健康食品中特定胺基酸檢驗方法之建立   全文下載

Analytical Method Development for Specific Amino Acids in Health Foods

胺基酸為蛋白質結構之基本單位，目前已有多件健康食品以纈胺酸（valine）、白胺酸（leucine）、異白胺酸（isoleucine）、甘胺酸（glycine）、丙胺酸（alanine）及精胺酸（arginine）等6項特定胺基酸作為保健功效或品管指標成分。由於雞精、魚精、蜆精中含蛋白質，需經水解方可進行胺基酸定量分析，故本研究參考衛生福利部食品藥物管理署公開之「食品中游離胺基酸、葡萄糖胺及牛磺酸之檢驗方法（TFDAA0060.00）」，並進一步探討線上衍生化（on-line derivatization）及水解處理步驟，研發可同步檢測食品中6項特定胺基酸之檢驗方法。檢體經15 M甲烷磺酸於160℃加熱水解45分鐘，經去離子水定容後，採用線上衍生化方式，並結合上述公開方法之分析條件，以高效液相層析儀搭配光電二極體陣列檢出器（HPLC-PDA）進行胺基酸定量分析。本研究經測試發現，使用線上衍生化可改善甘胺酸及丙胺酸衍生化後不穩定之現象，並可使得胺基酸定量分析之操作更準確及便利。以SRM 3252（protein drink mix）標準參考物質進行確效試驗，同日間6項胺基酸之回收率介於85.6-98.6%，變異係數介於1.27-2.35%；異日間6項胺基酸之回收率介於86.0-98.1%，變異係數介於1.67-2.44%，以上結果均符合本署食品化學檢驗方法之確效規範，顯示本方法之準確度與精密度良好。以本研究建立之檢驗方法針對6件市售雞精、魚精、蜆精等健康食品中6項胺基酸含量進行方法適用性驗證，結果均與原廠標示相符。本研究建立之方法可做為健康食品查驗登記及後市場產品監測之檢驗依據，保障國人消費之權益，並促進國內食品生技產業之發展。

Amino acids, including valine, isoleucine, leucine, glycine, arginine, and alanine, often work as functional ingredients or quality control indicators of health foods. In order to analyze amino acids in related products with protein ingredients, hydrolysis prior to amino acids analysis is necessary. In this study, we developed a novel hydrolysis process together with on-line derivatization and based on the existing testing method, the“Method of Test for Free Amino Acids, Glucosamine, and Taurine (TFDAA0060.00)”published by TFDA. Samples with protein ingredients were hydrolyzed with 15 M methanesulfonic acid (MSA) at 160℃for 45 minutes, then diluted to fixed volume with deionized water, and analyzed with a high performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA). We noticed more stable and better detection of glycine and alanine with on-line derivatization, which made it a more accurate and convenient quantification operation. We used SRM 3252 for method validation. The intra-day recoveries of the 6 amino acids were between 85.6-98.6%, and the coefficients of variation were between 1.27-2.35%. The inter-day recoveries of the 6 amino acids were between 86.0-98.1%, and the coefficients of variation were between 1.67-2.44%. All of the above results were in compliance with the validation guideline of food chemical testing methods published by TFDA, which indicated excellent accuracy and precision. Six commercial health food products, including chicken essence, fish essence, and clam essence, were examined using this method. The results showed the detected values of the 6 amino acids in the products were consistent to their labeled values. In summary, the developed method can be used as a reliable reference method for health food registration and post market surveillance to protect consumer rights and to facilitate the development of biotechnology industry.