微胞電動層析結合線上掃略式濃縮分析技術之酵母菌立體選擇性還原類固醇應用

On-line Sweeping Concentration Technique Coupled for Micellar Electrokinetic Chromatography for Analysis of Yeast Mediated Steroids Stereo-Selective Reduction

吳忠穎(Chung-Ying Wu)

指導教授 ： 鄭建業

中原大學/理學院/化學系/碩士(2008年)

https://doi.org/10.6840/cycu200900195

摘要

雌性與雄性類固醇荷爾蒙是人類生長、發育、行為、繁殖所需要之重要內分泌物質，在製藥工業上也佔有重要地位。在本實驗室中，利用酵母菌Saccharomyces cerevisiae，同時將雌固酮和雄烯二酮經由立體選擇性還原反應生成β-雌二醇和睪固酮。此種酵母菌立體選擇性還原反應可經由發展一個可靠和靈敏的微胞電動層析技術來偵測相關雌性和雄性類醇固荷爾蒙而予以證實。所發展的微胞電動層析，使用含有50 mM 陰離子介面活性劑十二烷基磺酸鈉鹽、10 mM β-環糊精和4 %甲醇之10 mM磷酸緩衝溶液(pH 7.0)，再結合毛細管微胞電動層析掃略式線上濃縮技術，可同時分離與分析酵母菌立體選擇性還原反應系中六種微量的荷爾蒙。定量分析使用外標準檢量線法，各分析物之檢量線的線性範圍為0.01-0.08 ppm，其相對應的線性迴歸係數(r2)皆可達到0.9969以上。經由紫外線偵測器所得偵測極限值分別為8.2 ppb (β-雌二醇)、7.8 ppb (α-雌二醇)、8.3 ppb (雌素酮)、7.1 ppb (雄烯二酮)、8.7 ppb (表睪酮)、9.1 ppb (睪固酮)。由各分析物定量結果的相對標準偏差值(RSD)均小於3.5%，以及分析樣品標準添加回收率介於96-99%，可知所發展的毛細管微胞電層析線上濃縮技術的確是高靈敏度精確度與準確度的分析技術。

關鍵字

酵母菌 ； 睪固酮 ； 表睪酮 ； 磷酸緩衝溶液 ； 十二烷基磺酸鈉鹽 ； β-環糊精 ； 微胞電動層析 ； 雌素酮 ； α-雌二醇 ； β-雌二醇 ； 雄烯二酮

並列摘要

Estrogens and androgens are steroid hormones that influence the growth, development, behavior, and regulation of reproductive tissues and are important in pharmaceutical industry. The stereoselective reduction of 17-ketosteroid estrone and androstenedione to the corresponding 17β-estradiol or testosterone has been performed with Saccharomyces cerevisiae in our laboratory. This study demonstrates a reliable and sensitive micellar electrokinetic chromatography (MEKC) on-line concentration technique for the determination of estrogenic and androgenic compounds in the yeast mediated cell culture. An efficient and simultaneous separation of estrogenic and androgenic compounds with MEKC was performed by using 10 mM phosphate buffer (pH 7.0) that contains 50 mM sodium dodecyl sulfate (SDS), 10 mM β-cyclodextrin (β-CD), and 4% methanol. On-line sweeping concentration technique was then coupled to MEKC for the anslysis of trace amount of the steroid hormones in the yeast mediated cell culture. The quantitative analysis of the steroid hormones was by the external staudard calibration curve. All the external standard calibration curves of the analytes have a linear range from 0.01 to 0.08 ppm and their corresponding linear correlation coefficients were all over 0.9969. The detection limits of the UV detector for the six tested estrogens and andrognens were 8.2 ppb (β-estradiol)、7.8 ppb (α- estradiol)、8.3 ppb (estrone)、7.1 ppb (androstenedione)、8.7 ppb (epitestosterone)、9.1 ppb (testosterone). The quantitative results of the analytes showed all the relative standard deviations were below 3.5% and the recovery rates were between 96% and 99%. Therefore, the developed MEKC on-line concentration technique for the steroid hormones analysis was high secsiteve, precision, and high accuracy.

並列關鍵字

Epitestosterone ； phosphate buffer ； sodium dodecyl sulfate ； β-cyclodextrin ； micellar electrokinetic chromatography ； Estrone ； α-Estradiol ； β-Estradiol ； Adnrostenedione ； Testosterone ； Saccharomyces cerevisiae

參考文獻

26. 黃悉雅，邱貞文，毛細管電泳技術的方法開發與應用，中原大學化學系博士論文，中華民國，台灣，中壢，九十五年六月。

61. 鄭建業，蔡向榮，結合性雙液相層析管柱系統分析酵母菌催化前掌性酮類於二相反應液中之掌性二級醇，中原大學化學系碩士論文，中華民國，台灣，中壢市，九十二年六月。

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