- 學位論文
衍生化高效能液相層析法測定肌醇之研究
Study for the determination of inositol by derivatization and high performance liquid chromatography
摘要
本研究利用紫外光偵測導向之衍生反應，配合高效能液相層析法，對具有生物活性之六碳糖醇類 myo-inositol（hexahydroxycyclohexane），建立簡單之分析方法。 本法利用 4-[N-methyl, N-(1-naphthylmethyl)]-amino-4-oxo-butanoic acid為衍生試劑，以乙腈（acetonitrile）為反應溶媒，於4-dimethylaminopyridine 及 N, N’-dicyclohexylcarbodiimide 之催化下，將 myo-inositol 加以衍生成具有紫外光吸光性之 4-[N-methyl, N-(1-naphthylmethyl)]-amino-4-oxo-butanoic acid 衍生物，利用逆向層析 C-18 管柱及乙腈：水：冰醋酸 ＝ 85：15：0.3（v/v/v）混合液為移動相進行分離，對 myo-inositol 之衍生物和內部標準品的波峰面積比值以紫外分光光度偵測器（281 nm）進行定量分析。 所得結果顯示本法具有良好之線性關係（R2 = 0.9998）， myo-inositol 之定量範圍為 0.111 ~ 11.1 mM，偵測極限 4.44 μM（S/N = 3，injection volume：20 μL）。 本研究對影響 myo-inositol 衍生化反應之諸多重要因素，包括衍生試劑、鹼性催化劑、偶合試劑需要量，反應溫度及時間等皆詳加探討。本研究對市售複方產品中 myo-inositol 之含量分析具有良好之回收率（≧ 96.4 ％）。
並列摘要
A simple method of analytical derivatization coupled with high-performance liquid chromatography was developed for the analysis of myo-inositol, which was known biologically active compound of the C6 sugar alcohol. The method was based on the derivatization of myo-inositol with 4-[N-methyl, N-(1-naphthylmethyl)]-amino-4-oxo-butanoic acid ( NAOB ) in acetonitrile, using 4-dimethylaminopyridine ( DMAP ) and N, N’- dicyclohexylcarbodiimide ( DCC ) as reaction activators. The resulting NAOB derivatives of myo-inositol were separated on a reversed phase C18 column, using a mixed solvent of acetonitrile：water：glacial acetic acid ＝ 85：15：0.3 ( v/v/v ) , as the mobile phase. The separated myo-inositol derivatives were monitored with an ultraviolet detector ( 281 nm ) , and the peak areas ratio of myo-inositol derivative to internal standard was used for quantitative analysis. The linear range of the method for the determination of myo-inositol derivative was over 0.111 ~ 11.1 mM with good linearity ( R2 = 0.9998 ). The detection limit ( signal to noise ratio = 3, injection volume：20 ?尳 ) of myo-inositol derivative was 4.44 μM. Several parameters effecting the derivatization of myo-inositol derivative were examined including the amount of derivatizing reagent, base catalyst and coupling reagent, reaction temperature, reaction time. Application of the method to analyze myo-inositol in pharmaceuticals contained one to five active ingredients had fine recovery ( ≧ 96.4 ％ ).