毛細管電泳對多樣胺基酸分析法之建立及應用

Development and Application of Capillary Electrophoresis Method in the Analysis of Multiple Amino Acids

何宇祥(Yu-Hsiang Ho)

指導教授 ： 吳信隆

高雄醫學大學/藥學院/藥學研究所/博士(2006年)

摘要

實物樣品分析的主要困難，在於複雜基質共存中微量待測物之檢測，基本上分析方法應具高解析力及高感度。本研究利用具高呈色性的試劑 (2-naphthoxy)acetyl chloride，經衍生反應將naphthoxy呈色基團標示到胺基酸待測物上，使其對紫外光呈高度吸收之衍生物，並利用高解析之毛細管電泳法，建立了多樣胺基酸高解析度及感度分析法。此衍生反應除提升胺基酸的分析感度外，亦可改變其親脂性，達到易分離的效果。本研究主要結果如下：
1. 利用經濟性 (2-naphthoxy)acetic acid為起始物，與oxalyl chloride於65℃下迴流反應，可得 (2-naphthoxy)acetyl chloride，首度由我們應用於多樣胺基酸之毛細管電泳分析。
2. (2-Naphthoxy)acetyl chloride於鹼性緩衝溶液中，對不具呈色基團之胺基酸進行衍生，所得複雜多樣衍生物，以膠束毛細管電動層析法 (micellar electrokinetic chromatography, MEKC) 進行分離，配合紫外光偵測器 (230 nm) 加以檢測；因所得衍生物導入親脂性之naphthoxy基團，適合利用MEKC進行分離，可在40分鐘內同時分析十七種胺基酸衍生物 (alanine, arginine, aspartic acid , cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine及valine)，其偵測極限為約在3.0-8.0 μM (S/N = 3, 注入時間5秒, 施注壓力1.0 psi)；此法之精密度 (precision, RSD) 及準確度 (accuracy, RE) 分別小於4.8%及4.9%，再現性及誤差性良好。
3. 本法已成功應用於胺基酸之實物檢品，包括注射液、運動飲料、藥酒及果汁中多樣胺基酸之分析，本法解析力佳，適用於複雜檢體中胺基酸之分析，其在胺基酸之臨床診斷分析上，值得推展。

關鍵字

胺基酸 ； 膠束毛細管電泳

並列摘要

The problem in real sample analysis is usually to determine trace target analytes coexisted in complicate matrices. To address the difficulties, high resolution method with good sensitivity is basically required. In this study, multiple amino acids were derivatized with a highly chromophoric reagent (2-naphthoxy)acetyl chloride. The resulting naphthoxy derivatives were analyzed by high resolution capillary electrophoresis with ultraviolet detection. The analytical derivatization makes the amino acid derivatives sensitive to a UV detector as well as easily resolvable by micellar electrokinetic capillary chromatography (MEKC). The main results of the study are summarized as follows:
1. (2-Naphthoxy)acetyl chloride was easily synthesized by reacting (2-naphthoxy)acetic acid with oxalyl chloride and it was first used to derivatize multiple amino acids for their quantitative analysis.
2. Multiple amino acids were derivatized with (2-naphthoxy)acetyl chloride in alkaline media, and the resulting derivatives were analyzed by MEKC (borate 35 mM, pH 9.50 and SDS 150 mM) with UV detection (230 nm). Seventeen amino acid derivatives (from alanine, arginine, aspartic acid, cystine, glutaminc acid, glycine, histidine, isoleucine, leucine, lysine methionine, phenylalanine, serine tryptophan, tyrosine and valine) can be completely separated in 40 min. The detection limits of the analytes were in the range of 3-8 μM (S/N = 3, injection 5 s, 1.0 psi). The precision (relative standard deviation, RSD) and accuracy (relative error, RE) of the method for intra- and inter-day analyses of the analytes were respectively below 4.8% and 4.9%.
3. Application of the method to the analysis of amino acids in real samples proved feasible including pharmaceutical preparation, sports drink, amino acids fortified liquor, and fruit juice. Further development of the method for analyzing related amino acids in biosamples for clinical study could be interesting.

並列關鍵字

amino acids ； micellar electrokinetic capillary electrophoresis

參考文獻

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