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.