以液相層析串聯式質譜儀檢測尿液中鄰苯二甲酸酯代謝物、雙酚A葡萄糖苷酸、1-羥基芘葡萄糖苷酸、全氟碳化物及白三烯E4

劉信宏

doi:10.6342/NTU.2012.02058

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以液相層析串聯式質譜儀檢測尿液中鄰苯二甲酸酯代謝物、雙酚A葡萄糖苷酸、1-羥基芘葡萄糖苷酸、全氟碳化物及白三烯E4

Determination of phthalate metabolites, bisphenol A glucuronide, 1-hydroxypyrene glucuronide, perfluorinated chemicals and leukotriene E4 in urine

劉信宏 , Masters　　Advisor：陳家揚　　

英文 DOI： 10.6342/NTU.2012.02058

鄰苯二甲酸酯；雙酚A ； 1-羥基芘；全氟碳化物；離子交換樹脂；葡萄糖苷酸； phthalate ； bisphenol A ； 1-hydroxypyrene ； perfluorinated chemicals ； WAX ； μElution ； glucuronide

Abstract │ Reference (56)

Abstract 〈TOP〉

鄰苯二甲酸酯（Phthalates）、雙酚A（Bisphenol A）、全氟碳化物（PFCs）和多環芳香烴等汙染物普遍存於日常生活環境當中。前三者為大量使用的工業原料，常見於個人保健用品、食品包裝和餐具之中，多環芳香烴則經由不完全燃燒所產生，汽機車引擎是日常暴露的主要來源。過往流行病學的文獻曾經報導個別類別汙染物的暴露與過敏之間的關聯性，但尚無相關研究同時評估或監測所有這些汙染物的暴露，以釐清何種汙染物對於健康效應最具關鍵性，抑或是共暴露效應之發生。本研究開發並驗證以負離子模式之電灑游離法（ESI-）作為質譜儀的游離源，搭配極致效能液相層析（UPLC-MS/MS）與同位素稀釋技術來定量尿液中四種鄰苯二甲酸酯代謝物、雙酚Ａ葡萄糖苷酸、1-羥基芘葡萄糖苷酸、九種全氟碳化物及白三烯E4之檢驗方法，共16種化合物。
尿液樣本在經過離心後取150微升上清液以同體積之10 mM醋酸銨水溶液進行稀釋，並加入20微升的同位素標定內標準品（配製於甲醇中）。總共320微升的樣品通過96孔弱陰離子交換樹脂（WAX）固相萃取盤（μElution plate）進行萃取（預先通過100微升含有0.1%氨水的甲醇兩次以及200微升的Milli-Q純水），接著以200微升的2%甲酸水溶液和70%甲醇水溶液依序通過吸附劑，沖提出無法與離子交換樹脂產生交互作用的化合物，並以空氣通過吸附劑持續五分鐘使其乾燥；最後使用25微升含有0.1%氨水的甲醇進行四次沖提，沖提液以空氣吹至近乾（20分鐘）後使用100微升的甲醇回溶。最後進樣兩微升的樣品以UPLC-MS/MS進行分析，使用Ascentis Express C18管柱（50 x 2.1 mm, 2.6μm）搭配以下兩種流動相進行梯度流析：(A) 乙腈：水 = 90 : 10 (v/v), 0.04%乙酸、(B) 乙腈：水 = 5 : 95 (v/v), 0.04%乙酸。
所有待測物的偵測極限為 0.23 ng/mL – 6.54 ng/mL，此方法達到十億分之一的敏感度足以偵測大多數一般大眾之暴露量。大多數待測物的基質效應介於95% 到132%之間，除了雙酚Ａ葡萄糖苷酸（637%）、1-羥基芘葡萄糖苷酸（189%）及白三烯E4（242%）三者較高，可能有離子增強（ion enhancement）之現象。半數代測物之萃取效率達60%以上，較低的待測物包括白三烯E4 （27.3%）、鄰苯二甲酸單乙酯（31.8%）與1-羥基芘葡萄糖苷酸（46.3%）。本研究成功開發使用離子交換樹脂從尿液中同時萃取具有強酸及弱酸性質的化合物之前處理方法，平均每個樣本只需要三分鐘，有效節省分析過程所需的時間和人力資源。
本研究利用此新研發之方法分析了350個學童的真實尿液樣本。鄰苯二甲酸酯代謝物、雙酚A葡萄糖苷酸和1-羥基芘葡萄糖苷酸可以在大多數的樣本中被偵測到；全氟己酸和白三烯E4只能在少數幾個樣本中被偵測到，檢出率分別為21%和2%，而其他全氟碳化物在大部分的尿液樣本中均小於偵測極限。
本研究開發了可以同時偵測16種與過敏相關的汙染物在尿液中的本體或代謝物之檢測方法，除了利於探討各個物種對於健康效應之貢獻程度，鑒於這些汙染物在生活環境中分佈廣泛，亦可作為監測一般大眾暴露的有效工具。本方法每個樣本平均只需要10分鐘就可以完成分析，並且在前處理不需要使用β-葡萄糖醛酸酶（β-glucuronidase）進行水解反應，將可以快速處理大量的樣本，適合應用於大規模的暴露評估計畫或是流行病學研究所需的生物偵測資料。

