适配体荧光共振能量转移法检测雌二醇
蒋晓华 丁文捷 栾崇林
摘要建立了一种同时检测鸡蛋中四溴双酚A(TBBP A)、六溴环十二烷(HBCD) 和多溴联苯醚(PBDEs)及其衍生物羟基多溴联苯醚(OHPBDEs)和甲氧基多溴联苯醚(MeOPBDEs)的凝胶渗透色谱(GPC)分散固相萃取(DSPE)液相色谱串联质谱(HPLCMS/MS)和气相色谱负化学源质谱(GCNCI/MS)的检测方法。样品经正己烷、二氯甲烷 (1∶1, V/V)加速溶剂萃取, 凝胶渗透色谱净化后, 经100 mg十八烷基键合硅胶(C18)分散固相萃取吸附剂去除杂质, 液相色谱串联质谱和气相色谱负化学源质谱方法测定,外标法定量。在蛋白和蛋黄样品中添加1.0或5.0 μg/kg的目标物,其回收率分别为64.5%~97.2%和65.6%~109.2%(除BDE85为54.8%,OHBDE137为47.4%外),相对标准偏差小于20.2%,定量限为0.01~0.2 μg/kg。
关键词多溴联苯醚及其衍生物;四溴双酚A;六溴环十二烷;鸡蛋;凝胶渗透色谱;分散固相萃取
1引言
多溴联苯醚(Polybrominated diphenyl ethers, PBDEs)、四溴双酚A(Tetrabromobisphenol A, TBBP A)和六溴环十二烷(Hexabromocyclododecane,HBCD)是目前广泛应用于电子设备、塑料、建筑材料、装饰材料及纺织品等的溴代阻燃剂(Brominated flame retardants, BFRs),近年来,人们逐渐注意到这三类溴代阻燃剂对环境及人类健康的影响。2009年,全球关于持久性有机污染物的《斯德哥尔摩公约》第四次缔约国大会上,商业化的五溴联苯醚及八溴联苯醚被列为全球控制和消减的持久性有机污染物(POPs),该公约提出在全球范围内禁止使用HBCD。现有研究表明,这三类阻燃剂普遍具有内分泌毒性、生殖毒性、肝脏毒性和神经毒性,威胁到人类的健康和生命安全。虽然目前对PBDEs在生物体内的代谢产物羟基代谢物(Hydroxylated polybrominated diphenyl ethers, OHPBDEs)及甲氧基代谢物(Methoxylated polybrominated diphenyl ethers, MeOPBDEs)的来源还有争议\[1~3\], 但MeOPBDEs和OHPBDEs可能具有更高的毒性\[4,5\],且在生物体内OHPBDEs与MeOPBDEs可以相互转化\[1\]。人体血液中MeOPBDEs的含量高于PBDEs\[6\],因此有必要同时测定生物体内的PBDEs、MeOPBDEs和OHPBDEs。
随着电子业的发展,环境中BFRs的污染报道也日益增加\[7,8\]。PBDEs、TBBPA和HBCD三类溴代阻燃剂的辛醇/水分配系数(lgKow)约为4.5~10.0,具有持久性和脂溶性,可通过生物富集作用累积在生物组织内,近年来关于三类溴代阻燃剂在生物组织中污染水平的报道日益增多\[9~17\]。动物性产品成为BFRs进入人体的重要途径之一\[18,19\],根据文献\[9\]的风险评估报告,淡水鱼、肉和鸡蛋等富含脂质的食品是人类膳食摄入PBDEs的重要来源,其中淡水鱼占12%~98%,猪肉占1%~20%,而鸡蛋占1%~15%。电子垃圾回收区鸡蛋的∑PBDEs和∑HBCD检出浓度甚至高达14100 ng/g (脂肪重量,fat weight, fw)和350 ng/g (fw) \[11\]。
鸡蛋样品脂肪含量高,色素重,目前常用的提取方法是索氏提取和超声提取,净化步骤多采用凝胶渗透色谱(Gel permeation chromatography,GPC)结合多层硅胶柱净化,或酸性硅胶柱结合酸性氧化铝柱净化,或直接采用多层硅胶柱净化等\[11,12,14\],前处理方法较为繁琐。而分散固相萃取法(Dispersive solid phase extraction,DSPE)具有简单、快速、廉价等特点,能有效去除有机酸、糖、色素及脂肪等杂质,然而,目前PBDEs, MeOPBDEs, OHPBDEs, TBBPA及HBCD同时检测方法未见报道。本研究建立了以加速溶剂萃取(Accelerated solvent extraction,ASE)提取,GPC及DSPE净化的快速前处理方法,结合液相色谱串联质谱(LCMS/MS)及气相色谱负化学源质谱(GCNCI/MS)检测包括PBDEs, MeOPBDEs和OHPBDEs, TBBPA及HBCD在内的30种溴代阻燃剂方法。
2实验部分
2.1仪器与试剂
Varian300气相色谱质谱/质谱仪(美国瓦里安公司);安捷伦1200液相色谱仪(美国Agilent公司);API5000三重四级杆串联质谱仪,Analyst 1.4.2工作软件(美国应用生物系统公司);ASE 300加速溶剂萃取仪(美国戴安公司); NE1101旋转蒸发仪(日本EYELA公司); FD1A50冷冻干燥机(上海博医康实验仪器有限公司); 凝胶渗透色谱净化系统(美国J2 scientific公司); GPC凝胶材料为聚苯乙烯二乙烯基苯微球(美国BioRad公司); 高速冷冻离心机(德国Biofuge Stratos公司); MilliQ超纯水器(美国Millipore公司)。
二氯甲烷、正己烷(色谱级,美国Fisher公司);乙酸乙酯、环己烷、乙腈(农残级,美国Honeywell公司); N丙基乙二胺吸附剂(Primary secondary amine,PSA)、石墨化碳黑(Graphitized carbon black,GCB)、十八烷基键合硅胶(Octadecyl bonded silica,C18)吸附剂(天津博纳艾杰尔公司);三硅酸二镁(Florisil,弗罗里硅土,美国Sigma公司)。
