柱前衍生RP-HPLC法测定肽藻营养粉中游离氨基酸的含量

Determination of Amino Acids in Peptide Nutrition Powder by RP-HPLC with Pre-column Derivatization

摘要:

本研究建立了柱前衍生化反向高效液相色谱法（RP-HPLC）测定肽藻营养粉中游离氨基酸的方法，并确认了肽藻营养粉中的氨基酸种类以及含量。通过研究确定了高效液相色谱法的最佳条件：流动相A：醋酸钠-三乙胺缓冲液（pH 7.20±0.05），加入0.5%四氢呋喃调节峰型；流动相B：醋酸钠缓冲液（pH 7.20±0.05）-乙腈-甲醇（20:40:40），以邻苯二甲醛（OPA）和氯甲酸芴甲脂（FOMC-Cl）为衍生剂，使用Agilent Hypersil ODS色谱柱（5 μm，4.0×250 mm），柱温40 ℃，自动在线衍生，检测波长为338 nm/262 nm。利用该方法检测出17种氨基酸，其中包含8种必需氨基酸，另外结果显示17种氨基酸均呈现良好的线性关系（r>0.9990），平均回收率为85%~115%（RSD为1.15%~5.23%，n=6）。10个批次样品中分别检测到至少15种游离氨基酸。表明该方法简便、准确且重复性好，适用于肽藻营养粉游离氨基酸含量测定。

Abstract:

The method for determining the type and content of free amino acids in the peptide nutrition powder was established by using the column derivatization reverse high performance liquid chromatography (RP-HPLC) for the first time. The optimal mobile phase A were determined as sodium acetate - triethylamine buffer (pH 7.20 ± 0.05) with tetrahydrofuran (0.5%) to adjust the peak type. And the best mobile phase B was sodium acetate buffer (pH 7.20 + 0.05) - acetonitrile-methanol (20:40:40). O-phthalaldehyde (OPA) and Fluorene methyl chloroformate (FOMC-Cl) were used as the pre-column derivatization reagent. Separation was performed on an Agilent Hypersil ODS column (4.0 mm×250 mm, 5μm) with column temperature of 40 ℃. The detection wavelength was set at 338 nm /262 nm. 17 kinds of amino acids, including 8 essential amino acids, were determined by using the former method. The results showed that all the 17 amino acids were in good liner relationship. And the average recovery rates of the hydrolyzed amino acids in peptide nutrition powder of Perfect Brand ranged from 85% to 115% (RSD 1.15%~5.23%, n=6). At least 15 kinds of amino acids were detected in the powder. The results also indicated that the method was accurate, sensitive and simple, and proved to be suitable for the quantification of each amino acid in peptide nutrition powder of Perfect Brand.