尿液中环境内分泌干扰物暴露水平检测方法建立及应用

Establishment of a method to detect exposure levels of environmental endocrine disruptors in urine

目的建立尿液中15种环境内分泌干扰物(EEDs)暴露水平的超高效液相色谱串联质谱检测方法,并应用于可能EEDs暴露的低龄人群的尿液样本检测,以助于临床诊断及暴露预防。方法取500μL尿液样本,用缓冲盐调节pH值为6.5,加入20μL内标,在37℃条件下经β-葡萄糖醛酸苷酶酶解90 min,酶解产物进行酸化、乙酸乙酯萃取,取乙酸乙酯层冻干,用甲醇复溶;使用Acquity UPLC BEH C18,2.1 mm×100 mm,1.7μm色谱柱,以0.1%乙酸/水溶液-0.01%氨水/乙腈溶液为流动相梯度洗脱,使用ESI离子源负离子模式,进行选择性离子扫描。应用所建立方法对614例0~12岁有临床症状人群的样本进行检测。结果15种环境内分泌干扰物在0.1~100 ng/mL浓度范围内均有良好的线性相关性(均r 2>0.999),检出限(S/N=3)为0.01~0.05 ng/mL,定量限(S/N=10)为0.01~0.1 ng/mL;检测结果重复性(n=18,连续3天)RSD值均小于15%,低、中、高浓度加标回收率在80%~120%之间。临床样本的MP、EP、MMP、MEP、MBP、MEHP检出率为75%~100%,PP检出率为53.85%~96.3%,BPA检出率为25%~56%,MBzP检出率为5.88%~87.88%。结论所建立方法具有样本使用量少、检测灵敏度高、方法重复性准确性良好、分析时间短、分析通量高的优势,适用于临床样本暴露筛查检测。

Objective A method utilizing ultra-high performance liquid chromatography tandem mass spectrometry was established to detect the exposure level of 15 environmental endocrine disruptors(EEDs)in urine samples from young individuals who may have been exposed to EEDs,with the aim of aiding clinical diagnosis and preventing further exposure.Methods Urine samples of 500μL were collected and adjusted to a pH value of 6.5 with buffer salt,followed by the addition of an internal standard(20μL)and enzymatic digestion usingβ-glucuronidase for 90 minutes at 37℃.The resulting products were acidified,extracted with ethyl acetate,freeze-dried,and reconstituted in methanol.The chromatography was conducted using an Acquity UPLC BEH C18,2.1mm×100 mm,1.7μm column with a mobile phase gradient elution of 0.1%acetic acid/aqueous solution and 0.01%ammonia/acetonitrile solution.Selective ion scanning was performed in negative ion mode using an ESI ion source.The established method was used to detect 614 samples from 0-12 years old with clinical symptoms.Results All 15 environmental endocrine disruptors exhibited excellent linear correlation(r 2>0.999)within the concentration range of 0.1-100 ng/mL.The limits of detection(S/N=3)ranged from 0.01 to 0.05 ng/mL,and the limits of quantification(S/N=10)ranged from 0.01 to 0.1 ng/mL.The test results showed a reproducibility rate of less than 15%over three consecutive days(n=18),with recoveries for low,medium,and high concentrations ranging between 80%to 120%.In clinical samples,the detection rates of MP,EP,MMP,MEP,MBP and MEHP ranged from 75%to 100%,while the detection rates of PP ranged from 53.85%to 96.3%.The detection rates of BPA were found to be between 25%and 56%,whereas those for MBzP varied from as low as 5.88%up to a maximum of 87.88%.Conclusions The established method offers several advantages,including reduced sample consumption,heightened detection sensitivity,improved reproducibility,shortened analysis time and increased analytical throughput.As such,it is well-suited for clinical sample exposure screening.