CN108614041A - 一种定量检测水中内分泌干扰物不同赋存形态含量的方法 - Google Patents
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Abstract
本发明涉及一种环境分析化学领域的水体有机污染物不同形态分离及检测方法，具体涉及一种定量检测水中内分泌干扰物(EDCs)不同赋存形态含量的方法。由于以往研究普遍假定经过滤膜处理后污染物在水体中呈溶解态，忽略了污染物与胶体微颗粒的结合而表现出的不同形态。本发明通过采用陶瓷膜过滤‑超声辅助萃取‑固相萃取进行分级提取的前处理方法，结合高效液相色谱‑质谱联用(LC‑MS/MS)分析技术，检测水中颗粒相、胶体结合相及真溶相EDCs含量。该方法可准确定量分析在水中EDCs的不同赋存形态含量，在水中内分泌干扰物风险评价与控制工作中有很好的应用价值。
Description
技术领域
本发明涉及一种环境分析化学领域的水体有机污染物不同形态分离及检测方法，具体涉及一种定量检测水中内分泌干扰物(EDCs)不同赋存形态含量的方法。
背景技术
内分泌干扰物(EDCs)污染已经成为继温室效应、臭氧层破坏之后的第三大全球性环境公害问题。据报道，美国每年需要耗费3400亿美金来解决因长期接触低浓度内分泌干扰化合物带来的健康问题，这相当于全美GDP的2.3％。欧洲的情况同样不容乐观，为此需要花费2170亿美元/年，占欧洲GDP的1.3％。大量研究表明，在全世界范围内的城市污水、地表水、地下水和饮用水中都能检出种类繁多的EDCs。即使在很低的环境浓度(ng/L)下，EDCs仍可引起雌激素、甲状腺激素等内分泌干扰毒性效应，长期暴露会严重影响鱼类、两栖类等水生生物正常的发育和繁殖，甚至导致种群的灭绝；而且还可通过食物链传递并影响到更高营养级的陆生动物和人类的健康。研究报告显示，不育症、复发性流产、乳腺癌、前列腺癌、甲状腺疾病、糖尿病、肥胖症、男性精液质量下降以及女性早熟等多种人类疾病的发生与EDCs暴露存在相关性。比如，美国每年因双酚A暴露引起儿童肥胖症的病例数达33000例，医疗花费达24亿美元/年。雌酮、雌二醇、雌三醇、双酚A(BPA)、烷基酚(AP)等内分泌干扰物是典型的全球性环境内分泌干扰物。2014年全世界双酚A产能达717.5万吨/年，总消费量超过560万吨/年；我国双酚A产能已超过180万吨/年，消费总量约为124万吨/年。烷基酚的世界年产量约为70万吨，其中约60％进入环境水体。我国由人和畜牧养殖动物排出的雌酮、雌二醇、雌三醇等类固醇内分泌干扰总量为3071吨/年，其中约有2486吨/年排放进入环境中，仅有少部分在污水处理中得到去除。
以往研究通常习惯简单地把水样过0.45μm或1μm孔径滤膜水样中EDCs浓度来评价水体污染情况，然后假定污染物在水相中呈“真溶态”，忽略了它们由于与胶体与细微颗粒结合而表现出不同的存在形态，没有充分考虑它们在水体中液相-胶体-悬浮颗粒物等连续微界面的赋存形态变化。已有研究表明，水体由一个复杂的分散体系组成，其组分通常可分为悬浮颗粒相(颗粒直径大于0.45μm)，胶体相(粒径大小介于1KDa～0.45μm)和真溶相(相对分子质量小于1KDa)。由于胶体颗粒具有特殊的物理化学性质，如较高的比表面积和较强的吸附能力以及较多的静电与化学结合位，从而使得各种毒害污染物容易与胶体颗粒结合。Hutchins等(2007)研究发现，咸水湖水体中的雌性激素污染物大部分存在于胶体物质，只有小部分处于“真溶态”(过1nm孔径的滤膜)。Zhao等(2010)也得到类似规律，发现河流水体中雌性激素污染物大部分存在0.7-1.2μm粒径的胶体与悬浮颗粒物上。Gong等发现珠江水体中约4-74％的双酚A以及21-67％的壬基酚存在胶体相中。从技术原理上而言，不同水体的污染程度及对水生生物的毒害作用很大程度取决于污染物在水体中的分配形态。
内分泌干扰物在水体的颗粒态、胶体结合态及自由态三种不同赋存状态决定着它在水处理过程的迁移转化及其生态风险。然而，目前国内外对EDCs在水体中颗粒相、胶体和真溶相三种不同相态的分配行为研究很少，对其形态分配机制及调控原理尚不清楚。因此，非常有必要科学分离出水体中不同赋存形态的EDCs，并建立对应的预处理及分析方法，这对掌握它们的环境行为及其生态风险有着重要作用。