2.2实验方法
2.2.1样品前处理(1)提取称取1.0 g(精确至0.01 g)冻干蛋白粉或蛋黄粉,加入1.0 g(精确至0.01 g)硅藻土混匀后,装入34 mL的ASE萃取池中,以正己烷二氯甲烷 (1∶1, V/V) 混合溶剂,温度100 ℃,压力10.4 MPa, 预热5 min,静态提取10 min,60%萃取池体积溶剂快速冲洗样品,氮气吹扫90 s等条件进行加速溶剂萃取,收集全部萃取液。将萃取液转入100 mL旋转蒸发瓶中,30 ℃减压旋转蒸发浓缩至近干, 转换溶剂为乙酸乙酯环己烷(1∶1, V/V) 混合溶剂,定容至10 mL,过0.45 μm尼龙膜后进行GPC净化。(2)净化GPC采用50g BioBeads SX3(400 mm×30 mm)聚苯乙烯树脂凝胶色谱柱,流动相为乙酸乙酯环己烷(1∶1, V/V),流速4.7 mL/min。收集18~30 min组分于100 mL旋转蒸发瓶中,在30 ℃以120 r/min旋转蒸发至近干,用乙腈润洗3次转入KD浓缩管,并旋蒸至1 mL以下,用乙腈定容至1 mL。用玻璃吸管将其转入装有100 mg C18分散固相萃取吸附剂的10 mL玻璃离心管中,高速涡旋1 min后,在4 ℃以3000 r/min离心5 min,上清液过0.22 μm有机相滤膜后进行测定,其中GCNCI/MS测定PBDEs和MeOPBDEs,LCMS/MS测定OHPBDEs, TBBPA和HBCD。
2.2.2色谱和质谱条件(1)GCNCI/MS气相色谱条件DB5 MS UI(30 m×0.25 mm×0.25 μm)毛细管柱;载气为高纯氦气(≥99.999%),恒流,柱流量为1.0 mL/min;进样口温度:280 ℃;不分流进样,进样体积: 1 μL。程序升温:初始温度180 ℃,保持2 min; 以15 ℃ /min升至205 ℃,保持2 min; 以3 ℃ /min升至251 ℃,保持1 min; 以5 ℃ /min升至310 ℃,保持10 min,共计41.83 min。质谱条件:离子源温度:280 ℃;电离模式:负化学电离源(NCI),电子能量:70 eV;溶剂延迟时间:6.00 min;反应气:甲烷(≥99.999%),压力为0.666 kPa;数据采集模式:选择离子监测(SIM)。12种PBDEs和5种MeOPBDEs的监测离子为79.0和80.9,选择79.0做定量离子。(2)LCMS/MS采用XBrige C18色谱柱(150 mm×2.1 mm,3.5 μm);柱温:40 ℃;进样体积:5 μL。流动相A为0.1%甲酸,流动相B为乙腈,流速为0.20 mL/min,梯度洗脱程序: 0 min, 60% B; 0~5 min, 60%~70% B; 5~15 min, 70%~90% B; 15~17 min, 90%~95% B; 17~18 min, 95%~60% B; 18~23 min, 60% B。
电喷雾离子源温度(TEM):500 ℃; 雾化气压力:448.5 kPa; 辅助气压力:310.5 kPa; 气帘气压力:172.5 kPa; 电喷雾电离负离子模式喷雾电压(IS
Symbolm@@ 4500 V; 监测离子对、碰撞能量(Collision energy,CE)和去簇电压(Declustering potential,DP)、碰撞室入口电压(Entrance potential,EP)和碰撞室出口电压(Collision cell exit potential,CXP)如表1所示。
3结果与讨论
3.1凝胶渗透色谱净化采用乙酸乙酯环己烷(1∶1, V/V)为流动相,流速为4.7 mL/min,将一定浓度的PBDEs(2 μg/L)、MeOPBDEs(2 μg/L)及OHPBDEs(0.2 μg/L)、TPBBA(2 μg/L)和HBCD(2 μg/L)的混合标准溶液通过凝胶渗透色谱系统,以紫外检测器确定各组分的采集时间。混合标准溶液的紫外吸收图中PBDEs及MeOPBDEs的出峰时间为23~29 min,而OHPBDEs、TPBBA和HBCD出峰时间为16~30 min。图1为蛋白和蛋黄加标后的ASEGPC紫外色谱图,蛋白萃取液中大分子类杂质浓度较低,蛋黄萃取液中大分子类杂质明显,流出时间约在8~15 min,目标物的流出时间在18~30 min之间。此外,本研究设置了不同的收集时间段的馏分,用回收率评价,在保证更好地去除脂肪的前提下,进一步确定目标化合物的最佳收集时间。实验表明,21~30 min采集时间内,TBBPA和αHBCD的平均回收率仅为26.3%和43.0%; 20~30 min采集时间内,TBBPA的平均回收率仅为57.7%;18~30 min 目标物质的平均回收率均在90.4%~114.5%,满足检测需要。故选择18~30 min作为样品GPC净化过程中PBDEs及其衍生物、TPBBA和HBCD的收集时间段。
3.2分散固相萃取吸附剂的选择
本实验选择PSA、GCB、C18和弗罗里硅土分散固相萃取吸附剂,比较了它们对目标物净化回收效果的影响。以50 mg GCB为分散固相萃取吸附剂时,发现其对目标物有较强的吸附作用,回收率仅为6.6%~85.8%, 这是由于GCB对平面分子具有极强的亲和力,而目标物分子含有平面苯环被部分吸附,从而导致回收率较低。PSA主要是通过离子交换作用除去样品中的金属离子、部分脂肪酸和糖,以50 mg PSA为分散固相萃取吸附剂时,目标物PBDEs、MeOPBDEs及HBCD的平均回收率为94.0%~128.3%,但对OHPBDEs及TBBPA有较大吸附,3OHBDE47, 5OHBDE47, 6OHBDE47, 4′OHBDE49, 2′OH6′ClBDE68, 6OHBDE90, 2OHBDE123, 6OHBDE137及TBBPA的回收率均低于10%。以50 mg弗罗里硅土为分散固相萃取吸附剂时,目标物PBDEs, MeOPBDEs及HBCD的平均回收率为78.1%~116.8%,但对四溴羟基代谢物及TBBPA有很大程度的保留,3OHBDE47, 5OHBDE47, 6OHBDE47, 4′OHBDE49及TBBPA的回收率均小于60%。而以50 mg C18为分散固相萃取吸附剂时,所有目标物的回收率在85.9%~120.2%之间,满足分析需要。本实验中选择100 mg C18作为分散固相萃取吸附剂。
