以即時直接分析質譜術與極致液相層析/串聯式質譜術定量血清及尿液中環境荷爾蒙

Determination of endocrine disruptors in serum and urine with direct analysis in real time/tandem mass spectrometry and ultra-performance liquid chromatography/ tandem mass spectrometry

廖士翔(Shih-Hsiang Liao)

指導教授 ： 陳家揚 教授

國立臺灣大學/公共衛生學院/環境與職業健康科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202104231

摘要

內分泌干擾物質是一群會影響人體內分泌系統正常運作的化合物。全氟烷基化合物(perfluoroalkyl substances, PFASs)、鄰苯二甲酸酯類(phthalate esters, PAEs)、羥基苯甲酸酯防腐劑類(parabens)、雙酚A及其替代物(BPs)這四群化合物，被廣泛運用在日常用品或食品包裝之中的內分泌干擾物質。人體暴露到這四類化合物的途徑主要來自飲食，其次為皮膚接觸或是呼吸到附著化合物的灰塵顆粒。本研究中使用血清以及尿液樣本，建立以生物偵測方法檢測體內總濃度；血清能夠反映化合物在體內總量之平衡濃度，尿液能顯示暴露後短時間代謝之化合物情況，兩者一起評估人體所有暴露來源之目標化合物累積濃度。生物偵測技術為建立人體全面暴露的方法，能夠得知可能的內在濃度，使評估暴露與健康效應之間的關係更加清楚。
本研究中血清以及尿液樣本來源為國立臺灣大學醫學院附設醫院兒童醫院，於2018年針對7至11歲世代追蹤孩童以及其兄弟姊妹取樣。本研究中共有303位研究個案，並收取265份血清樣本以及298份尿液樣本做定量分析。
本研究定量化合物為：17種全氟烷基化合物、8種鄰苯二甲酸酯類代謝物、4種羥基苯甲酸酯防腐劑類、雙酚A以及5種替代物。使用Waters I-Class極致液相層析(ultra-performance liquid chromatography, UPLC)搭配Waters Xevo TQ-XS串聯式質譜儀，以UniSpray游離源定量分析。14種全氟烷基化合物、8種鄰苯二甲酸酯類代謝物、4種羥基苯甲酸酯防腐劑類、雙酚S和雙酚AF使用Waters CORTECS (30 × 2.1 mm, 1.6 μm)層析液相管柱，有機動相為甲醇，水性動相為0.1%醋酸水溶液(pH 3.26)，梯度流析流速0.4 mL/min，管柱溫度為40°C，層析時間連同管柱再平衡總需10.3分鐘；剩餘3種全氟烷基化合物、雙酚A、雙酚F、雙酚B、雙酚A二環氧甘油醚二水合物等7種化合物，使用Waters BEH C18(50 × 2.1 mm, 1.7 μm)層析液相管柱，有機相仍為甲醇，水性動相為10 mM N-甲基嗎啡林水溶液(pH 9.65)，梯度流析流速0.4 mL/min，管柱溫度為55°C，層析時間連同管柱再平衡總需10.3分鐘。質譜儀端使用多重反應偵測模式(multiple reaction monitoring, MRM)，定量分析以內部標準品校正之標準品回歸線線性範圍在0.5–500 ng/mL，判定係數R2有0.995以上，儀器偵測極限為0.1–1126 fg，儀器定量極限為0.3–8071 fg，顯示有良好靈敏度。在半定量分析中，使用即時直接進樣游離源(direct analysis in real time, DART)搭配Waters Xevo TQ-XS串聯式質譜儀，優化參數包含游離源到MS端入口的距離為3.7 cm，柵電壓400 V，游離源溫度400°C，每次分析皆以同一樣本三重複，並計算其平均值，以校正訊號變動幅度大的問題，每次分析三重複樣本時間為3.35分鐘。