发明内容
1.发明要解决的技术问题
为科学、准确分析水中内分泌干扰物的赋存形态，本发明采用陶瓷膜分离-超声提取-有机溶剂萃取-固相萃取的方法进行分级提取，结合高效液相色谱-质谱联用(LC-MS/MS)检测技术，建立一种适用定量检测水中内分泌干扰物(EDCs)不同赋存形态含量的方法。
2.本发明的技术方案
本发明基本原理是利用陶瓷膜装置把城市污水按粒径尺度分成3种组分：粒径1μm的组分以上为颗粒，粒径1nm-1μm的组分为胶体相，小于1nm的组分为真溶相。颗粒相EDCs采用超声提取；胶体相EDCs采用液液萃取法提取；真溶相EDCs采用HLB固相萃取柱富集后测定。然后采用LC-MS/MS仪器对三种赋存形态EDCs进行准确定量分析。
本发明的技术方案为：
一种定量检测水中内分泌干扰物不同赋存形态含量的分析方法，其步骤为：
第一步、水中颗粒相、胶体相和真溶相组分的分离：取一定量待测水样，采用1μm孔径陶瓷超滤膜，截留相液体经真空冷冻干燥后作为颗粒相进一步分析；上述所得滤出液进一步经1nm孔径陶瓷纳滤膜高压分离，滤出液作为真溶相，保存截留液作为胶体相分析。
第二步、颗粒相EDCs的提取：称取2克真空冷冻干燥的颗粒相样品放置30mL圆底试管中，加入100μL内标(1.0mg/L)，然后加入10mL甲醇以及10mL二氯甲烷在涡旋混合器上振荡10min。振荡结束后，超声15min，最后3024g离心10min，分离出的上层清液装入200mL圆底烧瓶，在50℃条件蒸干大部分有机溶剂后用200mL纯水稀释，最后用阴离子交换树脂柱(SAX)以及HLB固相萃取柱富集并采用10mL甲醇溶剂洗脱，氮气吹近干，甲醇定容至1mL，待测。
第三步、胶体相EDCs的提取：取10mL第一步分离出的胶体相浓缩液用纯水稀释至500mL，同时取100mL甲基叔丁基醚一起倒入分液漏斗中，上下摇晃100次，分层后倒出底部水相，重复提取3次；将有机相放置于旋转蒸发仪上浓缩至0.5mL，再溶于20mL甲醇，继续置于旋转蒸发仪上浓缩至1mL，待测。
第四步、真溶相EDCs的富集分离：取1L第一步分离出的真溶相液体，用HCl调节pH至3,加入100μL内标(1.0mg/L)，采用500mL，HLB固相萃取柱对样品以10ml/min的速率进行富集，氮气吹脱HLB小柱30min后用8ml甲醇以及8ml二氯甲烷溶剂洗脱，氮吹仪吹干后加入1ml甲醇并置于涡旋仪震荡2min，最后转移至液相小瓶待测。
第五步、EDCs含量测定方法：采用LC-MS/MS测定EDCs含量。①色谱条件：色谱柱规格为2.1×100mm，1.7μm BEH C18柱；流动相为甲醇和水；柱流速为0.35mL/min。液相色谱方法为15％甲醇保持1min，3min时甲醇比例升至60％，到4min时甲醇比例升至90％并维持1min，到6min时甲醇比例降至15％并维持1min。②质谱条件：气帘气,30；离子喷雾电压(IS)：-4500V；温度：450℃。
3.有益效果
内分泌干扰物在水体中的不同赋存形态决定了它们在水体中迁移转化过程及其生物毒性。本发明通过采用陶瓷膜分离-甲醇超声提取-MTBE液液萃取-固相萃取进行分级提取的前处理方法，结合液质联用(LC-MS/MS)分析技术，检测水中颗粒相、胶体相及真溶相EDCs含量。该方法可准确定量分析EDCs在水体中的不同赋存形态，可有效解决采用EDCs总浓度评价水体污染程度不够科学的问题，将在水体EDCs污染风险评价、污染治理与毒性控制工作中有很好的应用价值。
具体实施方式
以下结合实施例进一步说明本发明
实施例1：某城市污水生化尾水中颗粒相、胶体相及真溶相炔雌醇(EE2)含量测定
1)不同形态EE2的分离：取10L城市污水生化尾水，采用1μm孔径陶瓷超滤膜，截留相液体经真空冷冻干燥后作为颗粒相进一步分析；上述所得滤出液进一步经TiO2陶瓷纳滤膜(1nm孔径)高压分离，滤出液作为真溶相，保存截留液作为胶体相分析。
