以液相層析/質譜/質譜儀配合化學衍生偵測水體中類固醇雌激素

Abstract

存在於環境中的內生性類固醇雌激素（steroid estrogens）與相關的人工合成化合物，包含動情激素（17β-estradiol, E2）、雌素酮（estrone, E1）、雌素醇（estriol, E3）和乙炔動情激素（17α-ethinyl estradiol, EE2）等，主要經由動物及人類代謝產物進入環境水體，濃度雖低 (pg/L~ng/L)，其生態毒性卻足以致使環境失衡，並比其他內分泌干擾物質更具效力。回顧目前應用於偵測環境水體中類固醇類雌激素的分析方法，液相層析搭配串連式質譜儀（LC/MS/MS）具有良好的靈敏度和選擇性；然而當環境基質複雜，且樣本量有限時，分析結果卻未必能到達所需的偵測極限。環境基質不但增加背景雜訊，也減低離子化效率，抑制分析物在質譜儀中產生的訊號。因此，除了加強分析真實樣品時的淨化步驟之外，利用化學衍生法提升離子化效率亦為可行之道。 本研究以LC/MS/MS配合化學衍生，改進類固醇雌激素在質譜儀中的離子化效率，並評估不同衍生方法在環境基質影響下之表現。衍生試劑係根據類固醇雌激素結構具有酚類官能基之特性，分別選用dansyl chloride、2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS)、pentafluorobenzyl bromide (PFBBr) 三種化學衍生試劑，進行管柱前衍生，並就其訊號強度和訊雜比(S/N)，與未衍生之化合物進行比較。 結果顯示，在未受基質干擾下，比較相同濃度的dansyl chloride衍生產物和未衍生之雌激素本體，前者之訊號強度可較後者高達一至二個數量級之譜；FMPTS之衍生反應具有選擇性，在相同的反應條件下，E1、E2和EE2可見訊號之提升，E3衍生產物之訊號卻明顯低於其他三者，甚至低於未衍生之E3；PFBBr 衍生方法對於四種待測物之訊號提升則有一致性。當衍生方法應用於河水、自來水、污水處理廠之放流水等實際環境水體時，使用電灑游離 (ESI) 為離子源之操作模式受到顯著的基質效應作用，而大氣壓化學游離 (APCI) 則較不受影響。綜合訊號提升與基質效應兩項因子之考量，dansyl chloride衍生方法適用於基質效應較少的水體，如飲用水之檢測；基質複雜的環境水體，如河水與污水處理廠之放流水則建議應用PFBBr 衍生方法。 本研究僅就衍生產物進行定性分析，後續之方法驗證及定量則需使用類固醇雌激素之內標準品，以同位素稀釋法建立檢量線及確定方法偵測極限，方能完整建立定量環境水體中類固醇雌激素之分析方法。

Environmental endocrine disruptors, including natural and synthetic steroid estrogens such as 17β-estradiol (E2), estrone (E1), estriol (E3), and 17α-ethinyl estradiol (EE2), have caused concerns for the disruption of the aquatic ecosystem and human health effects. Although the concentrations of steroid estrogens (pg/L~ng/L) are lower than those of other compounds known as feminizing agents, their effects may be more significant because of their high potencies. Due to their chemical properties and trace levels in the environment, LC/API/MS may not achieve sufficient sensitivity, especially for complex water matrixes. To enhance the signals of the analytes, structure modifications of estrogens with appropriate derivatization reagents are promising to improve detection limits by increasing the ionization efficiency on MS. In this study, three selected derivatization reagents of different modification mechanisms were used to enhance the detection of steroid estrogens in waters, and the objective was to evaluate the impact of environmental matrixes on their performance. Based on the structural feature of steroid estrogens, which contain a phenolic group, dansyl chloride, 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS), and pentafluorobenzyl bromide (PFBBr) were selected as the derivatization reagents. The derivatized products of dansyl chloride provided higher signal intensity up to one or two orders than that of underivatized estrogens under the conditions without matrix effects. The derivative signals of FMPTS seemed to be analyte-dependent; derivatized products of E1, E2, and EE2 gained signal enhancement ranged from 2.19 to 12.1 times, whereas derivatized products of E3 showed poor signal intensity that was even worse than underivatized E3. Signal intensities of the four target compounds were enhanced consistently up to 5.81 times with PFBBr derivatization. When the derivatization methods were applied to real water samples, including river water, drinking water and effluents from the sewage treatment plant (STP), severe matrix effects were observed in the procedures using electrospray ionization (ESI), but were insignificant in atmospheric pressure chemical ionization (APCI) operation. Considering both sensitivity enhancement and matrix effect, this study suggested utilizing the dansyl chloride derivatization method in samples with less matrix effect such as drinking water, and the PFBBr derivatization method for complex environmental matrixes such as river water and STP effluents. This study presented qualitative comparisons of these derivatization methods. Further method validation and quantitative analysis is desired to analyze steroid estrogens in environmental waters.