3.3方法的线性范围与检出限
取不含目标分析物的空白蛋白蛋黄样品,按照前处理步骤处理,用空白样品提取液配制0.1, 0.2, 0.5, 1.0, 2.0, 5.0和10.0 μg/kg的标准工作液,以定量离子的峰面积对浓度进行回归分析绘制标准曲线,结果表明,目标物在0.1~10 μg/kg浓度范围内线性关系良好,相关系数(r2)大于0.99。以10倍信噪比计算定量限(LOQ),目标物的定量限为0.01~0.2 μg/kg。
3.4方法的回收率和精密度
采用蛋白和蛋黄空白样品,进行添加回收和精密度实验。空白样品添加不同浓度的标准溶液,按本实验方法进行处理,用GCNCI/MS和HPLCMS/MS进样测定,外标法定量。蛋白和蛋黄样品的平均回收率(每个添加浓度平行测定3次)分别为64.5%~97.2%和65.6%~109.2%(除BDE85为54.8%,OHBDE137为47.4%外),相对标准偏差小于20.2%,结果见表2。蛋黄加标色谱图见图2。
3.5基质效应
用乙腈配制了0.1, 0.2, 0.5, 1.0, 2.0, 5.0和10.0 μg/L的系列混合标准溶液,同时称取空白蛋白和蛋黄,按照前处理步骤提取净化,并对空白样品进行分析,确定其不含痕量目标物后,再用空白样品液稀释系列同溶剂标样浓度的基质标样,分别以标样浓度为横坐标、峰面积为纵坐标进行线性回归。基质标样和溶剂标样的斜率列于表2。溶剂和基质标样曲线线性良好,相关系数均大于0.99。基质与溶剂标准曲线斜率的比值可反映基质效应的强弱。对同一物质,蛋白和蛋黄中基质效应有所不同,如TBBPA在蛋白基质中呈现微弱的基质增强效应,而在蛋黄基质中却呈现明显抑制效应。
3.6实际样品分析
本研究测定了采自北京批发农贸市场、大型超市及农村等91个批次鸡蛋样品。PBDEs在蛋白和蛋黄中的检出率分别为4.48%和10.45%,∑PBDEs浓度水平分别为0.40~1.86 μg/kg和0.42~33.57 μg/kg; MeOPBDEs在蛋白和蛋黄中的检出率分别为1.49%和4.48%,∑MeOPBDEs浓度水平分别为0.46 μg/kg和0.46~13.57 μg/kg,其中PBDEs和MeOPBDEs在蛋黄中的检出种类及浓度大于蛋白。TBBPA在蛋白和蛋黄中的检出率分别为50.55%和25.70%,浓度水平分别为0.02~1.27 μg/kg和0.08~0.68 μg/kg。αHBCD, βHBCD和γHBCD在蛋白中的检出率分别为23.08%, 5.49%和89.01%,浓度水平分别为0~4.44 μg/kg, 0.24~1.61 μg/kg和0.10~32.28 μg/kg;在蛋黄中的检出率分别为96.70%, 18.68%和87.91%,含量分别为0.10~360.40 μg/kg, 0.22~4.16 μg/kg和0.20~71.20 μg/kg。而样品中未检出OHPBDEs。图3为典型蛋黄样品中目标物的色谱图。
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AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction
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14Hu X, Hu D, Song Q, Li J, Wang P. Chemosphere, 2011, 82(5): 698-707
15HE SongJie, LI MingYuan, JIN Jun, WANG Ying, HE Chang, BU YunJie, TIAN Yang. Chinese J. Anal. Chem., 2012, 40(10): 1519-1523
何松洁, 李明圆, 金 军, 王 英, 何 畅,卜云洁, 田 旸. 分析化学, 2012, 40(10): 1519-1523
16Sjdin A, Wong L Y, Jones R S, Park A, Zhang Y, Hodge C, Dipietro E, McClure C, Turner W, Needham L L, Patterson D G Jr. Environ. Sci. Technol., 2008, 42(4): 1377-1384
17Lankova D, Lacina O, Pulkrabova J, Hajslova J. Talanta, 2013, 117: 318-325
18Rayne S, Ikonomou M G, Antcliffe B. Environ. Sci. Technol., 2003, 37: 2847-2854
19Ni K, Lu Y, Wang T, Kannan K, Xu L, Li Q, Wang L, Liu S. Int. J. Hyg. Environ. Health, 2013, 216(6): 607-623
AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction
