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有机磷酸酯(OPEs)具有良好的阻燃作用,增塑和润滑效果,在建材工业、电子设备、食品包装、室内装饰和纺织品中得到了广泛应用. 随着多溴联苯醚等溴系阻燃剂逐步禁止使用[1-3],作为重要替代品的OPEs ,其生产量和使用量显著增长[4]. 2006年,OPEs的全球使用量为46.5万吨[5], 2015年达到68万吨,呈以7.9%逐年递增[6]. 据估计,中国的OPEs产量每年将增加15%[4].OPEs以物理方式添加于各种材料,而不是化学键结合的形式[7], 随着时间的推移它们易通过挥发和磨损、渗漏而进入环境介质,如水体、土壤、大气及生物体内[8-13],从而导致人体通过多种途径暴露.OPEs具有潜在生物累积性,长期暴露于人体会对其健康产生不利影响. 大量研究证实,OPEs具有甲状腺和内分泌干扰作用[14-15]、神经毒性[16]和生殖毒性[17],并且具有致癌性[4-5]. 由于其潜在的毒性作用,以及在环境和生物体内的广泛存在,日益引起人们的关注[4-5].
目前已有大量的研究报道了OPEs在空气、室内灰尘、土壤、水体和沉积物等环境介质中的赋存情况[18-20]. 但是针对生物样品而言,其脂肪含量相对较高,前处理过程相对复杂,对于生物体内OPEs的研究仍比较有限[4-5, 21-22]. 与此同时,由于OPEs在生物体内能够发生快速的代谢转化[23-24],近年来越来越多的研究开始关注OPEs的代谢产物(mOPEs),并将其作为暴露标志物研究人体对OPEs的暴露情况[25-30]. 然而,Liu等[31]在室内灰尘中同时检出了OPEs和mOPEs,表明环境介质中同时存在OPEs和mOPEs. 因此,mOPEs除源自生物体对OPEs的代谢转化外,也可能来自外源直接摄入. 膳食摄入被认为是人体暴露阻燃剂的重要途径[32] ,此前的研究在北美洲休伦湖银鸥蛋[33]和广东清远电子废弃物回收区域鸡蛋中[34]均检测到了一定程度的OPEs. 有机磷酸酯具有神经、生殖毒性及致癌作用已被广泛报道,目前对于OPEs的研究较多,而关于代谢产物mOPEs的研究较少. 研究表明,部分mOPEs的毒性很可能比其母体化合物更高,如Su等[35]采用鸡胚胎细胞实验发现,DPHP(磷酸二苯酯)对神经系统发育的负面作用比其母体化合物TPHP(磷酸三苯酯)更强,这也提示我们需要更加关注mOPEs的毒性效应. 由于鸡蛋是一种高脂肪含量的生物样本,从前检测鸡蛋中OPEs的方法在萃取、纯化和测定过程中存在一定不足,如萃取和纯化过程复杂,且少有能同时萃取、纯化和测定鸡蛋中OPEs和mOPEs的方法. 因此,建立能同时提取鸡蛋中OPEs和mOPEs的高效、灵敏、准确的分析方法对研究OPEs和mOPEs的生物富集和人体暴露评估都具有十分重要的意义.
本研究建立了基于固相萃取和LC-MS/MS同步检测鸡蛋中16种OPEs和mOPEs的分析方法,并利用建立的方法分析了12例鸡蛋中OPEs和mOPEs的含量水平,本研究的结果为准确测定高脂肪含量生物样品中的OPEs和mOPEs提供了有效的支撑.