本研究使用的血清及尿液前處理方法皆為實驗室先前開發之方法加以改良。100 μL血清加入含1%甲酸之乙腈(acetonitrile)，經過Ostro 96孔盤萃取後加入20 μL二甲基亞砜(dimethyl sulfoxide)，再經濃縮與甲醇回溶步驟得以上機；150 μL尿液經過β-glucuronidase以及arylsulfatase混合酵素於37℃培養40分鐘，再加入乙腈沉澱蛋白質後，經過Sirocco 96孔盤過濾後加入20 μL二甲基亞砜，再經濃縮與甲醇回溶步驟得以上機。方法確效部分，血清基質效應為68–136%，萃取效率為50–134%；尿液基質效應為45–134%，萃取效率為59–143%。同日和異日之準確度在80–120%，相對偏差大部分皆在20%以內。血清方法偵測極限範圍與定量極限範圍分別為0.5–521 pg/mL與2.1–844 pg/mL (不包含8:2 diPAP 1480 pg/mL以及MEP 1709 pg/mL)；尿液方法偵測極限範圍及定量極限範圍為1.6–344 pg/mL與20.4–926 pg/mL(不包含6:2 PAP 1479 pg/mL以及8:2 PAP 1506 pg/mL)。
定量結果顯示血清中各化合物檢測率大於80%，除PFHxA (19.2%)、PFDoDA (76.2%)、6:2 PAP (71.7%)、8:2 PAP (63.4%、6:2 diPAP (65.7%)、OH-MINCH (0.75%), BPAF (45.7%)、BPB (42.6%)以及BADGE-2H2O (63%)。尿液中各化合物陽性率高於90%，除了PFBA (85.6%)、PFDoDA (59.4%)、PFHxS (81.9%)、6:2 diPAP (80.9%)、8:2 diPAP (40.9%)、OH-MINCH (48.7%)、BPAF (59.4%) 以及BPB (61.7%)。短碳鏈全氟烷基化合物(PFBA、PFPeA、PFBS、PFHxS)在尿液中相較其他全氟烷基化合物有較高的幾何平均濃度(1.24–13.2 μg/g-cr)。PFOA、PFNA、PFHxS以及PFOS在血中相較其他全氟烷基化合物有較高的幾何平均濃度(1.97–4.63 ng/mL)。將四大類內分泌干擾物質各自做血中相關性統計與尿中相關性統計，發現同一類化合物間有著低度至中度正相關存在，表示這群受試者有共同暴露這些化合物的可能性。在同一化合物在血與在尿的濃度相關性中，PAE代謝物與paraben有著顯著的低度正相關，可能表示尿為這兩大類內分泌干擾物質代謝途徑。使用問卷與化合物濃度做單變項分析，結果發現外出使用塑膠餐具、海水魚、牡蠣以及海帶與PFASs有著正相關，包裝飲料和PFASs有負相關；使用乳液、外出使用塑膠餐具與PAE代謝物有正相關，牡蠣和PAE代謝物有負相關；外出使用塑膠餐具、包裝飲料、牡蠣和海帶與BPs有正相關；使用乳液、外出使用塑膠餐具和海帶與parabens有正相關。本研究定量孩童血中與尿中各類化合物濃度結果以及相關性分析，並與問卷內容作單變相分析找出可能的相關暴露來源，提供未來對內分泌干擾物質管控之優先項目，以降低孩童暴露風險。