12Qin X F, Qin Z F, Li Y, Zhao Y X, Xia X J, Yan S S, Tian M, Zhao X R, Xu X B, Yang Y J. J Environ Scichina, 2011, 23(1): 133-138
13Covaci A, Roosens L, Dirtu A C, Waegeneers N, Overmeire I V, Neels H, Goeyens L. Sci. Total Environ., 2008, 407(15): 4387-4396
14Hu X, Hu D, Song Q, Li J, Wang P. Chemosphere, 2011, 82(5): 698-707
15HE SongJie, LI MingYuan, JIN Jun, WANG Ying, HE Chang, BU YunJie, TIAN Yang. Chinese J. Anal. Chem., 2012, 40(10): 1519-1523
何松洁, 李明圆, 金 军, 王 英, 何 畅,卜云洁, 田 旸. 分析化学, 2012, 40(10): 1519-1523
16Sjdin A, Wong L Y, Jones R S, Park A, Zhang Y, Hodge C, Dipietro E, McClure C, Turner W, Needham L L, Patterson D G Jr. Environ. Sci. Technol., 2008, 42(4): 1377-1384
17Lankova D, Lacina O, Pulkrabova J, Hajslova J. Talanta, 2013, 117: 318-325
18Rayne S, Ikonomou M G, Antcliffe B. Environ. Sci. Technol., 2003, 37: 2847-2854
19Ni K, Lu Y, Wang T, Kannan K, Xu L, Li Q, Wang L, Liu S. Int. J. Hyg. Environ. Health, 2013, 216(6): 607-623
AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction
摘要建立了一种同时检测鸡蛋中四溴双酚A(TBBP A)、六溴环十二烷(HBCD) 和多溴联苯醚(PBDEs)及其衍生物羟基多溴联苯醚(OHPBDEs)和甲氧基多溴联苯醚(MeOPBDEs)的凝胶渗透色谱(GPC)分散固相萃取(DSPE)液相色谱串联质谱(HPLCMS/MS)和气相色谱负化学源质谱(GCNCI/MS)的检测方法。样品经正己烷、二氯甲烷 (1∶1, V/V)加速溶剂萃取, 凝胶渗透色谱净化后, 经100 mg十八烷基键合硅胶(C18)分散固相萃取吸附剂去除杂质, 液相色谱串联质谱和气相色谱负化学源质谱方法测定,外标法定量。在蛋白和蛋黄样品中添加1.0或5.0 μg/kg的目标物,其回收率分别为64.5%~97.2%和65.6%~109.2%(除BDE85为54.8%,OHBDE137为47.4%外),相对标准偏差小于20.2%,定量限为0.01~0.2 μg/kg。
关键词多溴联苯醚及其衍生物;四溴双酚A;六溴环十二烷;鸡蛋;凝胶渗透色谱;分散固相萃取
1引言
多溴联苯醚(Polybrominated diphenyl ethers, PBDEs)、四溴双酚A(Tetrabromobisphenol A, TBBP A)和六溴环十二烷(Hexabromocyclododecane,HBCD)是目前广泛应用于电子设备、塑料、建筑材料、装饰材料及纺织品等的溴代阻燃剂(Brominated flame retardants, BFRs),近年来,人们逐渐注意到这三类溴代阻燃剂对环境及人类健康的影响。2009年,全球关于持久性有机污染物的《斯德哥尔摩公约》第四次缔约国大会上,商业化的五溴联苯醚及八溴联苯醚被列为全球控制和消减的持久性有机污染物(POPs),该公约提出在全球范围内禁止使用HBCD。现有研究表明,这三类阻燃剂普遍具有内分泌毒性、生殖毒性、肝脏毒性和神经毒性,威胁到人类的健康和生命安全。虽然目前对PBDEs在生物体内的代谢产物羟基代谢物(Hydroxylated polybrominated diphenyl ethers, OHPBDEs)及甲氧基代谢物(Methoxylated polybrominated diphenyl ethers, MeOPBDEs)的来源还有争议\[1~3\], 但MeOPBDEs和OHPBDEs可能具有更高的毒性\[4,5\],且在生物体内OHPBDEs与MeOPBDEs可以相互转化\[1\]。人体血液中MeOPBDEs的含量高于PBDEs\[6\],因此有必要同时测定生物体内的PBDEs、MeOPBDEs和OHPBDEs。
随着电子业的发展,环境中BFRs的污染报道也日益增加\[7,8\]。PBDEs、TBBPA和HBCD三类溴代阻燃剂的辛醇/水分配系数(lgKow)约为4.5~10.0,具有持久性和脂溶性,可通过生物富集作用累积在生物组织内,近年来关于三类溴代阻燃剂在生物组织中污染水平的报道日益增多\[9~17\]。动物性产品成为BFRs进入人体的重要途径之一\[18,19\],根据文献\[9\]的风险评估报告,淡水鱼、肉和鸡蛋等富含脂质的食品是人类膳食摄入PBDEs的重要来源,其中淡水鱼占12%~98%,猪肉占1%~20%,而鸡蛋占1%~15%。电子垃圾回收区鸡蛋的∑PBDEs和∑HBCD检出浓度甚至高达14100 ng/g (脂肪重量,fat weight, fw)和350 ng/g (fw) \[11\]。