Parallel Abstract 〈TOP〉

Phthalates, bisphenol-A and perfluorinated chemicals (PFCs) are emerging contaminants that are used in many consumer products, and polycyclic aromatic hydrocarbons (PAHs) are common pollutants exist in the ambient air. People are continuously exposed to these ubiquitous chemicals through various pathways in daily life. In addition, many previous researches have reported the associations between adverse immune effects and the exposure to the above chemicals; however there has not a research to explore them simultaneously yet. To discover the crucial chemicals or their interactions, and to monitor the exposure in general population, biomonitoring of the internal dose of these pollutants is a suitable approach to evaluate their health effects from different sources.
This study developed and validated a method to simultaneously determine four phthalate metabolites, bisphenol A glucuronide (BPA-G), 1-hydroxypyrene glucuronide (1-OHP-G), nine perfluorinated chemicals, and leukotriene E4 (LTE4) in urine using ultra-high performance liquid chromatography/tandem mass spectrometry at selected-reaction monitoring with isotope-labeled techniques, and was applied on analyzing human urine samples. 
A high-throughput sample preparation was optimized on a 96-well weak anion ion-exchange (WAX) μElution plate. One milliliter of urine sample was centrifuged at 3600 rpm for 10 minutes, then an aliquot of 150-μL supernant was diluted with the same volume of 10 mM ammonium acetate in water, and was spiked with 20 uL of isotope-labeled internal standards. The total 320-μL sample was passed through the pre- conditioned sorbent with two times of 100μL 0.1% NH4OH in methanol and 200 μL of Milli-Q water; the sorbent were washed by 200 μL of 2% formic acid in Milli-Q water (v/v) and 50 μL of 70% methanol (v/v). The sorbent was dried by vacuum and analytes were eluted with four times of 25 μL of 0.1% NH4OH in methanol. The collected eluents were evaporated to barely dry by air, and were reconstituted with 100 μL of methanol. Two microliters of sample were inject onto the UPLC-MS/MS; the analytes were separated with an Ascentis Express C18 column with mobile phases composed of (A) 0.04% acetic acid in acetonitrile/water = 90:10 (v/v) and (B) 0.04% acetic acid in acetonitrile/water = 5:95 (v/v).
Limits of detection of all analytes were ranged from 0.23 ng/mL to 6.54 ng/mL. The matrix effects of most analytes were 95% - 132%, except for BPA-G, 1-OHP-G, and LTE4 were 637%, 189% and 242%, respectively. Only half of the analytes performed extraction efficiencies more than 60%, the lowest ones were LTE4 (27.3%), monoethyl phthalate (MEP) (31.8%), and 1-OHP-G (46.3%). However, this method was able to extract both strong and weak acidic compounds simultaneously from complex urine matrix instead of analyzing separately, and also the sample preparation was fast and efficient needs only 3 min per sample.
Totally 350 real samples from children were analyze by the new assay. Phthalate metabolites, BPA-G, and 1-OHP-G were detected in most samples. In contrast, PFCs and LTE4 were only detected in few samples with positive rate 21% (perfluorohexanoic acid) and 2%, respectively; most of the PFCs in samples were below LODs. 
This method can analyze 16 analytes relating to hypersensitization in human urine, and only took totally 10 min in sample preparation and in the instrument analysis, plus no need of enzymatic hydrolysis to cleave the glucuronide group. This method is able to process a large number of urine samples shortly, and provides biomonitoring data for epidemiological studies or exposure assessment.

Reference ( 56 ) 〈TOP〉

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