關鍵字

極致液相層析/串聯式質譜術 ； UniSpray游離源 ； 即時直接進樣游離源 ； 尿液 ； 血清 ； 內分泌干擾素

並列摘要

Endocrine disrupting chemicals (EDCs) are a group of substances that interfere the function of human systems. Perfluoroalkyl substances (PFASs), phthalate esters (PAEs), parabens, bisphenol A and its analogs (BPs) were EDCs, which are ubiquitous in our daily life. Ingestion from diet is the primary exposure route to these EDCs, and other exposure routes include dermal contact and inhalation or ingestion of contaminated dust. In this study, we used biomonitoring analysis of serum and urine to establish the concentration of these four kinds of EDCs in children in Taiwan. The concentration in serum provided the total balanced amount in body; the concentration in urine reflected the metabolism in body for a short time.
The serum and urine in this study were from National Taiwan University Children’s Hospital. They were collected in 2018 from a cohort study in which the subjects were children from 7 to 11 years old in 2018. In this study, the total subjects are 303, and we have analyzed the 265 serum samples and 298 urine samples out of them.
This study developed a method to quantify 17 PFASs, eight PAE metabolites, six BPs and four parabens in serum and urine by using Waters ultra-performance liquid chromatography coupled with tandem mass spectrometer (UPLC-MS/MS) at UniSpray ionization source (USI) in negative mode. Three PFASs and four BPs were analyzed by Waters BEH C18 column (50 × 2.1 mm, 1.7 μm) with 10 mM N-methylmorpholine as aqueous mobile phase (pH 9.65) and MeOH as organic mobile phase. The remaining 28 chemicals were analyzed by Waters CORTECS column (30 × 2.1 mm, 1.6 μm) with 0.1% acetic acid as aqueous mobile phase (pH 3.26) and MeOH as organic mobile phase. The flow rate of chromatography was 0.4 mL/min and the temperature of columns were 55℃ and 40℃, respectively. For semi-quantifying, we used direct analysis in real time (DART) ionization source coupled with tandem mass spectrometer. The optimized DART parameters include 3.7 cm from DART nuzzle to MS inlet, 400 V for grid voltage, and 400 °C for source temperature. We did triplicate for each sample to calibrate the varied signal intensity, and the analysis time for each triplicate sample is 3.35 minutes.
In serum preparation steps, we added acetonitrile with 1% formic acid into 100 μL serum samples and extracted the solution by Ostro plates. The extract was added 20 μL dimethyl sulfoxide and were concentrated to approximate 20 μL, and we reconstituted the residue with methanol and centrifuged it. As for urine preparation steps, we added enzyme mixture into 150 μL urine samples and incubated at 37℃ for 40 minutes. We added acetonitrile and filtrated the solution by Sirocco plate. The filtrate was added 20 μL dimethyl sulfoxide and were concentrated to approximate 20 μL, and we reconstituted the residue with methanol and centrifuged it. The matrix effect and extraction efficiency were 68–136% and 50–134% for serum, and 45–134% and 59–143% for urine. The %bias and %RSD of inter-day and intra-day of both matrixes were lower than 20% for most compounds. The limit of detections (LOD) in serum were 0.5–520.9 pg/mL, and the limit of quantitations (LOQ) were 2.1–844 pg/mL (except for 8:2 diPAP 1480 pg/mL and MEP 1709 pg/mL)；the LODs and LOQs in urine were 1.6–344 pg/mL and 20.4–926 pg/mL (except for 6:2 PAP 1479 pg/mL and 8:2 PAP 1506 pg/mL).
The positive rates of most analytes in serum were above 80%, and the positive rates of most analytes in urine were above 90%. In the test of Pearson correlation within four groups of EDCs in the same matrix, there were weakly to moderately positive correlation in some chemicals. This finding may indicate the possibility of exposure of these correlated chemicals at the same time. In the two-sample t-test, PFASs had positive correlation with the usage of plastic tableware when eating out and consumption of sea fish, oyster and kelp once a week or more. PAE metabolites had positive correlation with the usage of lotion and the usage of plastic tableware when eating out. As for BPs, they had positive correlation with the usage of plastic tableware when eating out and consumption of packaged beverage, oyster, and kelp once a week or more. Parabens had positive correlation with the usage of lotion, the usage of plastic tableware when eating out and the consumption of kelp once a week or more. These findings provide possible exposure sources that we may give priority to when we supervised these EDCs.

並列關鍵字

ultra-performance liquid chromatograph/tandem mass spectrometer ； UniSpray ionization ； DART ； urine ； serum ； endocrine disrupting chemicals

參考文獻

1. Agency for Toxic Substances and Disease Registry. Draft toxicological profile for perfluoroalkyls. 2021 May; Available from: https://www.atsdr.cdc.gov/toxprofiles/tp200.pdf. Accessed 16 June 2021.

2. Zota, A.R., Calafat, A.M., and Woodruff, T.J., Temporal trends in phthalate exposures: findings from the National Health and Nutrition Examination Survey, 2001-2010. Environmental Health Perspectives, 2014. 122(3): p. 235-41.

3. Wang, Z., Dewitt, J.C., Higgins, C.P., and Cousins, I.T., A never-ending story of per- and polyfluoroalkyl substances (PFASs)? Environmental Science Technology, 2017. 51(5): p. 2508-2518.

4. Lau, C., Perfluorinated compounds: an overview, in Toxicological Effects of Perfluoroalkyl and Polyfluoroalkyl Substances, Jamie C. DeWitt, Editor. 2015, Springer International Publishing: Cham. p. 1-21.

5. Wang, Z., Cousins, I.T., Scheringer, M., Buck, R.C., and Hungerbühler, K., Global emission inventories for C4-C14 perfluoroalkyl carboxylic acid (PFCA) homologues from 1951 to 2030, Part I: production and emissions from quantifiable sources. Environment International, 2014. 70: p. 62-75.