水包油型微乳液電動層析於皮質類固醇化合物之應用

本研究首先利用油溶性染劑-雙硫腙 (dithizone) 的輔助，藉由偵測其在不同組成溶液中的光吸收強度，發現在不同油相對界面活性劑及共界面活性劑混合物之相對比例下(Ro/s)，當微乳液結構開始改變時，會有一明顯的轉折點，可藉此建立微乳液的類三相圖，得知各組成份在微乳液狀態下可變動的比例範圍，有助於進行微乳液電動層析 (microemulsion electrokinetic chromatography, MEEKC) 時，各比例調整的依據。隨後將微乳液應用於毛細管電泳所無法分離的中性皮質類固醇化合物，這類化合物常應用於治療類風濕性關節炎、過敏、氣喘、癌症等疾病。由研究發現以陰離子界面活性劑 (SDS) 所形成的微乳液，各組成比例為0.8% (w/w) n-octane，3.6% (w/w) SDS，6.6% (w/w) 1-butanol及89% (w/w) phosphate buffer (40 mM, pH 8.0)，分析電壓 + 7 kV時，可使六種結構相似的皮質類固醇達到基線分離，包括prednisone (Ps)、hydrocortisone (H)、hydrocortisone acetate (HA)、prednisolone (P)、prednisolone acetate (PA) 及cortisone acetate (CA)。但加入過多的SDS會使得遷移時間過長，雖可藉由添加有機修飾劑如乙腈、甲醇降低分析物與油滴的作用力以縮短整體分析時間，但反而會造成分析物訊號的重疊而降低選擇性，且無法容許加入較大量的有機修飾劑。因此改選用具有較低表面張力的L-酒石酸二乙酯 (diethyl L-tartrate) 作為油相，微乳液比例為0.5% (w/w) diethyl L-tartrate，1.7% (w/w) SDS，1.2% (w/w) 1-butanol，89.6% (w/w) phosphate buffer (40 mM, pH 7.0) 及7% (w/w) ACN，分析電壓 + 10 kV下，可將前述六種皮質類固醇Ps、H、HA、P、PA及CA，甚至包含aldosterone (A)、dexamethasone (D)、triamcinolone (T) 及triamcinolone acetonide (TA) 共十種結構非常相似的類固醇作完全分離，除了 T、Ps 及 A 外，RSD 值均 < 0.8 %，平均理論板數為 18800 m-1。此研究顯示即使是不帶電荷且構造相似的物種，仍可藉由奈米級微乳液的輔助，透過調整其各項參數來達到高分離效率。本研究進而以尿液為基質進行皮質類固醇之直接分析，發現其對疏水性較低的七種類固醇可完全分離，顯示此系統對皮質類固醇之臨床分析是一值得開發的高效率且簡便的分析方法。

關鍵字

微乳液；微乳液電動層析；皮質類固醇；陰離子界面活性劑

並列摘要

Generally microemulsion electrokinetic chromatography (MEEKC) using SDS as surfactant was difficult to separate neutral, highly hydrophobic and structure-related corticosteroids. In this study, for better understanding of the microemulsion property, dithizone was used as an index to measure the absorbance of various compositions of oil, surfactant, cosurfactant and aqueous buffer.Therefore the pseudoternary phase diagram could be constructed. Hereafter we found at the condition of 0.8% (w/w) n-octane, 3.6% (w/w) SDS, 6.6% (w/w) 1-butanol, 89% (w/w) 40 mM phosphate buffer (pH 8.0), and applied voltage of + 7 kV, six corticosteroids including, prednisone (Ps), hydrocortisone (H), hydrocortisone acetate (HA), prednisolone (P), prednisolone acetate (PA), and cortisone acetate (CA) could be completely separated. But the higher SDS concentration resulted in longer retention time. Even by the addition of organic modifier, the separation could not be improved. To reduce SDS content used we turned to use diethyl L-tartrate having less surface tension as oil phase. Under the microemulsion compositions of 0.5% (w/w) diethyl L-tartrate, 1.7% (w/w) SDS, 1.2% (w/w) 1-butanol, 89.6% (w/w) 40 mM phosphate buffer (pH 8.0), 7% (w/w) ACN, and applied voltage of + 10 kV, ten structure-related steroids including Ps, H, HA, P, PA, CA and aldosterone (A), dexamethasone (D), triamcinolone (T), and triamcinolone acetonide (TA) could be completely baseline separated. This indicates that SDS-containing microemulsions also have good selectivity for corticosteroids. The reproducibility of the retention time (N=4) for most of the compounds was less than 0.8% except that for T, Ps, and A. The average theoretical plate was 18800 m-1. To evaluate the validation of the system, corticosteroids in urine was also studied. It was demonstrated that the developed method was highly promising in the clinical analysis of these compounds.

並列關鍵字

Microemulsion ； Microemulsion electrokinetic chromatography ； MEEKC ； Corticosteroids

參考文獻

1. Langevin, D., Low interfacial tensions in microeulsion

3. Prince, L. M., Emulsions and emulsion technology,

4. Danielsson, J., Lindman, B., Colloid and Surface 1981,

5. De Gennes, P. G., Taupin, C., J. Phys. Chem. 1982, 86,

6. Pileni, M. P., J. Phys. Chem. 1993, 97, 6961.

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水包油型微乳液電動層析於皮質類固醇化合物之應用

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