三黃瀉心湯、固醇類荷爾蒙及維生素E之毛細管電泳分析

The Analysis of San-huang-xie-xin-tang, Steroid Hormones, and Tocopherols by Capillary Electrophoresis

張琳巧(Lin-Chau Chang)

指導教授 ： 李水盛 ； 張煥宗 ； 郭錦樺

國立臺灣大學/醫學院/藥學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.02604

摘要

為了解應用毛細管電泳於藥物分析之可行性，本研究以膠束電動層析法及微乳劑電動層析法兩種模式為探討之核心。中草藥萃取物的混合物，因成分複雜，須要高解析度之分析方法加以分離。本研究以膠束電動層析法，分析常見中藥材黃連、黃芩及大黃之13種指標成分的混合物。結果發現，有機修飾劑乙腈之添加百分比，會造成解析度、峰形和遷移窗域顯著的改變。藉由調整緩衝溶液酸鹼值、界面活性劑之濃度及乙腈之添加百分比，13種指標成分得以成功分離。其緩衝溶液之組成為，在pH值7.3下，3 mM di-sodium tetraborate、10 mM sodium dihydrogen phosphate、50 mM sodium deoxycholate與30%乙腈之混合溶液。應用此方法分析含上述三種藥材之三黃瀉心湯時，可以定量萃取物中所含之八種成分，此方法可作為分析其他混合此三種藥材之起始條件。
由於固醇類荷爾蒙之分析，對於診斷、預後及藥物品質管制等皆具重要性，因此，本研究利用含陽離子界面活性劑cetyltrimethylammonium bromide (CTAB) 之膠束電動層析法，同時分析分屬雄性素、雌性素、助孕素及糖皮質素之固醇類荷爾蒙，以期增進對分析此類物質時所可能產生的各種狀況之了解。結果顯示，重要影響因子包含CTAB之濃度、有機修飾劑之種類和比例及樣品間質等。其中，尤以有機修飾劑之影響最為顯著，其於緩衝溶液中影響解析度，而於樣品間質中則影響峰形。在施加電壓-25 kV下，以含有pH 9.0之100 mM Tris-boric acid buffer、40 mM CTAB 及 20% 2-propanol之混合溶液，得以成功分離cortisone、hydrocortisone、estriol、testosterone、estrone、progesterone及estradiol。
對於分析結構相似度高、且厭水性高之各種型式的tocopherols，本研究採用峰效率及溶解能力高之微乳劑電動層析法進行分析，並探討緩衝溶液系統、環糊精的種類與濃度、溫度及樣品間質等，對於分析效果之影響。在-26 kV、25 °C下，利用含有4% (w/w) sodium dodecyl sulfate (SDS)、6.6% (w/w) 1-butanol、0.8% (w/w) n-octane、20% (w/w) 2-propanol、68.6% (w/w) phosphate (25 mM, pH 2.5) 及25 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) 之微乳劑系統，得以成功分離α-、γ-、δ-tocopherol、α-tocopherol acetate及抗氧化劑butylated hydroxytoluene (BHT)。由以上之結果得知，在適當之條件控制下，具高解析度、高分析效能及低樣品與溶媒消耗量之毛細管電泳，確實具有應用於藥物分析之價值和前景。

關鍵字

毛細管電泳 ； 膠束電動層析法 ； 微乳劑電動層析法 ； 三黃瀉心湯 ； 固醇類荷爾蒙 ； 維生素E

並列摘要

The aim of the present study was to elucidate the feasibility of capillary electrophoresis, with focus on micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) in pharmaceutical analysis. The components of the mixture of Chinese herbal extracts were usually complex, which necessitated the use of analytical methods with high resolution power. Therefore, MEKC was applied to analyze a mixture of 13 bioactive components of common Chinese herbs including Coptidis Rhizoma, Scutellariae Radix, and Rhei Rhizoma. Acetonitrile percentage was found to significantly influence the resolution, peak shape, and elution window. By adjusting buffer pH, the concentration of surfactant, and acetonitrile proportion, 13 bioactive components could be successfully separated at pH 7.3 using a buffer mixture of 3 mM di-sodium tetraborate, 10 mM sodium dihydrogen phosphate, and 50 mM sodium deoxycholate with 30% (v/v) acetonitrile. Eight components of San-huang-xie-xin-tang, which contained all three herbs, could be determined using the developed method. The separation condition could serve as a starting point to evaluate other related formulae containing these herbs.
Since the analysis of steroid hormones was important for diagnosis, prognosis, and quality control of pharmaceutical products, MEKC with a cationic surfactant, cetyltrimethylammonium bromide (CTAB), was used to simultaneously analyze the steroid hormones which belonged to androgens, estrogens, progestins, and glucocorticoids. The aim of the present study was to expand the understanding of the separation phenomena which would be encountered regarding the analyses of steroid hormones. Influential parameters included the concentration of CTAB, type and proportion of organic modifiers, and sample matrix. Organic modifier was the most prominent parameter, since it affected the resolution through the presence in separation buffer and the peak shape though the existence in sample matrix. Successful separation of cortisone, hydrocortisone, estriol, testosterone, estrone, progesterone, and estradiol was achieved at -25 kV using 100 mM Tris-boric acid buffer (pH 9.0) with 40 mM CTAB and 20% 2-propanol.
MEEKC, claimed to attain high peak efficiency with great solubilization power, was also utilized in the present study to separate different forms of tocopherols with highly structural similarities and high hydrophobicities. We investigated the effects of various parameters, such as buffer system, type and concentration of cyclodextrins, temperature, and sample matrix on the separation of tocopherols. By using a buffer mixture of 4% (w/w) sodium dodecyl sulfate (SDS), 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane, 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25 mM, pH 2.5), and 25 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD), the separation of α-, γ- and δ-tocopherol, α-tocopherol acetate, as well as the antioxidant butylated hydroxytoluene (BHT) at -26 kV, 25 °C was succeeded. The results suggested that under proper control of separation conditions, capillary electrophoresis, due to its high resolution power, high separation efficiency, and low sample and solvent consumption, was indeed of great values to be applied in pharmaceutical analysis.

並列關鍵字

capillary electrophoresis ； micellar electrokinetic chromatography ； microemulsion electrokinetic chromatography ； San-huang-xie-xin-tang ； steroid hormones ； tocopherols

參考文獻

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