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有机亚磷酸酯抗氧化剂(organophosphite antioxidants, OPAs)被广泛用于印刷油墨、塑料、石油、个人护理产品甚至食品中,以延缓氧化反应并延长其保质期[1]。其中,亚磷酸三(2,4-二叔丁基苯基)酯(tris(2,4-di-tert-butylphenyl) phosphite,AO168)能够显著提高聚合物的稳定性并延长使用寿命,被广泛应用于聚丙烯(PP)、聚乙烯(PE)、苯乙烯及其共聚物等高分子材料中[2-3]。在美国,AO168的年产量为4536—22680 t[4]。2020年,在中国的PP和PE塑料制品中,AO168的添加量为63 t[3]。研究表明,AO168的大鼠急性注射毒性LD50≥2000 mg·kg−1,并且在生产和使用过程中,AO168主要转化为三(2,4-二叔丁基苯基)-磷酸酯(tris(2,4-di-tert-butylphenyl)-phosphate,AO168O)和2,4-二叔丁基苯基(2,4-di-tert-butylphenyl,2,4DtBP)(结构式见图1)[5]。
近年来,AO168O和2,4DtBP在大气颗粒物[6]、室内灰尘[7]和日常用品[8-9]中广泛检出,受到了越来越多的关注[10]。AO168作为抗氧化剂[11],在塑料生产过程使用过程中不断被消耗[12-13],AO168O的含量逐渐上升[14]。进入环境中的AO168也可能通过生物和非生物途径降解,导致AO168O和2,4DtBP在环境中检出[14]。毒理学研究表明,2,4DtBP能够与雌激素受体结合,减少17β-E刺激细胞的生长,具有内分泌干扰作用[15-16]。虽然AO168O的毒理学机理尚未明确,但通过呼吸暴露和皮肤暴露途径进入人体的每日摄入量(estimated daily intake, EDI)计算结果表明,其对人体健康产生的潜在风险仍不容忽视[4, 17]。
血清作为评估有机污染物人体内暴露的重要介质,可以反映污染物对人体长时间的暴露水平[18]。目前,有关血清中AO168O和2,4DtBP的研究较少,相应的分析方法及人体健康风险也未见报道。因此,开发血清中AO168O和2,4DtBP的分析方法,对评估AO168O和2,4DtBP人体暴露水平及潜在的健康风险具有重要意义。
目前,AO168O和2,4DtBP的分析方法主要有气相色谱-质谱法(GC-MS/MS)[5-6]和液相色谱-质谱法(LC-MS/MS)[19]。本研究基于GC-MS/MS检测,通过对液相萃取和固相萃取方法的优化和比较,建立了血清中AO168O和2,4DtBP的前处理方法,并对采集自天津市部分青年人群的66份血清样品进行测定,验证了方法在实际样品分析中的可行性。该方法可为评估AO168O和2,4DtBP的人体健康风险提供技术支持。
液相萃取-气相色谱-质谱法快速分析血清中有机磷酸酯类三(2,4-二叔丁基苯基)-磷酸酯(AO168O)和2,4-二叔丁基苯基(2,4DtBP)
Rapid determination of tris(2,4-di-tert-butylphenyl)-phosphate (AO168O) and 2,4-di-tert-butylphenyl (2,4DtBP) in human serum by liquid phase extraction - gas chromatography - mass spectrometry
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摘要: 本研究建立了液相萃取-气相色谱-质谱法快速测定血清中三(2,4-二叔丁基苯基)-磷酸酯(tris(2,4-di-tert-butylphenyl)-phosphate, AO168O)和2,4-二叔丁基苯基(2,4-di-tert-butylphenyl, 2,4DtBP)。血清样品经乙腈提取,离心分离,浓缩定容后进行仪器分析。采用DB-5MS(30 m × 0.25 mm, 0.25 μm)色谱柱进行分离,多重反应监测模式(MRM)进行测定。结果表明,AO168O在0.8—100 ng·mL−1范围内线性关系良好,2,4DtBP在0.6—100 ng·mL−1范围内线性关系良好,相关系数R2分别为0.9992和0.9995;方法检出限分别为0.4 ng·mL−1和0.2 ng·mL−1;加标回收率分别为103%和95%,相对标准偏差(n=5)分别为8.9%和4.7%。采用本方法对天津市青年人群的66份血清样品进行检测,AO168O的检出率为33%,2,4DtBP的检出率为98%,浓度范围分别为ND(未检出)—492.33 ng·mL−1和ND—21.63 ng·mL−1。本研究所建立的方法适用于快速测定血清中的AO168O和2,4DtBP,对进一步评估AO168O和2,4DtBP的人体暴露风险具有重要意义。
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关键词:
- 气相色谱-串联质谱 /
- 液相萃取 /
- 三(2,4-二叔丁基苯基)-磷酸酯 /
- 2,4-二叔丁基苯基 /
- 血清
