建立毛細管電泳與高效能液相層析平台應用於食品安全檢測塑化劑與瘦肉精

Development of capillary electrophoresis and high performance liquid chromatography for detection of phthalates and β-agonists in food-safety assay

謝松裕(Sung-Yu Hsieh)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學系博士班/博士(2018年)

摘要

本研究係建立毛細管電泳與高效能液相層析檢測平台，來對食品中的塑化劑與瘦肉精進行分析檢測。第一部分的研究，以毛細管電泳中的微乳化電動層析法(Microemulsion electrokinetic chromatography, MEEKC)搭配線上掃集濃縮技術(Sweeping)，並在微乳化電動層析法中的微乳液，加入具有高分子與界面活性劑特性的Pluronic® F-127做為共界面活性劑。研究結果證明，Pluronic® F-127能穩定微乳液的微胞並增加脂溶性化合物Bis(2-ethylhexyl) phthalate與Dibutyl phthalate的分離效率。而此兩種脂溶性化合物被Sodium dodecyl sulfate的微胞包覆後，比較電動取樣與壓力進樣之感度，結果指出，電動取樣能增加25倍的感度。方法確效方面，此兩種脂溶性化合物的檢量線之相關係數皆大於0.999，而相對標準偏差值與相對誤差值均小於4.9%。本方法也順利應用於分析六種不同品牌的市售運動飲料，測定是否含有此兩種脂溶性化合物，其中一種市售運動飲料中被發現含有Bis(2-ethylhexyl) phthalate，並經液相層析串聯質譜儀驗證此結果。第二部分之研究，以高效能液相層析儀搭配固相萃取，建立同時分離九種鄰苯二甲酸酯類之分析方法，其偵測極限為0.5-10 ng/mL。檢量線分析之相關係數皆大於0.990，而相對標準偏差值與相對誤差值均小於9.5%。將此方法應用於分析兩種不同品牌的市售茶包袋，發現此兩種市售茶包袋在熱水浸泡後含有Dibutyl phthalate，並以液相層析串聯質譜儀進行定性。第三部分之研究，為建立微胞電動層析法(Micellar electrokinetic chromatography, MEKC)搭配電場放大樣品堆積與掃集濃縮的兩種線上濃縮技術，來對動物飼料中的瘦肉精進行分析。利用陰離子界面活性劑Sodium di(2-ethylhexyl) sulfosuccinate能形成類似脂雙層微囊的結構來做為擬靜相，可對動物飼料中的八種瘦肉精完全分離，而與傳統的微胞電動層析法方法比較，感度可增加400-2000倍。方法確效方面，八種瘦肉精在動物飼料中的偵測極限為5-20 ng/mL，檢量線分析之相關係數皆大於0.994，而相對標準偏差值與相對誤差值均小於12.7%。將本分析方法應用於分析四種不同品牌的市售動物飼料，並未監測到含有瘦肉精。

關鍵字

微乳化電動層析 ； 鄰苯二甲酸酯 ； Pluronic F-127 ； 高效能液相層析 ； 固相萃取 ； 瘦肉精 ； 磺基琥珀酸鈉二辛酯(AOT)

並列摘要

The study developed three detection methods for analyzing phthalate esters and β-agonists in foods by using capillary electrophoresis and high-performance liquid chromatography. The first study developed an on-line stacking method by using microemulsion electrokinetic chromatography combined with sweeping to separate phthalate esters. This method used Pluronic® F-127 as co-surfactant to stabilize microemulsion and enhance the separation of bis(2-ethylhexyl) phthalate and dibutyl phthalate. The sensitivity of the two hydrophobic phthalate esters were compared between electrokinetic and hydrodynamic injection. The result indicated that electrokinetic injection could enhanced 25-fold sensitivity compared with hydrodynamic injection. For validation, all of the coefficient of correlation were more than 0.999. The relative standard deviation and relative error were all less than 4.9%. This method was also successfully applied to analyze six different commercial soft drinks. One of them was found to has bis(2-ethylhexyl) phthalate and confirmed by liquid chromatography coupled with mass spectrometry. The second study developed high-performance liquid chromatography combined with solid-phase extraction for analyzing nine phthalate esters. The limit of detection was 0.5 to 10 ng/mL for nine phthalate esters, individually. The coefficient of correlation for all the calibration curves were more than 0.990. The relative standard deviation and relative error were all less than 9.5%. This method was utilized to analyze two different commercial tea bags and found the presence of dibutyl phthalate after soaking with hot water. The results were confirmed by liquid chromatography coupled with mass spectrometry. The last study developed micellar electrokinetic chromatography combined with field-amplified sample injection and sweeping to separate β-agonists in animal feeds. The method used sodium di(2-ethylhexyl) sulfosuccinate to form a lipid bilayer structure as pseudostationary phase and separated eight β-agonists in animal feeds. In comparison of the sensitivity in traditional micellar electrokinetic chromatography method, the sensitivity of this method could enhanced 400 to 2000 folds. The limit of detection of eight β-agonists were from 5 to 20 ng/mL. The coefficient of correlation of all calibration curves were more than 0.994. The relative standard deviation and relative error were all less than 12.7%. The method was used for detection of four commercial animal feeds and no any β-agonists was found.

並列關鍵字

microemulsion electrokinetic chromatography ； phthalate ester ； Pluronic F-127 ； high-performance liquid chromatography ； solid-phase extraction ； β-agonists ； sodium di(2-ethylhexyl) sulfosuccinate (AOT)

參考文獻

[1] Martino-Andrade, A. J., & Chahoud, I. (2010). Reproductive toxicity of phthalate esters. Molecularnutrition & Food Research, 54(1), 148-157.

[2] Frederiksen, H., Skakkebaek, N. E., & Andersson, A. M. (2007). Metabolism of phthalates in humans. Molecular Nutrition & Food Resarch, 51(7), 899-911.

[3] Koch, H. M., & Calaft, A. M. (2009). Human body burdens of chemicals used in plastic manufacture. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences, 364(1526), 2063-2078.

[4] Waring, R. H., & Harris, R. M. (2011). Endocrine disrupters--a threat to women’s health? Maturitas, 68(2), 111-115.

[5] Stales, C. A., Peterson, D. R., Parkerton, T. F., & Adams, W. J. (1997). The environmental fate of phthalate esters: a literature review. Chemosphere, 35(4), 667-749.