鸡蛋样品脂肪含量高,色素重,目前常用的提取方法是索氏提取和超声提取,净化步骤多采用凝胶渗透色谱(Gel permeation chromatography,GPC)结合多层硅胶柱净化,或酸性硅胶柱结合酸性氧化铝柱净化,或直接采用多层硅胶柱净化等\[11,12,14\],前处理方法较为繁琐。而分散固相萃取法(Dispersive solid phase extraction,DSPE)具有简单、快速、廉价等特点,能有效去除有机酸、糖、色素及脂肪等杂质,然而,目前PBDEs, MeOPBDEs, OHPBDEs, TBBPA及HBCD同时检测方法未见报道。本研究建立了以加速溶剂萃取(Accelerated solvent extraction,ASE)提取,GPC及DSPE净化的快速前处理方法,结合液相色谱串联质谱(LCMS/MS)及气相色谱负化学源质谱(GCNCI/MS)检测包括PBDEs, MeOPBDEs和OHPBDEs, TBBPA及HBCD在内的30种溴代阻燃剂方法。
2实验部分
2.1仪器与试剂
Varian300气相色谱质谱/质谱仪(美国瓦里安公司);安捷伦1200液相色谱仪(美国Agilent公司);API5000三重四级杆串联质谱仪,Analyst 1.4.2工作软件(美国应用生物系统公司);ASE 300加速溶剂萃取仪(美国戴安公司); NE1101旋转蒸发仪(日本EYELA公司); FD1A50冷冻干燥机(上海博医康实验仪器有限公司); 凝胶渗透色谱净化系统(美国J2 scientific公司); GPC凝胶材料为聚苯乙烯二乙烯基苯微球(美国BioRad公司); 高速冷冻离心机(德国Biofuge Stratos公司); MilliQ超纯水器(美国Millipore公司)。
二氯甲烷、正己烷(色谱级,美国Fisher公司);乙酸乙酯、环己烷、乙腈(农残级,美国Honeywell公司); N丙基乙二胺吸附剂(Primary secondary amine,PSA)、石墨化碳黑(Graphitized carbon black,GCB)、十八烷基键合硅胶(Octadecyl bonded silica,C18)吸附剂(天津博纳艾杰尔公司);三硅酸二镁(Florisil,弗罗里硅土,美国Sigma公司)。
2.2实验方法
2.2.1样品前处理(1)提取称取1.0 g(精确至0.01 g)冻干蛋白粉或蛋黄粉,加入1.0 g(精确至0.01 g)硅藻土混匀后,装入34 mL的ASE萃取池中,以正己烷二氯甲烷 (1∶1, V/V) 混合溶剂,温度100 ℃,压力10.4 MPa, 预热5 min,静态提取10 min,60%萃取池体积溶剂快速冲洗样品,氮气吹扫90 s等条件进行加速溶剂萃取,收集全部萃取液。将萃取液转入100 mL旋转蒸发瓶中,30 ℃减压旋转蒸发浓缩至近干, 转换溶剂为乙酸乙酯环己烷(1∶1, V/V) 混合溶剂,定容至10 mL,过0.45 μm尼龙膜后进行GPC净化。(2)净化GPC采用50g BioBeads SX3(400 mm×30 mm)聚苯乙烯树脂凝胶色谱柱,流动相为乙酸乙酯环己烷(1∶1, V/V),流速4.7 mL/min。收集18~30 min组分于100 mL旋转蒸发瓶中,在30 ℃以120 r/min旋转蒸发至近干,用乙腈润洗3次转入KD浓缩管,并旋蒸至1 mL以下,用乙腈定容至1 mL。用玻璃吸管将其转入装有100 mg C18分散固相萃取吸附剂的10 mL玻璃离心管中,高速涡旋1 min后,在4 ℃以3000 r/min离心5 min,上清液过0.22 μm有机相滤膜后进行测定,其中GCNCI/MS测定PBDEs和MeOPBDEs,LCMS/MS测定OHPBDEs, TBBPA和HBCD。
2.2.2色谱和质谱条件(1)GCNCI/MS气相色谱条件DB5 MS UI(30 m×0.25 mm×0.25 μm)毛细管柱;载气为高纯氦气(≥99.999%),恒流,柱流量为1.0 mL/min;进样口温度:280 ℃;不分流进样,进样体积: 1 μL。程序升温:初始温度180 ℃,保持2 min; 以15 ℃ /min升至205 ℃,保持2 min; 以3 ℃ /min升至251 ℃,保持1 min; 以5 ℃ /min升至310 ℃,保持10 min,共计41.83 min。质谱条件:离子源温度:280 ℃;电离模式:负化学电离源(NCI),电子能量:70 eV;溶剂延迟时间:6.00 min;反应气:甲烷(≥99.999%),压力为0.666 kPa;数据采集模式:选择离子监测(SIM)。12种PBDEs和5种MeOPBDEs的监测离子为79.0和80.9,选择79.0做定量离子。(2)LCMS/MS采用XBrige C18色谱柱(150 mm×2.1 mm,3.5 μm);柱温:40 ℃;进样体积:5 μL。流动相A为0.1%甲酸,流动相B为乙腈,流速为0.20 mL/min,梯度洗脱程序: 0 min, 60% B; 0~5 min, 60%~70% B; 5~15 min, 70%~90% B; 15~17 min, 90%~95% B; 17~18 min, 95%~60% B; 18~23 min, 60% B。
电喷雾离子源温度(TEM):500 ℃; 雾化气压力:448.5 kPa; 辅助气压力:310.5 kPa; 气帘气压力:172.5 kPa; 电喷雾电离负离子模式喷雾电压(IS