固相萃取-液相色谱串联质谱(LC-MS/MS)同步测定鸡蛋样品中有机磷酸酯及其代谢产物
Simultaneous determination of organophosphate esters and their specific metabolites in egg samples by solid-phase extraction with LC-MS/MS
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摘要: 本研究基于固相萃取和液相色谱串联质谱(LC-MS/MS)建立了同步提取、净化和检测高脂肪含量生物样品中7种有机磷酸酯及其9种代谢产物的分析方法. 取鸡蛋样品用体积比为9 : 1乙腈 : 甲苯混合溶剂超声提取,用Florisil-PR 固相萃取柱初步净化,将含有目标物的溶液经Z-Sep/C18除脂,并采用Strata X-AW柱进一步净化,使用LC-MS/MS进行分析. 目标化合物在0.2—500.0 ng·mL−1范围内呈良好线性关系,相关系数R2介于0.997—0.999之间. OPEs(有机磷酸酯)和它们的代谢物(mOPEs)的加标回收率范围分别在69.9%—81.3%和61.2%—82.5%之间,相对标准偏差(RSDs)分别介于7.9%—20%和11%—24%之间. 利用本文建立的分析方法对12例鸡蛋样品中的16种OPEs和mOPEs进行同步检测,鸡蛋样品中Σ7OPEs的浓度范围为1.43—5.9 ng·g−1,TCPP浓度最高(浓度中值为2.13 ng·g−1),占OPEs总浓度的58.8%. 7种OPEs除TEHP外,其他OPEs均有检出. Σ9mOPEs的浓度范围为2.81—11.8 ng·g−1,DPHP(磷酸三(2-氯丙基)酯)为主要检出单体(浓度中值为3.14 ng·g−1),浓度贡献占比为54%. 9种mOPEs除BCEP(二-β,β'-氯乙基磷酸)和BCPP(双-(1-氯-2-丙基)磷酸)外,其他mOPEs均有检出.Abstract: An effective method was established for the analysis of organophosphorus flame retardants and their diesters in organisms by liquid-liquid extraction and solid-phase extraction, followed by triple quadrupole liquid chromatography-tandem mass spectrometry(LC-MS/MS)for quantification. The organisms sample were extracted with mixed acetonitrile : Toluene(9:1, V/V)and purified by Florisil-PR cartridges. The phase was loaded onto Z-Sep/C18 tube and Strata X-AW cartridges for extraction and further clean-up, Then the extracts were analyzed by LC-MS/MS. Under the optimized conditions, good linearities for both OPEs(organophosphate esters) and their metabolites (mOPEs) were achieved as the analytes in the corresponding concentration range(0.2—500.0 ng·mL−1) and the correlation coefficients(R2)are 0.997—0.999.The average recoveries of OPEs and mOPEs in organisms range from 69.9% to 81.3% and 61.2% to 82.5%, with RSDs of 7.9% to 20% and 11% to 24%, respectively. Sixteen OPEs and mOPEs in 12 egg samples were detected synchronically using the method established. The concentration of ΣOPEs in egg samples ranged from 1.43 ng·g−1 to 5.9 ng·g−1, and the concentration of TCPP was the highest(2.13 ng·g−1, median concentration), accounting for 58.8% of total OPEs. All OPEs except TEHP and EHDPP were detected. ΣmOPEs ranged from 2.81 ng·g−1 to 11.8 ng·g−1, and DPHP was the main detected analyte(3.14 ng·g−1, median concentration), accounting for 54% of the concentration contribution. Except BCEP and BCPP, all other mOPEs were detected.
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表 1 有机磷酸酯及其代谢产物的中文全称、英文全称、简称及CAS
Table 1. Chinese name,English full name,abbreviation and CAS number of OPEs and their specific metabolites
中文全称
Chinese name英文全称
English full name简称
AbbreviationCAS 内标
