基于AQbD的培植牛黄中18种氨基酸含量测定方法的建立与优化

目的 基于分析方法质量源于设计理念，建立与优化同时测定培植牛黄中18种氨基酸含量的分析方法。方法 以峰对的最低分离度为指标，采用Hunter筛选试验设计筛选影响色谱分离的关键色谱参数；然后采用中心复合响应曲面实验设计建立分析方法关键连续变量与5个关键响应指标之间的多变量回归函数，再将5个多变量回归函数整合至1个设计空间中，应用设计空间确定这些关键色谱参数的最优值和控制范围。结果 Hunter筛选试验设计确定色谱参数柱温、pH值和缓冲盐类型对响应值有显著性影响(P<0.05)。同时实验中发现有机相中甲醇和乙腈分别对不同的色谱峰对有不同的分离效果。应用中心复合响应面实验设计确定3个连续色谱变量(柱温、pH值和甲醇-乙腈比例)的最优值和控制范围。柱温接受范围为46~58 ℃，最优值为50 ℃；流动相pH可接受范围为7.7~8.7，最优值为8.2；流动相中甲醇-乙腈比例可接受范围为0.7~1.28，最优值为1.0。结论 所建立的分析方法可靠，可用于同时测定培植牛黄中18种氨基酸的含量。

Establishment and Optimization of Simultaneous Determination of 18 Kinds of Amino Acids in Cultured Bezoar Based on AQbD

OBJECTIVE To establish and optimize the determination method of 18 kinds of amino acids in cultured bezoar based on analytical quality by design. METHODS The least resolution between peak pair was taken as response, the Hunter screening trial was used to screen the critical chromatographic parameters; next, central composite response surface design was employed to set up the multivariate regression functions between the critical chromatographic parameters screened by Hunter screening trial and 5 chromatographic responses, respectively. Five regression functions were incorporated into a design space to determine the optimum and acceptable range of 3 critical chromatographic parameters. RESULTS The column temperature, pH and type of buffer salt were identified as the key factors that could affect significantly the response by Hunter screening trial design(P<0.05). Additionally, it was found that methanol and acetonitrile in mobile phase had distinct separation effect on different peak pairs. Central composite was utilized to determine the optimum and control arrange of 3 key paramters(column temperaure, pH value and methanol-to-acetonitrile ratio), column temperature acceptable range of 46-58℃, optimum value of 50℃; pH acceptable range of 7.7-8.7, optimum value of 8.2; and acceptable range of methanol-to-acetonitrile ratio of 0.7-1.28, optimum value of 1.0. CONCLUSION The established determination method is reasonable, which can be used to determine the contents of 18 kinds of amino acids in cultured bezoar simultaneously.