Symbolm@@ 4500 V; 监测离子对、碰撞能量(Collision energy,CE)和去簇电压(Declustering potential,DP)、碰撞室入口电压(Entrance potential,EP)和碰撞室出口电压(Collision cell exit potential,CXP)如表1所示。
3结果与讨论
3.1凝胶渗透色谱净化采用乙酸乙酯环己烷(1∶1, V/V)为流动相,流速为4.7 mL/min,将一定浓度的PBDEs(2 μg/L)、MeOPBDEs(2 μg/L)及OHPBDEs(0.2 μg/L)、TPBBA(2 μg/L)和HBCD(2 μg/L)的混合标准溶液通过凝胶渗透色谱系统,以紫外检测器确定各组分的采集时间。混合标准溶液的紫外吸收图中PBDEs及MeOPBDEs的出峰时间为23~29 min,而OHPBDEs、TPBBA和HBCD出峰时间为16~30 min。图1为蛋白和蛋黄加标后的ASEGPC紫外色谱图,蛋白萃取液中大分子类杂质浓度较低,蛋黄萃取液中大分子类杂质明显,流出时间约在8~15 min,目标物的流出时间在18~30 min之间。此外,本研究设置了不同的收集时间段的馏分,用回收率评价,在保证更好地去除脂肪的前提下,进一步确定目标化合物的最佳收集时间。实验表明,21~30 min采集时间内,TBBPA和αHBCD的平均回收率仅为26.3%和43.0%; 20~30 min采集时间内,TBBPA的平均回收率仅为57.7%;18~30 min 目标物质的平均回收率均在90.4%~114.5%,满足检测需要。故选择18~30 min作为样品GPC净化过程中PBDEs及其衍生物、TPBBA和HBCD的收集时间段。
3.2分散固相萃取吸附剂的选择
本实验选择PSA、GCB、C18和弗罗里硅土分散固相萃取吸附剂,比较了它们对目标物净化回收效果的影响。以50 mg GCB为分散固相萃取吸附剂时,发现其对目标物有较强的吸附作用,回收率仅为6.6%~85.8%, 这是由于GCB对平面分子具有极强的亲和力,而目标物分子含有平面苯环被部分吸附,从而导致回收率较低。PSA主要是通过离子交换作用除去样品中的金属离子、部分脂肪酸和糖,以50 mg PSA为分散固相萃取吸附剂时,目标物PBDEs、MeOPBDEs及HBCD的平均回收率为94.0%~128.3%,但对OHPBDEs及TBBPA有较大吸附,3OHBDE47, 5OHBDE47, 6OHBDE47, 4′OHBDE49, 2′OH6′ClBDE68, 6OHBDE90, 2OHBDE123, 6OHBDE137及TBBPA的回收率均低于10%。以50 mg弗罗里硅土为分散固相萃取吸附剂时,目标物PBDEs, MeOPBDEs及HBCD的平均回收率为78.1%~116.8%,但对四溴羟基代谢物及TBBPA有很大程度的保留,3OHBDE47, 5OHBDE47, 6OHBDE47, 4′OHBDE49及TBBPA的回收率均小于60%。而以50 mg C18为分散固相萃取吸附剂时,所有目标物的回收率在85.9%~120.2%之间,满足分析需要。本实验中选择100 mg C18作为分散固相萃取吸附剂。
3.3方法的线性范围与检出限
取不含目标分析物的空白蛋白蛋黄样品,按照前处理步骤处理,用空白样品提取液配制0.1, 0.2, 0.5, 1.0, 2.0, 5.0和10.0 μg/kg的标准工作液,以定量离子的峰面积对浓度进行回归分析绘制标准曲线,结果表明,目标物在0.1~10 μg/kg浓度范围内线性关系良好,相关系数(r2)大于0.99。以10倍信噪比计算定量限(LOQ),目标物的定量限为0.01~0.2 μg/kg。
3.4方法的回收率和精密度
采用蛋白和蛋黄空白样品,进行添加回收和精密度实验。空白样品添加不同浓度的标准溶液,按本实验方法进行处理,用GCNCI/MS和HPLCMS/MS进样测定,外标法定量。蛋白和蛋黄样品的平均回收率(每个添加浓度平行测定3次)分别为64.5%~97.2%和65.6%~109.2%(除BDE85为54.8%,OHBDE137为47.4%外),相对标准偏差小于20.2%,结果见表2。蛋黄加标色谱图见图2。
3.5基质效应
用乙腈配制了0.1, 0.2, 0.5, 1.0, 2.0, 5.0和10.0 μg/L的系列混合标准溶液,同时称取空白蛋白和蛋黄,按照前处理步骤提取净化,并对空白样品进行分析,确定其不含痕量目标物后,再用空白样品液稀释系列同溶剂标样浓度的基质标样,分别以标样浓度为横坐标、峰面积为纵坐标进行线性回归。基质标样和溶剂标样的斜率列于表2。溶剂和基质标样曲线线性良好,相关系数均大于0.99。基质与溶剂标准曲线斜率的比值可反映基质效应的强弱。对同一物质,蛋白和蛋黄中基质效应有所不同,如TBBPA在蛋白基质中呈现微弱的基质增强效应,而在蛋黄基质中却呈现明显抑制效应。
3.6实际样品分析
本研究测定了采自北京批发农贸市场、大型超市及农村等91个批次鸡蛋样品。PBDEs在蛋白和蛋黄中的检出率分别为4.48%和10.45%,∑PBDEs浓度水平分别为0.40~1.86 μg/kg和0.42~33.57 μg/kg; MeOPBDEs在蛋白和蛋黄中的检出率分别为1.49%和4.48%,∑MeOPBDEs浓度水平分别为0.46 μg/kg和0.46~13.57 μg/kg,其中PBDEs和MeOPBDEs在蛋黄中的检出种类及浓度大于蛋白。TBBPA在蛋白和蛋黄中的检出率分别为50.55%和25.70%,浓度水平分别为0.02~1.27 μg/kg和0.08~0.68 μg/kg。αHBCD, βHBCD和γHBCD在蛋白中的检出率分别为23.08%, 5.49%和89.01%,浓度水平分别为0~4.44 μg/kg, 0.24~1.61 μg/kg和0.10~32.28 μg/kg;在蛋黄中的检出率分别为96.70%, 18.68%和87.91%,含量分别为0.10~360.40 μg/kg, 0.22~4.16 μg/kg和0.20~71.20 μg/kg。而样品中未检出OHPBDEs。图3为典型蛋黄样品中目标物的色谱图。