Internal standard有机磷酸酯(OPEs) 磷酸三丁酯 Tributyl Phosphate TBP 126-73-8 d27-TBP 磷酸三(2-氯乙基)酯 Tris(2-Chloroethyl)Phosphate TCEP 115-96-8 d12-TCEP 磷酸三(2-乙基己基)酯 Tris(2-Ethylhexyl)Phosphate TEHP 78-42-2 d27-TBP 磷酸三(1,3-二氯-2-丙基)磷酸酯 Tris(1-Dichloro-2-propyl)Phosphate TDCPP 13674-87-8 d18-TCPP 磷酸三苯酯 Triphenyl Phosphate TPHP 115-86-6 d15-TPHP 磷酸三(2-丁氧乙基)酯 Tris(2-Butoxyethyl)Phosphate TBOEP 78-51-3 d27-TBOEP 磷酸三(2-氯丙基)酯 Tris(1-Chloro-2-propyl)Phosphate TCPP 13674-84-5 d18-TCPP 有机磷酸酯代谢产物(mOPEs) 磷酸二正丁酯 Dibutyl Phosphate DBP 107-66-4 d10-BDCIPP 二-β,β'-氯乙基磷酸 Di-β,β'-Chloroethylphosphoric Acid BCEP 3040-56-0 d8-BCEP 双 - (1-氯-2-丙基)磷酸 Bis-(1-chloro-2-propyl)phosphate BCPP 789440-10-4 d10-DPHP 双(1,3-二氯-2-丙基)磷酸 Bis(1,3-dichloro-2-propyl) Phosphate BDCPP 72236-72-7 d10-BDCIPP 磷酸二苯酯 Diphenyl phosphate DPHP 838-85-7 d10-DPHP 双(2-丁氧基乙基)2-羟基乙基磷酸三酯 Bis(2-butoxyethyl) 2-Hydroxyethyl
Phosphate TriesterBBOEHEP 1477494-86-2 d4-BBOEHEP 双(丁氧乙基)磷酸酯 Bis(butoxyethyl) Phosphate BBOEP 14260-97-0 d8-BBOEP 二(2-乙基己基)磷酸酯 Di(2-ethylhexyl) phosphate BEHP 298-07-7 d8-BBOEP 双(2-丁氧基乙基)2-(羟基丁氧基)乙基磷酸三酯 Bis(2-butoxyethyl) 2-(3-Hydroxybutoxy)
ethyl Phosphate TriesterOH-TBOEP 1477494-87-3 d8-BBOEP 表 2 流动相洗脱梯度
Table 2. Gradient elution of mobile phase
时间/min
Time(A)/% (B)/% 流速/(μL·min−1)
Velocity0 99 1 500 7.5 1 99 500 11.5 1 99 500 15.0 99 1 500 表 3 ESI-MS/MS参数
Table 3. ESI-MS/MS Parameter
分析物
Analytes母离子
Precursor ion(m/z)子离子
Product ion(m/z)时间 /min
Time去簇电压 /V
DP碰撞能/V
CE正离子模式 TBP 267.3 98.7/154.9 9.29 92/90 20/15 TCEP 289.0 65.1 7.06 78 46 TCPP 327.0 99.8/250.9 8,36 33/66 42/13 TDCPP 421.4 147.2/221.1 9.47 123/113 31/31 TPHP 327.4 152.4/215.0 9.03 93/112 45/33 TBOEP 399.2 299.2/199.3 9.13 115/75 19/21 TEHP 435.4 99.2/211.1 12.15 116/116 17/11 BBOEHEP 342.9 243.1/101.1 8.15 75/59 16/21 OH-TBOEP 415.1 199.1/243.1 8.57 82/85 21/23 负离子模式 DBP 209 153/79 7.6 −70/−70 −20/−45 BCEP 221.1 35 4.53 −39 −24 BCPP 249.0/250.9 35.1/37.1 6.57 −30/−13 −21/−21 BDCPP 316.8 35.1 7.94 −42 −31 DPHP 249.2/265.1 93.1/96.7 7.05 −87/−97 −36/−38 BBOEP 296.9 182.5 8.3 −76 −46 BEHP 321.1 209.2/79 13.39 −97/−102 −28/−45 表 4 不同萃取溶剂空白加标回收率(%)
Table 4. Blank standard recovery of different extraction solvents(%)
分析物
Analytes萃取溶剂
Extraction solvents乙腈 甲醇 :甲苯 乙腈 :甲苯 OPEs TCEP 65 66 67 TCPP 90 88 88 TDCPP 48 50 51 TBP 41 48 52 TBOEP 105 111 109 TEHP 31 57 90 TPHP 89 88 90 mOPEs DBP 61 10 98 BCEP 88 83 94 BCPP 86 118 50 BDCPP 88 91 94 DPHP 74 80 80 BBOEHEP 73 75 75 BBOEP 110 103 130 BEHP 60 16 88 OH-TBOEP 81 80 82 表 5 目标化合物的线性回归方程、相关系数及方法定量限
Table 5. Linear regression equations, correlation coefficients and method detection limit of quantification for analyte compounds
分析物
Analytes线性回归方程
Linear regression equations相关系数
Correlation coefficient空白浓度/(ng·g−1)
Blank方法定量限/( ng·g−1)
