OP and NP were 80.9-107%、80.4-108%and 80.4-107%.The precision was 4.24-11.5%、4.93-12.4%and 3.64-9.88%respectively.The experiment showed that the amount of migration is greatest when the sample was soaked in 65%alcohol（60°C）for 3 hours.The method was sensitivity、selectivity、accurate and credibility.（4） The determination and the exposure assessment of the BPA,NP and OP Using the improved methods established in this work,the BPA,NP and OP in vegetable,fish,meat can food samples from Beijing’s markets were determined.We collect 30 can food samples（10 vegetable samples、10 vegetable samples and 10 vegetable samples）.11 samples of the total were found to contain BPA,its level is 0.180-12.3μg/kg;27 samples of the total were found to contain NP,its level is 1.35-100μg/kg;6 samples of the total were found to contain OP,its level is 0.05 -23.5μg/kg.The results demonstrated the widespread occurrence of NP in can food.60 urine samples were determined by using the method,the method is sensitivity、selectivity and good reproducibility.8 samples of the total were found to contain BPA（13.3%）,its level is0.515-49.5μg/ml;29 samples of the total were found to contain NP（48.3%）,its level is 2.32-28.5 ng/m1;17 samples of the total were found to contain NP（28.3%）,its level is 0.720-15.2ng/ml.The female detection ratios was higher the male detection ratios.The detection ratios increased with the age increase.15 plastics product samples（plastics box、baby’s bottle、water cup and lubricator）were determined 13 samples of the total were found to contain BPA,its level is 10.3-1004ng/L;14 samples of the total were found to contain NP,its level is 103 -9875ng/L;5 samples of the total were found to contain OP,its level is 1.02-12.1 ng/L.The results demonstrated many samples contained BPA,NP and OP,and NP was resided widely in can food and plastic products.更多还原