以一維及二維氣相層析質譜儀檢測生物檢體中烷基酚之分析與研究

Analysis and Study of Alkylphenols in Biosamples by Gas Chromatography/Mass Spectrometry and Two-Dimensional Gas Chromatography/Mass Spectrometry

林維雋(Wei-chun Lin)

指導教授 ： 丁望賢

國立中央大學/理學院/化學系/碩士(2007年)

摘要

烷基酚聚乙氧基醇類化合物(alkylphenol polyeyhoxylates, APEOs)從1940年起被廣泛使用的一種非離子型介面活性劑，因其具有良好的浸透、洗淨和乳化能力，因此常使用在強去污效果之清潔劑或是紡織與染化工業的洗滌劑中。這類界面活性劑經使用後，排放至水環境中受到水中微生物的降解代謝後，會降解成烷基酚(alkylphenols)及相關結構之殘留物質。而由於烷基酚殘留物因其化學結構與動物雌性激素類似，具有荷爾蒙效應與不易再被降解之持久特性，已證實會干擾動物體內分泌的機制，造成動物內分泌失調，使雄性動物雌性化，此類化學物質被稱之為環境荷爾蒙(environmental hormones)，近年來引起了大眾的關注。因此本研究即為開發穩定及可靠檢測烷基酚殘留物質之方法，並觀察其在動物體內降解代謝情形。
本研究第一部份為檢測魚類中烷基酚殘留物之含量，所使用之前處理方法為利用蒸氣蒸餾萃取法搭配衍生化步驟並結合氣相層析質譜儀，進行對魚類中烷基酚殘留物質定性與定量的分析，所使用的衍生化試劑為BSTFA加上1% TMCS，溫度與反應時間分別為80°C及30分鐘。結果顯示在所檢測之魚類中均有OP與NP之殘留，濃度範圍分別介於13.0-202.9 ng/g、9.2-529.7 ng/g之間，OP添加回收率在66.8%以上，NP在67.4%以上，再現性介於2.1%-10.4%之間，顯示回收率與再現性皆相當不錯。第二部份為檢測市售牛奶與人體母乳中烷基酚殘留物之含量，首先對牛奶與母乳做液-液萃取，所使用的萃取溶劑為正己烷，接著迴旋濃縮至1毫升，以50毫升50% (v/v)甲醇水溶液潤洗後通過0.45 µm濾紙除去懸浮物質並定量至150毫升，最後通過固相萃取與淨化管柱，以氮氣吹乾內標回溶後便可注射至氣相層析質譜儀進行定性與定量的工作。在所檢測的20件母乳樣品中，有8件檢測出有4-octylphenol殘留，其濃度介於0.4-1.14 ng/g之間，19件檢測出有4-nonylphemol isomers殘留，其濃度介於1.74-11.58 ng/g之間；而在所檢測的7件市售牛奶樣品中，均發現有4-nonylphemol isomers殘留，濃度介於2.93-8.84 ng/g，而僅一件發現有4-octylphenol殘留，濃度為0.14 ng/g。研究顯示使用50% (v/v)甲醇水溶液作為潤洗溶液對於OP與NP之萃取回收率均相當良好，再現性在10%以下，而擬似標準品回收率也在80%以上，顯示此檢測方法良好之回收率與再現性，能有效地檢測母乳中烷基酚殘留物之濃度。
最後，由於每個壬基酚異構物其生物降解速率、荷爾蒙效應與生物累積效應均不相同，而只有特定四個結構之壬基酚異構物具有較高的荷爾蒙效應，因此我們使用了100 m氣相層析管柱與二維氣相層析儀(GC x GC)搭配四極矩質譜儀來進一步分離在魚類及母乳不同基質中的壬基酚化合物，探討其在生物體中降解代謝情形，結果發現直鏈式之壬基酚異構物可能較易為生物體所降解代謝。另外，也對於5個不同地方生產之壬基酚化合物標準品進行分析探討，結果顯示這5個壬基酚化合物之間是有所差異的，未來希望可以藉由化學指紋比對鑑定來辨認出在汙染或黑心商品裡的壬基酚化合物是屬於哪裡所生產的。

