柱前在线衍生-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

摘要

ABSTRACT:Objective To establish a method for determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar by reversed-phase high performance liquid chromatography (RP-HPLC). MethodsProteins in the samples were hydrolyzed into amino acids in 6 mol/L hydrochloric acid solution, o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were used as online pre-column deriva-tive reagents for the primary amino acids and secondary amino acids respectively, and the chromatographic experiments were performed on an Agilent Hypersil AA-ODS column. The 0.16% sodium acetate solution was used as mobile phase. Signals were detected by fluorescence detection (FLD) (excitation wavelength: 340 nm, emission wavelength: 450 nm, excitation wavelength changed to 266 nm and emission wavelength changed to 305nm at the 15 min). ResultsThe concentration of sixteen kinds of amino acids demonstrated a good linear-ity in the range of 5~250 pmol/μL(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 0.4~1.2 pmol.ConclusionThis method has ad-vantages of high sensitivity, precision and accuracy. It's suitable for the determination of sixteen kinds of amino acids in the capsule of sheep placenta and medlar.%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6 mol/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：340 nm,发射波长：450 nm；15 min时,激发波长：266 nm,发射波长：305 nm)。结果16种氨基酸在5～250 pmol/μL浓度范围内呈现良好的线性关系(r：0.99959～1.00000,n=5),平均加标回收率为91.6%～104.1%(n=9),相对标准偏差(RSD)为0.9%～3.7%,最低检测限为0.4～1.2 pmol。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。