衍生化液相層析螢光法對Isovaleric Acid與Valeric Acid之分析

Derivatization and Liquid Chromatographic Analysis of Isovaleric and Valeric Acids as Fluorescent Derivatives

鐘堂嘉(Tang-Chia Chung)

指導教授 ： 吳信隆 教授

高雄醫學大學/藥學院/藥學研究所/碩士(2002年)

摘要

本研究利用螢光偵測導向之衍生反應，配合高效能液相層析法，對代謝疾病或污臭有關指標物isovaleric acid與valeric acid，建立簡單高靈敏之分析方法。本法利用2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES)為衍生試劑，以甲苯為反應溶媒，於碳酸鉀及18-crown-6 ether之催化下，將isovaleric acid與valeric acid加以衍生成螢光性之naphthoxyethyl衍生物，利用C-18固定相及甲醇：水：四氫呋喃為72：22：6（v/v）混合液為移動相進行分離，對所得待測物以螢光偵測器（lex: 235nm; lem: 350nm）檢測。所得結果顯示本法靈敏性高，Isovaleric acid與valeric acid之定量範圍爲0.5 ~10.0 mM，偵測極限0.07 mM (S/N = 3；20 mL)。
本研究對影響isovaleric acid與valeric acid衍生化反應之重要因素，包括試劑需要量、鹼性催化劑、反應時間及溫度等，皆詳加探討。本法對病人尿液中微量isovaleric acid與valeric acid之分析應用正探討中。

關鍵字

異戊酸 ； 異戊酸血症 ； 尿液

並列摘要

Analytical derivatization coupled with high-performance liquid chromatography was used for the analysis of isovaleric and valeric acids as biomarkers in metabolic acidosis or as malodorant in organic pollution. The method is based on the derivatization of isovaleric acid and valeric acid with 2-(2-naphthoxy)ethyl 2-(piperidino) ethanesulfonate (NOEPES) in toluene, using potassium carbonate and 18-crown-6 ether as reaction activators. The resulting naphthoxyethyl derivative of isovaleric acid and valeric acid were separated on a C-18 column, using a mixed solvent of methanol : water : tetrahydrofuran (72：22：6, v/v) as the mobile phase. The separated isovaleric acid and valeric acid derivatives were monitored with a fluorimetric detector (lex: 235nm; lem: 350nm), and the peak-areas of isovaleric and valeric acids derivatives were used for quantitative analysis.
The linear range of the method for the determination of isovaleric acid and valeric acid derivative were over 0.5 ~ 10.0 mM. The detection limit (signal to noise ratio = 3, injected amount 20 mL) of isovaleric acid and valeric acid was about 0.07 mM. Several parameters affecting the derivatization of isovaleric and valeric acids were discussed including amount of derivatization reagent, reaction temperature, reaction time, and base catalyst. Application of the method to the analysis of isovaleric and valeric acids in urine is in procession.

並列關鍵字

isovaleric acid ； isovaleric acidemia ； urine

參考文獻

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