MDLOPEs TBP Y=0.07186x+0.00179 0.99716 0.044 ± 0.007 0.063 TCEP Y=0.33848x+0.00347 0.99928 0.044 ± 0.006 0.060 TCPP Y=2.01067x+0.03463 0.99875 0.22 ± 0.04 0.33 TDCPP Y=0.55785x+8.20943 0.99761 0.033 ± 0.006 0.051 TPHP Y=1.00853x+0.01098 0.99971 nd 0.0040 TBOEP Y=0.49885x+8.77055 0.99830 nd 0.045 TEHP Y=0.04196x+1.39158 0.99814 nd 0.0040 mOPEs DBP Y=34.50089x+0.02457 0.99871 0.0088±0.003 0.016 BCEP Y=0.38345x+0.00182 0.99942 nd 0.0040 BCPP Y=0.07190x+9.45927 0.99849 nd. 0.040 BDCPP Y=1.83710x+0.00469 0.99837 nd 0.0040 DPHP Y=0.72931x+7.46303 0.99945 0.018 ± 0.004 0.028 BBOEHEP Y=1.70772x+0.01410 0.99909 0.23 ± 0.03 0.33 BBOEP Y=0.45782x+0.01208 0.99659 nd 0.0040 BEHP Y=1.20436x+0.00717 0.99931 0.00089 ± 0.0002 0.0015 OH-TBOEP Y=31.84854x+0.02318 0.99878 0.041 ± 0.004 0.054 注:Y,峰面积比值;x,质量浓度,ng·g−1;nd,未检出. Note:Y,peak area ratio;x,concentration,ng·g−1;nd,not detected. 表 6 样品基质加标回收率
Table 6. Recovery rate of substrate
分析物
Analytes基质加标 Spiked substrate 回收率/%
Recovery rate相对标准偏差/%
RSDOPEs TCEP 75.4 19 TCPP 69.9 12 TDCPP 81.3 17 TBP 75.0 20 TBOEP 71.9 17 TEHP 78.4 7.9 TPHP 78.1 10 mOPEs DBP 71.9 23 BCEP 61.4 13 BCPP 62.1 15 BDCPP 61.8 18 DPHP 73.1 20 BBOEHEP 61.2 17 BBOEP 82.5 24 BEHP 75.9 16 OH-TBOEP 76.9 11 表 7 鸡蛋样品中OPEs和mOPEs的浓度(ng·g−1)
Table 7. Concentration of OPEs and mOPEs in egg samples(ng·g−1)
编号
No.OPEs TCEP TCPP TDCPP TBP TBOEP TEHP TPHP Σ7OPEs 1 0.126 2.14 0.152 0.891 0.134 nd nd 3.45 2 0.0972 1.93 nd 0.612 0.123 nd 1.46 4.22 3 0.208 2.72 nd 0.786 0.126 nd nd 3.84 4 0.078 1.66 nd nd 0.101 nd nd 1.84 5 0.139 1.63 nd 0.630 nd nd 1.54 3.95 6 0.142 2.90 0.141 1.01 0.104 nd nd 4.29 7 0.112 2.16 0.201 0.967 nd nd nd 3.45 8 0.170 2.84 nd nd 0.108 nd nd 3.12 9 0.171 2.81 nd 0.664 0.150 nd nd 3.79 10 0.147 2.11 nd 0.734 0.485 nd 2.41 5.90 11 0.101 1.74 nd 0.447 nd nd nd 2.28 12 0.192 0.988 nd nd 0.249 nd nd 1.43 均值 0.140 2.14 0.165 0.748 0.176 — 1.81 3.46 中值 0.141 2.13 0.152 0.734 0.126 — 1.54 3.62 检出率/% 100 100 25 75 75 0 25 — 编号
No.mOPEs DBP BCEP BCPP BDCPP DPHP BBOEHEP BBOEP BEHP OH-TBOEP Σ9mOPEs 1 0.0241 nd nd 1.05 1.13 nd 0.235 1.54 0.0278 4.01 2 0.102 nd nd 0.369 3.38 0.116 0.0762 1.03 nd 5.08 3 0.137 nd nd 5.62 3.32 nd 0.171 2.41 nd 11.7 4 0.0411 nd nd nd 1.97 nd 0.119 1.98 nd 4.11 5 0.0733 nd nd 0.726 4.39 nd 0.133 1.47 nd 6.80 6 0.0370 nd nd 0.0639 2.95 nd 0.272 6.27 nd 9.59 7 0.0291 nd nd 0.0409 2.17 nd 0.177 0.788 0.0487 3.25 8 0.0586 nd nd nd 1.61 nd nd 1.15 nd 2.81 9 0.0235 nd nd 0.562 1.63 nd 0.150 0.641 0.0973 3.10 10 0.153 nd nd 3.21 4.04 nd 0.187 2.34 nd 9.94 11 0.506 nd nd 0.0486 11.1 nd nd 0.149 nd 11.8 12 0.148 nd nd nd 6.90 nd 0.122 2.86 nd 10.0 平均值 0.111 — — 1.30 3.72 0.116 0.164 1.89 0.0579 6.85 中值 0.0660 — — 0.562 3.14 0.116 0.160 1.51 0.0487 5.94 检出率/% 100 0 0 75 100 8.3 83.3 100 25 — 注:nd,未检出; Σ7OPEs,7种OPEs的总和. Σ9mOPEs,9种OPEs代谢产物的总和.
Note:nd,not detected;Σ7OPEs,sum of 7 OPEs. Σ9mOPEs,sum of 9 OPEs metabolites. -
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