固相萃取-超高效液相色谱-串联质谱法测定斑马鱼暴露体系4种双酚类环境激素的研究

为评估双酚类环境激素对水环境可能造成的环境影响，建立固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)法测定斑马鱼暴露体系中的双酚C(BPC)、双酚F(BPF)、双酚S(BPS)、双酚Z(BPZ). 对前处理条件进行优化，斑马鱼样品依次采用6 mL乙腈溶液提取，30 min超声萃取及振荡混合，8 000 r/min下离心10 min，重复2次，于?80 ℃冷冻除脂48 h，过滤并用超纯水稀释至500 mL. 采用Generik H2P柱萃取上述鱼样及养殖水体样品，依次用10 mL 10%甲醇水溶液(V/V)淋洗，10 mL甲醇溶液洗脱. 优化参数确定最佳质谱条件，以甲醇-水溶液为流动相进行梯度洗脱，采用电喷雾电离、负离子选择反应监控(SRM)模式、同位素内标法进行测定. 结果表明:①固相萃取-超高效液相色谱-串联质谱法的检出限为0.019~0.60 μg/L，定量限为0.06~1.89 μg/L，BPS在0.5~100 μg/L范围内线性关系良好，相关系数为0.999 0，BPZ、BPF和BPC在1~100 μg/L范围内线性关系良好，相关系数在0.998 9~0.999 8范围内. ②在1.5、4.5、15 μg/L双酚类环境激素的添加浓度下，养殖水体中目标物的回收率为91.45%~102.91%，相对标准偏差为1.47%~11.04%，斑马鱼体内目标物的回收率为85.95%~97.45%，相对标准偏差为4.63%~16.36%. ③高浓度暴露组中，鱼体内BPF、BPS、BPC含量约是低浓度暴露组的10倍，而BPZ含量在两组间无明显差异. 研究显示，BPC、BPF、BPS、BPZ短时间内在斑马鱼体内产生了富集，通过分析斑马鱼全鱼样品、养殖水样及实际景观水体样品，证明固相萃取-超高效液相色谱-串联质谱法样品回收率高、检出限低、灵敏度高、重现性好，具有较好的实用性. 

Determination of 4 Bisphenol Environmental Hormone in Zebrafish Exposure System by Solid Phase Extraction-Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

In order to evaluate the possible environmental impact of bisphenol environmental hormones on the water environment, a solid phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) method was established for the sample pretreatment and determination of bisphenol C (BPC), bisphenol F (BPF), bisphenol S (BPS) and bisphenol Z (BPZ) in zebrafish exposure system. The optimum sample pretreatment conditions for zebrafish were established as follows: 6 mL of acetonitrile solution as extractant, 30 min of ultrasonic extraction and shaking, 10 min of centrifugal at 8000 r/min repeated twice, 48 h of degreasing at ?80 ℃, filtration and dilution to 500 mL with ultra-pure water. Generik H2P column was used for solid phase extraction of zebrafish and culture water. 10 mL of 10% methanol-water (V/V) was used to rinse and 10 mL methanol solution was used to elute the target analyte. The optimum mass spectrometry conditions were determined through parameter optimization. Gradient elution was carried out with methanol aqueous solution as mobile phase. Electrospray ionization, negative ion selective reaction monitoring (SRM) mode and isotope internal standard method were used for determination of bisphenol hormones. The results show that: (1) The limits of detection and quantification of this method were 0.019-0.60 and 0.06-1.89 μg/L, respectively. The linearity of BPS ranged from 0.5 to 100 μg/L, and the correlation coefficient was 0.9990. The linearity of BPZ, BPF and BPC was in the range of 1-100 μg/L, and the correlation coefficients were in the range of 0.9989-0.9998. (2) When bisphenol concentration was 1.5, 4.5 and 15 μg/L, the recoveries of the target substances in aquaculture water were 91.45%-102.91%, with relative standard deviation of 1.47%-11.04%. The recoveries of target substances in zebrafish were 85.95%-97.45%. The relative standard deviations were 4.63%-16.36%. (3) The BPF, BPS, and BPC in zebrafish in the high-concentration exposure group were almost 10 times that of the low-concentration exposure group. There was no significant difference in BPZ content between the two groups. The study shows that BPC, BPF, BPS, BPZ were enriched in zebrafish in a short time. The method is suitable for the determination of bisphenol environmental hormones in zebrafish, culture water, and actual landscape water samples, indicating that the proposed method has the advantages of good recovery, low detection limit, high sensitivity, good reproducibility, and practicality.