RP-HPLC柱前在线衍生法测定羊胎盘枸杞胶囊中16种氨基酸的含量.docVIP

RP-HPLC柱前在线衍生法测定羊胎盘枸杞胶囊中16种氨基酸的含量 游景水 （汤臣倍健股份有限公司，珠海 519040） [摘要] 目的：建立一种采用反相高效液相色谱法测定羊胎盘枸杞胶囊中16种氨基酸的含量。方法：样品中的蛋白质在6mol/L盐酸溶液中水解为氨基酸，其中一级氨基酸用邻苯二甲醛（OPA）、二级氨基酸用9-芴甲基氯甲酸酯（FMOC）进行柱前在线衍生；用Agilent Hypersil AA-ODS柱（200mm×2.1mm,5μm）分离；用流动相(0.16%醋酸钠)进行梯度洗脱；经荧光检测器（FLD）检测(激发波长：340nm，发射波长：450nm；15min时，激发波长：266nm，发射波长：305nm)。结果：16种氨基酸在5pmol/ul～250pmol/ul浓度范围内呈现良好的线性关系（r:0.99959～1.00000)（n=5)，平均加标回收率（n=9）为91.6%～104.1%，相对标准偏差(RSD)为0.9%～3.7%,最低检测限为0.4pmol～1.2pmol。结论：该方法灵敏度高，具有良好的精密度与准确度，可以有效地控制产品质量。 [关键词] 羊胎盘枸杞胶囊；柱前在线衍生；氨基酸；反相高效液相色谱法； 邻苯二甲醛（OPA）；9-芴甲基氯甲酸酯（FMOC）； Online pre-column derivazation determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by RP-HPLC YOU Jing - shui (By-Health Co.Ltd,Zhuhai 519040,China) [Abstract] Objective:To establish a method for determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by RP-HPLC。Methods:Proteins in the samples were hydrolyzed into amino acids in 6mol/L hydrochloric acid solution， o-phthalaldehyde(OPA) and 9-Fluorenylmethyl chloroformate(FMOC) were used as online pre-column derivative reagents for the primary amino acids and secondary amino acids respectively,and the chromatographic experiments were performed on Agilent Hypersil AA-ODS column（200mm×2.1mm,5μm).The 0.16% sodium acetate solution was used as mobile phase.Signals were detected by fluorescence detection (FLD)(excitation wavelength: 340nm,emission wavelength:450nm),exciation wavelength changed to 266nm and emission wavelength changed to 305nm at the 15th minute.Results:The concentration of Sixteen kinds of Amino Acids demonstrated good linearity in the range of 5 pmol/ul～250 pmol/ul,（r:0.99959～1.00000),(n=5).The recoveries were 91.6%～104.1% (n=9).The RSD were 0.9%～3.7%.The limits of detection were 0.4pmol～1.2pmol.Conclusion:This method has advantages of high sensitivity and precision and accuracy.Its suitable for the determination of sixteen kinds of amino acids in the capsule of sheep placenta and