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8Lin Y, Qiu X H, Zhao Y F, Ma J, Yang Q Y, Zhu T. Environ. Sci. Technol., 2013, 47: 12761-12767
9Chan J K, Man Y B, Wu S C, Wong M H. Sci. Total Environ., 2013, 463464: 1138-1146
10Labunska I, Harrad S, Santillo D, Johnston P, Yun L. Environ. Sci. Technol., 2013, 47: 9258-9266
11Zheng X B, Wu J P, Luo X J, Zeng Y H, She Y Z, Mai B X. Environ. Int., 2012, 45: 122-128
12Qin X F, Qin Z F, Li Y, Zhao Y X, Xia X J, Yan S S, Tian M, Zhao X R, Xu X B, Yang Y J. J Environ Scichina, 2011, 23(1): 133-138
13Covaci A, Roosens L, Dirtu A C, Waegeneers N, Overmeire I V, Neels H, Goeyens L. Sci. Total Environ., 2008, 407(15): 4387-4396
14Hu X, Hu D, Song Q, Li J, Wang P. Chemosphere, 2011, 82(5): 698-707
15HE SongJie, LI MingYuan, JIN Jun, WANG Ying, HE Chang, BU YunJie, TIAN Yang. Chinese J. Anal. Chem., 2012, 40(10): 1519-1523
何松洁, 李明圆, 金 军, 王 英, 何 畅,卜云洁, 田 旸. 分析化学, 2012, 40(10): 1519-1523
16Sjdin A, Wong L Y, Jones R S, Park A, Zhang Y, Hodge C, Dipietro E, McClure C, Turner W, Needham L L, Patterson D G Jr. Environ. Sci. Technol., 2008, 42(4): 1377-1384
17Lankova D, Lacina O, Pulkrabova J, Hajslova J. Talanta, 2013, 117: 318-325
18Rayne S, Ikonomou M G, Antcliffe B. Environ. Sci. Technol., 2003, 37: 2847-2854
19Ni K, Lu Y, Wang T, Kannan K, Xu L, Li Q, Wang L, Liu S. Int. J. Hyg. Environ. Health, 2013, 216(6): 607-623
AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction
12Qin X F, Qin Z F, Li Y, Zhao Y X, Xia X J, Yan S S, Tian M, Zhao X R, Xu X B, Yang Y J. J Environ Scichina, 2011, 23(1): 133-138
13Covaci A, Roosens L, Dirtu A C, Waegeneers N, Overmeire I V, Neels H, Goeyens L. Sci. Total Environ., 2008, 407(15): 4387-4396
14Hu X, Hu D, Song Q, Li J, Wang P. Chemosphere, 2011, 82(5): 698-707
15HE SongJie, LI MingYuan, JIN Jun, WANG Ying, HE Chang, BU YunJie, TIAN Yang. Chinese J. Anal. Chem., 2012, 40(10): 1519-1523
何松洁, 李明圆, 金 军, 王 英, 何 畅,卜云洁, 田 旸. 分析化学, 2012, 40(10): 1519-1523
16Sjdin A, Wong L Y, Jones R S, Park A, Zhang Y, Hodge C, Dipietro E, McClure C, Turner W, Needham L L, Patterson D G Jr. Environ. Sci. Technol., 2008, 42(4): 1377-1384
17Lankova D, Lacina O, Pulkrabova J, Hajslova J. Talanta, 2013, 117: 318-325
18Rayne S, Ikonomou M G, Antcliffe B. Environ. Sci. Technol., 2003, 37: 2847-2854
