以4-[N-methyl, N-(1-naphthylmethyl)amino]-4-oxobutanoic acid 之可移除性醇基專一性衍生反應配合高效能液相層析分析醇類的方法及其應用

本研究之主要目的為以具有高感度發色基團(chromophore)之微量分析用可移除性衍生試劑，並藉由可移除之催化劑與偶合試劑的配合下，結合高效能液相層析法，對親核性能力相當弱之一級醇和二級醇類化合物，建立一個簡單、高感度、高選擇性且可應用在一些基質複雜之檢品的分析方法。衍生反應配合層析乃達成微量分析之常用技術，但是過量的試劑及檢品的複雜基質常會造成分析上定性及定量的困難；因此在一些衍生反應後，必須藉助額外純化待分析衍生物的方法處理，因而常增加分析步驟、時間及成本。本研究為解決此一困難而探討建立一種可移除性的衍生方法。本研究的基本設計為先合成一種可移除性衍生發色試劑4-[N-methyl, N-(1-naphthylmethyl)amino]-4-oxobutanoic acid (NAOB)，再配合可移除之催化劑4-dimethyl- aminopyridine (DMAP)及可移除之偶合試劑1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)，可在溫和的條件下對一些醇類化合物進行發色基團之衍生反應，並配合高效能液相層析方法進行分離，配合紫外光偵測器進行微量分析，並應用於吐根流浸膏、市售紅酒及中藥酒製品(含水量約45%~92%)中微量甲醇之測定;同時也將此衍生方法應用於市售營養產品中膽固醇的微量分析。本方法對短鏈醇(甲醇、乙醇、正丙醇)之線性範圍是0.5~10 mmol/mL,而對膽固醇的線性範圍為0.75~150 mg/mL; 其準確度和精密度皆小於3.9 %，可見本法具有良好的定量性和準確度。

關鍵字

可移除性衍生

並列摘要

A high performance liquid chromatography (HPLC) with chemically removable derivatizing reagents for the analysis of weak nucleophile, such as primary alcohols and secondary alcohols, has been developed. The alcohols was selectively derivatized with 4-[N-methyl, N- (1-naphthylmethyl)amino]- 4-oxobutanoic acid (NAOB), wich is a removable reagent with a highly sensitive chromophore, using 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as activators. NAOB and the activators are simplely removed by aqueous base and acid treatment after derivatization to minimize reagent interference in chromatography. In addition, the method can be useful for complicated matrix samples. Derivatization coupled with chromatography is frequently used for trace analysis. After derivatization, the excess amount of reagents and complicated matrix may seriously interfere in the separation of minor analyte from major reagent. Therefore additional treatments are often used to remove the excess reagents. The treatments are often tedious and time consuming so this study is to development a HPLC with chemically removeable derivitzing method, wich is coupling alcoholic compound with chromophore at the mild conidition. The method was not only applied to the analysis of shor-chain alcohols but also for trace methanol in complicated matrix, such as Ipecac fluidextract, wine and Chinses liquor medicine (water content 45%~92%). In addition, the method was also applied to the analysis for cholesterol in powder milk products.