2)颗粒相EE2的提取：称取2克真空冷冻干燥的颗粒相样品放置30mL圆底试管中，加入100μL内标(1.0mg/L)，然后加入10mL甲醇以及10mL二氯甲烷在涡旋混合器上振荡10min。振荡结束后，超声15min，最后3024g离心10min，分离出的上层清液装入200mL圆底烧瓶，在50℃条件蒸干大部分有机溶剂后用200mLminiQ纯水稀释，最后用阴离子交换树脂柱(SAX)以及HLB固相萃取柱富集并采用10mL甲醇溶剂洗脱，氮气吹近干，甲醇定容至1mL，待测。
3)胶体相EE2的提取：取10mL第一步分离出的胶体相浓缩液用miniQ纯水稀释至500mL，同时取100mL甲基叔丁基醚一起倒入分液漏斗中，上下摇晃100次，分层后倒出底部水相，重复提取3次；将有机相放置于旋转蒸发仪上浓缩至0.5mL，再溶于20mL甲醇，继续置于旋转蒸发仪上浓缩至1mL，待测。
4)真溶相EE2的富集分离：取1L第一步分离出的真溶相液体，用HCl调节pH至3,加入100μL内标(1.0mg/L)，采用500mL，6mg的HLB固相萃取柱对样品以10ml/min的速率进行富集，氮气吹脱HLB小柱30min后用8ml甲醇以及8ml二氯甲烷溶剂洗脱，氮吹仪吹干后加入1ml甲醇并置于涡旋仪震荡2min，最后转移至液相小瓶待测。
5)EE2含量测定方法：采用LC-MS/MS测定EDCs含量。①色谱条件：色谱柱规格为2.1×100mm，1.7μm BEH C18柱；流动相为甲醇和水；柱流速为0.35mL/min。液相色谱方法为15％甲醇保持1min，3min时甲醇比例升至60％，到4min时甲醇比例升至90％并维持1min，到6min时甲醇比例降至15％并维持1min。②质谱条件：气帘气,30；离子喷雾电压(IS)：-4500V；温度：450℃。
6)测定结果：采用内标法计算出某城市污水生化尾水中颗粒相EE2含量为19.9ng/g，胶体结合态EE2含量为12.6ng/L，真溶相EE2含量为27.4ng/L。
实施例2：某重污染河流水体中颗粒相、胶体相及真溶相双酚A(BPA)含量测定
1)不同形态BPA的分离：取10L河流水样，采用1μm孔径陶瓷超滤膜，截留相液体经真空冷冻干燥后作为颗粒相进一步分析；上述所得滤出液进一步经TiO2陶瓷纳滤膜(1nm孔径)高压分离，滤出液作为真溶相，保存截留液作为胶体相分析。
2)颗粒相BPA的提取：称取2克真空冷冻干燥的颗粒相样品放置30mL圆底试管中，加入100μL内标(1.0mg/L)，然后加入10mL甲醇以及10mL二氯甲烷在涡旋混合器上振荡10min。振荡结束后，超声15min，最后3024g离心10min，分离出的上层清液装入200mL圆底烧瓶，在50℃条件蒸干大部分有机溶剂后用200mLminiQ纯水稀释，最后用阴离子交换树脂柱(SAX)以及HLB固相萃取柱富集并采用10mL甲醇溶剂洗脱，氮气吹近干，甲醇定容至1mL，待测。
3)胶体相BPA的提取：取10mL第一步分离出的胶体相浓缩液用miniQ纯水稀释至500mL，同时取100mL甲基叔丁基醚一起倒入分液漏斗中，上下摇晃100次，分层后倒出底部水相，重复提取3次；将有机相放置于旋转蒸发仪上浓缩至0.5mL，再溶于20mL甲醇，继续置于旋转蒸发仪上浓缩至1mL，待测。
4)真溶相BPA的富集分离：取1L第一步分离出的真溶相液体，用HCl调节pH至3,加入100μL内标(1.0mg/L)，采用500mL，6mg的HLB固相萃取柱对样品以10ml/min的速率进行富集，氮气吹脱HLB小柱30min后用8ml甲醇以及8ml二氯甲烷溶剂洗脱，氮吹仪吹干后加入1ml甲醇并置于涡旋仪震荡2min，最后转移至液相小瓶待测。
5)BPA含量测定方法：采用LC-MS/MS测定EDCs含量。①色谱条件：色谱柱规格为2.1×100mm，1.7μm BEH C18柱；流动相为甲醇和水；柱流速为0.35mL/min。液相色谱方法为15％甲醇保持1min，3min时甲醇比例升至60％，到4min时甲醇比例升至90％并维持1min，到6min时甲醇比例降至15％并维持1min。②质谱条件：气帘气,30；离子喷雾电压(IS)：-4500V；温度：450℃。
6)测定结果：采用内标法计算该重污染河流中颗粒相BPA含量为89ng/g，胶体结合态BPA含量为45.4ng/L，真溶相BPA含量为167ng/L。
Claims (9)
1.一种定量检测水中内分泌干扰物不同赋存形态含量的方法，其步骤特征主要包括：