關鍵字

固相萃取法 ； 蒸氣蒸餾萃取法 ； 二維氣相層析質譜儀 ； 烷基酚 ； 環境荷爾蒙 ； 氣相層析質譜儀

並列摘要

Alkylphenol polyeyhoxylates (APEOs), one of the most important classes of nonionic surfactants, were introduced in the 1940s. Because of their great soaking, cleaning and emulsifying abilities, they are widely used in the household detergents, textile industries, dyeing industries and other commercial applications in Taiwan. APEOs in aquatic environment are biodegraded into alkylphenols (APs) and relative metabolites. Indeed, APs have been estimated that they are persistence organic pollutions which are not easily biodegraded by microorganisms and demonstrated as endocrine disruptors which can cause estrogenic effects in organisms. This effect has raised increasing concern about their impact on wildlife and human health. Consequently, it is necessary to develop analytical methods for routinely determining the levels of alkylphenolic compounds in the Taiwanese environment and observe the biodegradation of 4-nonylphenol isomers.
The first part of this study is using steam distillation extraction and derivatization methods coupled with gas chromatography/mass spectrometry (GC/MS) to determine the levels of alkylphenol compounds in fish. The silylating agent used was BSTFA+1% TMCS and the optimal reaction time and temperature were 30 min and 80°C. Under these conditions, the recovery of OP and NP was above 66.8% and 67.4%, respectively. RSD ranged from 2.1 to 10.4%. The concentration of 4-t-OP and 4-NPs ranged from 13-202.9 ng/g and from 9.2-529.7 ng/g, respectively. The second part of this study is deterimining the alkylphenols in commercial milk and breast milk. The method involves extracting a sample by liquid-liquid extraction and solid-phase extraction and then clean up by anhydrous sodium sulfate and aluminum oxide. Alkylphenols were identified and quantitated by gas chromatography /mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. Various solution compositions were evaluated as rising solution. The limit of quantitation (LOQ) were less than 0.2 ng/g for 4-t-OP and 1.5 ng/g for 4-NP in 20 g breast milk. In 20 breast milk samples, 4-t-OP was detected in 8 samples and concentration ranged from 0.4 to 1.14 ng/g, while 4-NPs was detected in 19 samples and concentration ranged from 1.74 to 11.58 ng/g. In 7 commercial milk samples, 4-t-OP was detected in 1 samples and concentration was 0.14 ng/g, while 4-NPs was detected in all testing samples and concentration ranged from 2.93 to 8.84 ng/g. The results of the recovery and reproducibility for the SPE method indicated that it’s better to use methanol:water=1:1 (v/v) solution than ethanol:water=1:1 (v/v) as rinsing solution. The spiked recovery of 4-t-OP and 4-NPs were above 77.5% while RSD was below 9% which indicated this method is reliable and sensitive for determining traces of alkylphenols residues in milk.
Finally, because of different biodegradation rates, estrogenic effect and bioaccumulation potentials of each 4-nonylphenol isomer, only a few 4-nonylphenol isomers have higher estrogenic effects. So we used 100 m GC column and two-dimensional gas chromatography (GC x GC) combined with quadrupole mass spectrometer to separate 4-nonylphenol isomers more further in fish and breast milk samples. The results indicated that the more branched isomers of 4-nonylphenol isomers which may be more resistant to biodegradation than the less branched isomers in organisms. Furthermore, we analyzed five technical 4-nonylphenol standards from different vendors by GC x GC-MS system. Different patterns were observed from these standards. Hence, the GC x GC-MS system can be used as a rapid screening tool to access nonylphenol pollution associated with chemical migration and source apportionment studies.

並列關鍵字

two-dimensional gas chromatography/mass spectrom ； steam distillation extraction ； environmental hormones ； alkylphenols ； gas chromatography/mass spectrometry ； solid-phase extraction

參考文獻

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