19Ni K, Lu Y, Wang T, Kannan K, Xu L, Li Q, Wang L, Liu S. Int. J. Hyg. Environ. Health, 2013, 216(6): 607-623
AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction
12Qin X F, Qin Z F, Li Y, Zhao Y X, Xia X J, Yan S S, Tian M, Zhao X R, Xu X B, Yang Y J. J Environ Scichina, 2011, 23(1): 133-138
13Covaci A, Roosens L, Dirtu A C, Waegeneers N, Overmeire I V, Neels H, Goeyens L. Sci. Total Environ., 2008, 407(15): 4387-4396
14Hu X, Hu D, Song Q, Li J, Wang P. Chemosphere, 2011, 82(5): 698-707
15HE SongJie, LI MingYuan, JIN Jun, WANG Ying, HE Chang, BU YunJie, TIAN Yang. Chinese J. Anal. Chem., 2012, 40(10): 1519-1523
何松洁, 李明圆, 金 军, 王 英, 何 畅,卜云洁, 田 旸. 分析化学, 2012, 40(10): 1519-1523
16Sjdin A, Wong L Y, Jones R S, Park A, Zhang Y, Hodge C, Dipietro E, McClure C, Turner W, Needham L L, Patterson D G Jr. Environ. Sci. Technol., 2008, 42(4): 1377-1384
17Lankova D, Lacina O, Pulkrabova J, Hajslova J. Talanta, 2013, 117: 318-325
18Rayne S, Ikonomou M G, Antcliffe B. Environ. Sci. Technol., 2003, 37: 2847-2854
19Ni K, Lu Y, Wang T, Kannan K, Xu L, Li Q, Wang L, Liu S. Int. J. Hyg. Environ. Health, 2013, 216(6): 607-623
AbstractA stimulate method for determination of polybrominated diphenyl ethers (PBDEs) and derivatives (OHPBDEs and MeOPBDEs), tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) in egg samples was developed by gel permeation chromatography (GPC) and dispersive solid phase extraction (DSPE) combined with liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS). The analytes were extracted with mixture of hexane and dichloromethane (1∶1, V/V) by accelerated solvent extraction (ASE), and purified by 100 mg C18 dispersive solid phase extraction (SPE) sorbents followed with gel permeation chromatography (GPC) , and then analyzed by liquid chromatography tandem mass spectrometric (HPLCMS/MS) and gas chromatographynegative chemical ionization mass spectrometry (GCNCI/MS), respectively. The quantitation was carried out external standard method. The recoveries of objects were 64.5%-97.2% and 65.6%-109.2% (except BDE85 was 54.8%, OHBDE137 was 47.4%) spiked at 1.0 μg/kg or 5.0 μg/kg in egg white and egg yolk, respectively. The relative standard deviations (RSDs) were less than 20.2%. The limits of quantitation (LOQ) for the object were 0.01-0.2 μg/kg.
KeywordsPolybrominated diphenyl ethers and derivatives; Tetrabromobisphenol A; Hexabromocyclododecane; Egg; Gel permeation chromatography; Dispersive solid phase extraction