HPLC-PAD法测定盐酸大观霉素含量及有关物质

Determination of spectinomycin and related substances by HPLC-PAD

目的建立了一种新的HPLC-PAD法测定盐酸大观霉素含量及其有关物质的分析方法。方法采用ApolloC18（250mm×4．6mm，5μm）色谱柱，以100mmol／L三氟乙酸为流动相，流速为1．0mL／min，柱温为30℃，柱后衍生氢氧化钠溶液浓度为21g／L，流速为0．5mL／min。脉冲安培检测器，工作电极为金电极（3mm），四电位工作模式。结果盐酸大观霉素在0．15～150μg／mL（r=0．9999）内线性关系良好，杂质A在0．05～50μg／mL（r=0．9999）内线性关系良好，杂质B在0．5～50μg／mL（r=1．0000）内线性关系良好，杂质C在0．5～50μg／mL（r=1．0000）内线性关系良好。大观霉素、杂质A、杂质B和杂质C的检测限分别为1．5、1．0、2．0和3．0ng。大观霉素、杂质A、杂质B和杂质C的精密度分别为1．5％、1．1％、1．6％和1．8％。大观霉素的回收率为96．7％～102．5％。结论该方法准确、灵敏，重复性和耐用性好，可用于盐酸大观霉素及其有关物质的实验室日常测定。

Objective To develop an HPLC coupled with pulsed amperometric detection （PAD） method for the determination of spectinomycin and its related substances. Methods The column was Apollo C18（250mm×4.6mm, 5 μm）. The mobile phase was 100mmol/L TFA with a flow rate of 1.0mL/min. The post-column derivatization solution was 21g/L sodium hydroxide solution with a flow rate of 0.5mL/min. The working electrode was golden electrode （a diameter of 3mm）, with a four potentials working mode. Results The linear ranges of spectinomycin, impurity A, impurity B and impurity C were 0.15-150μg/mL（r=0.9999）, including impurity A 0.05-50μg/mL（r-=0.9999）, impurity B 0.5-50μg/mL（r=1.0000） and impurity C 0.5-50μg/mL（r=1.0000）, respectively. The LODs of spectinomycin, impurity A, impurity B and impurity C were 1.5, 1.0, 2.0 and 3.0ng, respectively. The precisions of spectinomycin, impurity A, impurity B and impurity C were 1.5%, 1.1%, 1.6% and 1.8%, respectively. The recovery rate of spectinomycin was 96.7%-102.5%. Conclusion The established method is accurate, sensitive, repeatable, robust and suitable for routine analysis of spectinomycin in laboratory.