第一步、水中颗粒相、胶体相和真溶相组分的分离：取一定量待测水样，采用1μm孔径陶瓷超滤膜，截留相液体经真空冷冻干燥后作为颗粒相进一步分析；上述所得滤出液进一步经1nm孔径陶瓷纳滤膜高压分离，滤出液作为真溶相，保存截留液作为胶体相分析；
第二步、颗粒相EDCs的提取：称取2克真空冷冻干燥的颗粒相样品放置30mL圆底试管中，加入100μL内标(1.0mg/L)，然后加入10mL甲醇以及10mL二氯甲烷在涡旋混合器上振荡10min。振荡结束后，超声15min，最后3024g离心10min，分离出的上层清液装入200mL圆底烧瓶，在50℃条件蒸干大部分有机溶剂后用200mL纯水稀释，最后用阴离子交换树脂柱(SAX)以及HLB固相萃取柱富集并采用10mL甲醇溶剂洗脱，氮气吹近干，甲醇定容至1mL，待测；
第三步、胶体相EDCs的提取：取10mL第一步分离出的胶体相浓缩液用纯水稀释至500mL，同时取100mL甲基叔丁基醚一起倒入分液漏斗中，上下摇晃100次，分层后倒出底部水相，重复提取3次；将有机相放置于旋转蒸发仪上浓缩至0.5mL，再溶于20mL甲醇，继续置于旋转蒸发仪上浓缩至1mL，待测；
第四步、真溶相EDCs的富集分离：取1L第一步分离出的真溶相液体，用HCl调节pH至3,加入100μL内标(1.0mg/L)，采用500mL，HLB固相萃取柱对样品以10ml/min的速率进行富集，氮气吹脱HLB小柱30min后用8ml甲醇以及8ml二氯甲烷溶剂洗脱，氮吹仪吹干后加入1ml甲醇并置于涡旋仪震荡2min，最后转移至液相小瓶待测.
第五步、EDCs含量测定方法：采用LC-MS/MS测定EDCs含量。①色谱条件：色谱柱规格为2.1×100mm，1.7μm BEH C18柱；流动相为甲醇和水；柱流速为0.35mL/min。液相色谱方法为15％甲醇保持1min，3min时甲醇比例升至60％，到4min时甲醇比例升至90％并维持1min，到6min时甲醇比例降至15％并维持1min。②质谱条件：气帘气,30；离子喷雾电压(IS)：-4500V；温度：450℃。
2.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第一步分离水中不同组分的方法中，优选的采用1μm超滤陶瓷膜加压方法分离水中颗粒相组分。
3.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第一步分离水中不同组分的方法中，采用1nm纳滤陶瓷膜加压方法分离出水体中的胶体相组分，过滤液为真溶相组分。
4.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第二步的颗粒相EDCs提取中，采用甲醇/二氯甲烷混合溶剂进行超声方法萃取出颗粒相中的EDCs。
5.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第二步的颗粒相EDCs净化中，颗粒相EDCs提取液需要经过离子交换柱(SAX)及HLB柱净化，才能进行LC/MS检测。
6.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第三步的胶体相EDCs提取与净化中，最好采用甲基叔丁基醚为溶剂进行液液萃取的方法提取城市污水中的胶体结合态EDCs。
7.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第四步的真溶相EDCs提取中，是以经过1nm纳滤陶瓷膜分离的过滤液为提取对象，固相萃取前需调节pH至3并添加内标。
8.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第四步的真溶相EDCs提取中，采用甲醇/二氯甲烷混合溶剂洗脱富集于固相萃取柱的EDCs。
9.根据权利要求1所述的定量检测水中内分泌干扰物不同赋存形态含量的方法，其特征在于：在第五步的EDCs含量测定方法中，采用LC-MS/MS测定EDCs含量，色谱流动相为甲醇和水。
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