超声提取-气相色谱-三重四极杆串联质谱法测定斑马鱼胚胎中对苯二胺抗氧化剂

Determination of p-Phenylenediamine Antioxidants in Zebrafish Embryos by Gas Chromatography-Tandem Mass Spectrometry with Ultrasonic Extraction

本文建立了超声提取-气相色谱-三重四极杆串联质谱法测定斑马鱼胚胎中4种对苯二胺抗氧化剂(PPDs),优化了超声提取溶剂和吸附剂,最终选择5 mL二氯甲烷超声15 min(重复2次)和0.1 g N-丙基乙二胺(PSA)净化。结果表明:该方法的线性范围为0.1~200μg/L,线性相关系数优于0.99,方法检出限在0.38~0.68 ng/g之间。除N,N′-二甲苯基对苯二胺(DTPD)的加标回收率高于120%,其他PPDs,即N-(1,3-二甲基丁基)-N′-苯基对苯二胺(6PPD)、N-苯基-N′-环己基对苯二胺(CPPD)、N-异丙基-N′-苯基对苯二胺(IPPD)的加标回收率为73.1%~109%;日内精密度2.97%~15.0%,日间精密度4.76%~18.8%。应用本方法检测暴露于PPDs中的斑马鱼胚胎体内PPDs浓度为12.5~16.4 ng/g。该方法操作简单、灵敏度高,适用于斑马鱼胚胎中对苯二胺抗氧化剂的检测。

p-Phenylenediamine antioxidants(PPDs)are rubber antioxidants that can suppress oxidation,heat,or light radiation to delay the aging of polymer components and extend the service life of rubber products.In recent years,PPDs have been found in the environment,and their ecological risks remain largely unknown,which cause widespread concern.In this work,a method of gas chromatography-tandem mass spectrometry(GC-MS/MS)with ultrasonic extraction was developed for the determination of PPDs in zebrafish embryos.In order to have good performance,extraction solvents and sorbents were optimized.Under optimal conditions,embryonic samples were combined with 5 mL of dichloromethane and ultrasonically extracted for 15 min.The extraction procedure was repeated twice,after that the resulting extracts were purified by 0.1 g primary secondary amine(PSA).Based on internal standard method,the analytes were detected using GC-MS/MS in selective reaction monitoring(SRM)mode with optimal parameters.The results showed that the chromatographic peaks of analyte were completely separated,and the baseline was smooth.The correlation coefficient(R 2)of each analyte exceeded 0.99 in the concentration range of 0.1-200μg/L.Method detection limits(MDLs)and method quantification limits(MQLs)were in the range of 0.38-0.68 ng/g and 1.28-2.28 ng/g,respectively.Except for the average recovery of N,N′-bis(methylphenyl)-1,4-benzenediamine(DTPD),which was higher than 120%,the average recoveries of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine(6PPD),N-phenyl-N′-cyclohexyl-p-phenylenediamine(CPPD)and N-isopropyl-N′-phenyl-1,4-phenylenediamine(IPPD)were 73.1%-109%,and the intra-day and inter-day relative standard deviations(RSDs)were 2.97%-15.0%and 4.76%-18.8%,respectively.In order to demonstrate the feasibility of this method,it was applied to detect PPDs in zebrafish embryos exposed to PPDs.The results indicated that PPD concentrations were 12.5-16.4 ng/g.After calculation,the measured bioconcentration factors of each analyte within 48 h were 1.70-33.6 L/kg.These values were much lower than the corresponding predicted bioconcentration factors,because PPD concentrations in vivo had not reached equilibrium during the exposure time of 48 h.This method is simple,sensitive,and suitable for the detection of PPDs in zebrafish embryos,which facilitates future bioaccumulation and toxicity mechanism studies for a comprehensive assessment of the ecological risks of PPDs.