高效液相色谱-脉冲安培电化学检测器法替代微生物检定法测定硫酸新霉素效价的研究

目的建立用于测定硫酸新霉素效价的高效液相色谱-脉冲安培电化学检测器法(HPLC-PAD)。方法建立并验证HPLC-PAD法,测定硫酸新霉素各主要组分含量及相关物质;采用在线膜抑制脱盐技术辅助LC-IT-TOF法测定硫酸新霉素杂质谱,结合组分活性研究确定了硫酸新霉素的主要活性组分;采用半制备液相色谱-蒸发光散射检测器串联制备硫酸新霉素B及硫酸新霉素C精制品,采用波谱手段进行结构确证;采用质量平衡法测定二者的纯度;采用三剂量抗生素微生物检定法测定B、C精制品各自的效价值,推导公式计算出硫酸新霉素的量效转换系数,设计实验进行交互作用考察及实际样品的验证。结果建立的HPLC-PAD方法在分离能力及方法的稳定性上均优于《欧洲药典》(EP)方法,适用于硫酸新霉素各组分的准确定量及有关物质检查;在线膜抑制脱盐技术成功地去除了流动相中的三氟乙酸,对硫酸新霉素的杂质谱进行研究,结合活性研究结果,确定新霉素B和新霉素C为新霉素的主要活性组分;成功制备获得新霉素B、C精制品,通过实验和计算获得了硫酸新霉素的量效转换系数,并通过了验证。结论初步实现了仪器法对微生物生物检定法的替代,基本完成了多组分氨基糖苷类抗生素硫酸新霉素的量效一致性研究工作。

Transition of Determination Method of Potency of Neomycin Sulfate from Microbiological Assay to HPLC-PAD

OBJECTIVE To establish a high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD) method for determination of potency of neomycin sulfate.METHODS An improved HPLC-PAD method from EP method for determination of the content and related substances of neomycin sulfate was established and validated.The study of impurity profile of neomycin sulfate was completed by LC-IT-TOF method with the help of on-line desalination using a suppressor;and the main components in neomycin sulfate were clarified combining the results of impurity profile and minimum inhibitory concentrations of the main components and impurities.The semi-preparative liquid chromatography-evaporative light scattering detector(ELSD) was self-assembled,highly purified neomycin B and neomycin C were prepared and their structural confirmation was also conducted.The contents of highly purified neomycin B and neomycin C were determined by means of mass balance method.The potencies of highly purified neomycin B and neomycin C were determined by three-dose antibiotic microbial assay and the conversion factors between contents of neomycin B and neomycin C and their potencies were calculated separately and then a formula for the calculation of potency of neomycin sulfate from the content of main components of neomycin B and neomycin C was obtained.At last,a verification experiment for the accuracy of the conversion factor and the formula were designed and a serial of tests were carried out to investigate the interaction and the verification for the actual sample.RESULTS The improved HPLC-PAD method was superior to the European Pharmacopoeia method in the separation ability and stability,and was suitable for accurate quantification of various components of neomycin sulfate and related substance inspection.The successful removal of trifluoroacetic acid in the mobile phase by the technology of desalination on-line using a suppressor broke a new way for the study of impurity profile of aminoglycoside such as neomycin sulfate.Combining the impurity profile with the results of MIC it was clarified that the main activity components of neomycin sulfate were neomycin B and neomycin C.Highly purified neomycin B and neomycin C were successfully prepared.A conversion factor for the transition from potency to purity of neomycin sulfate was obtained through experiments and calculations and was verified successfully.CONCLUSION It is feasible to replace the microbial assay by HPLC-PAD method for determining the potency of neomycin sulfate.