Abstract: A liquid phase extraction-gas chromatography-mass spectrometry method was established for rapid determination of tris(2,4-di-tert-butylphenyl)-phosphate (AO168O) and 2,4-di-tert-butylphenyl (2,4DtBP) in human serum. The samples were extracted by acetonitrile and the supernatant was centrifuged and concentrated for further instrumental analysis. The separation was performed on a DB-5MS (30 m × 0.25 mm, 0.25 μm) column, and the determination was performed in multiple reaction monitoring mode (MRM). The results showed good linearity for AO168O and 2,4DtBP, in the range of 0.8—100 ng·mL−1 and 0.6—100 ng·mL−1, respectively. The correlation coefficient of AO168O and 2,4DtBP were 0.9992 and 0.9995, respectively. The limits of detection for AO168O and 2,4DtBP were 0.4 ng·mL−1 and 0.2 ng·mL−1, respectively. The spiked recoveries (n=5) of AO168O and 2,4DtBP were 103% and 95%, and the relative standard deviations (RSDs) were 8.9% and 4.7%, respectively. The method was applied to the determination of AO168O and 2,4DtBP in 66 serum samples from a young population in Tianjin, China. The detection frequencies of two analytes were 33% and 98%. The concentrations of AO168O and 2,4DtBP ranged from not detected (ND) to 492.33 ng·mL−1 and ND to 21.63 ng·mL−1, respectively. The method is suitable for rapid determination of AO168O and 2,4DtBP in human serum and is important for further risk assessment of human exposure.-
Key words:
- gas chromatography-mass spectrometry /
- liquid phase extraction /
- AO168O /
- 2,4DtBP /
- serum
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表 1 3种目标化合物的质谱参数
Table 1. GC - MS /MS conditions for 3 analytes
序号
No.化合物
Analytes保留时间/min
Retention time定性离子
Qualitative ion(m/z)定量离子
Quantitative ion(m/z)碰撞能/V
CE1 2,4DtBP 7.8 191.1/163.2 191.1/57.2 10 2 AO168O 20.7 662.2/647.3 662.2/316.3 35 3 TPHP-d15 12.4 341.1/339.2 341.1/243.2 10 2,4DtBP:2,4-二叔丁基苯酚(2,4-di-tert-butylphenyl);AO168O:三(2,4-二叔丁基苯基)-磷酸酯(tris(2,4-di-tert-butylphenyl)-phosphate);TPHP-d15:磷酸三苯酯-d15(Triphenyl phosphate-d15) 表 2 AO168O和2,4DtBP的全称、缩写、CAS号、相对分子质量、沸点和lg Kow
Table 2. Full name, abbreviation, CAS number, molecular weight, boiling point, and lg Kow of AO168O and 2,4DtBP
序号
No.化合物
AnalytesCAS号
CAS No相对分子质量
Molecular weight沸点/℃
Boiling point a辛醇/水分配系数
lg Kowa1 2,4DtBP 96-76-4 206.33 263.5 5.19 2 AO168O 95906-11-9 662.94 480 16.16 a由EPI Suite 4.1计算得出。aCalculated by EPI Suite 4.1 表 3 方法性能参数
Table 3. Method performance parameters
化合物
Analytes线性范围/(ng·mL−1)
Linear range相关系数
(R2)检出限/(ng·mL−1)
LOD定量限/(ng·mL−1)
LOQ回收率/%
Recovery相对标准偏差/%
RSD(n=5)2,4DtBP 0.6—100 0.9995 0.2 0.6 95 4.7 AO168O 0.8—100 0.9992 0.4 0.8 103 8.9 -
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