鄰苯二甲酸二異壬酯 (DINPs) 為廣泛被使用的塑化劑，是由一群支鏈上含有九個碳的雙烷基鄰苯二甲酸鹽類所構成的物質。在動物實驗上已經證實暴露到DINPs與抗雄性激素，以及肝臟和腎臟的毒性有關。由於DINPs是由同分異構物所構成的物質，因此進入人體後經由代謝後被排至尿液中的代謝物也是同分異構物。到目前為止，只有少數的代謝物是被經過鑑定後可被用來做為DINPs的生物指標。
本研究的研究目的運用液相層析質譜儀與運用肝臟酵素進行體外代謝後，鑑定出DINP的代謝物，尋找暴露指標。為了鑑定出DINPs的代謝物，本研究主要分成了化合物的合成，體外肝臟酵素代謝，以及運用質譜分析三個部份。本研究中選擇合成支鏈為4,4,5-trimethylhexanol-1的DINP一階代謝物 (MINP)，其原因此種物質其合成的步驟較簡單，可較快得到合成產物。再將合成得到的物質經由體外肝臟酵素被代謝後，得到含有代謝物的樣本。本研究運用質譜儀結合穩定的同位素標定物質做為追蹤物，以及本實驗室所開發的Signal Mining Algorithm with Isotope Tracing (SMAIT) 軟體，篩選出DINP代謝物的訊號。為了驗證整個代謝物鑑定程序的可行性，使用鄰苯二甲酸二乙基己基酯 (DEHP)，證實整個鑑定程序是可被使用於代謝物訊號的尋找。接著，再將合成後所得到的MINP經由體外代謝後置入液相層析質譜儀內進行分析。為了證實整個尋找代謝物程序的可行性，因此連續重複三次實驗。在此三次實驗中一共找到18個出現兩次以上可能是DINPs代謝物的訊號，因此這18個可能是代謝物的訊號需進一步利用MS/MS的分析。由MS/MS分析結果顯示，這18個訊號當中有5個訊號為過去研究當中DINP已知的代謝物，其結構分別為OH-MINP，與兩個 carboxy-MINP 的同分異構物， MINP，以及mono(carboxy-isopentyl)phthalate (MCiPeP)。另外有9個訊號判定與所添加的MINPs是有關的，但目前其結構仍是未知的。
本研究中一共鑑定出14個與DINPs有關的訊號。未來，期望可在尿液樣本中找出這些代謝物的訊號，更進一步找出適合做為DINPs暴露評估的生物指標。

Di-isononyl phthalates (DINPs), a mixture of widely used plasticizers, contain various isomeric nine-carbon branch chain dialkyl phthalates. The associations between DINPs exposure and antiandrogenic activity, liver, and kidney toxicity in animal experiments were reported. Due to isomeric nature of DINPs, a wide variety of isomeric metabolites is excreted in human urine. So far, few metabolites from DINP isomers are identified for exposure biomonitoring.
This study aims to identify DINP metabolites for exposure marker discovery using rat liver metabolizing enzymes and liquid chromatography mass spectrometry (LC-MS). Three experimental steps, including compound synthesis, in vitro rat liver enzyme incubation, and metabolite signal identifying by using an MS-based strategy, were designed for identification (ID) of DINP metabolites. A primary DINP metabolite with one type side-chain, 4,4,5-trimethylhexanol-1, was selected to be synthesized. The synthesized compound was in vitro incubated with liver enzymes to generate metabolites. An MS-based strategy coupled with stable isotope tracing was used for screening the metabolites. For validation of the metabolite identification strategy, di-2-ethylhexyl phthalate (DEHP) which generates well known metabolites was used. The synthesized MINP was generated by in vitro rat liver enzyme incubation and was screened its metabolite signals by the validated ID strategy. For examination of ID reliability of the DINP metabolites, the ID process was repeated for 3 times. Eighteen probable metabolite signals were presented at least two times in the triplet experiments. These 18 probable metabolite signals were selected as candidates for further tandem mass spectrometry analysis. We set three criteria for product ion selection to eliminate non-DINP metabolite signals. Among the 18 signals, five metabolite signals have been reported to be the DINP metabolites in the previous studies. The structures of five reported metabolites contain one OH-MINP, two carboxy-MINP isomers, one MINP and one mono(carboxy-isopentyl)phthalate (MCiPeP). Nine signals were associated with MINPs, but their structures are still unknown.
In this study, we successfully identified 14 signals associated with DINPs. In the future, the metabolite signals can be validated for DINP exposure markers for biological monitoring in urinary samples.

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