柱前在线衍生-反相高效液相色谱法测定羊胎盘

Determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by online pre-column derivazation-reversed-phase high

中文关键词:  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  

英文关键词::Capsule of sheep placenta and medlar  Online pre-column derivazation  Amino acid  RP-HPLC  O-phthaldialdehyde (OPA)  9-Fluorenylmethyl chloroformate(FMOC).

中文摘要:

      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。

英文摘要:

      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.It"s suitable for the determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar.