河豚毒素原料药及有关物质含量的高效液相色谱-蒸发光散射检测法的建立

Development of a high-performance liquid chromatography-evaporative light scattering detection method for the determination of tetrodotoxin and related substances

目的建立高效液相色谱-蒸发光散射检测器(HPLC-ELSD)方法,测定河豚毒素(TTX)原料药及有关物质的含量。方法选用TSK Gel amide-80(150 mm×2 mm,5μm)色谱柱,以乙腈-含5 mmol/L乙酸铵的0.1%甲酸溶液(70∶30,V/V)为流动相,流速0.2 ml/min,柱温40℃。ELSD漂移管温度105℃,雾化器温度80℃,气体流量1.8 L/min。结果TTX在10~100μg/ml浓度范围内,线性关系良好,相关系数R^(2)为0.9995,检测限(S/N=3)为5 ng,定量下限(S/N=10)为10 ng。准确度试验相对标准差为0.93%;日内/日间精密度试验相对标准差均<2%;破坏试验中降解产物与TTX的分离度均>2.0;耐用性试验相对标准差均<2%。结论本方法适用于TTX和有关物质的含量检测,可为TTX原料药质量控制及其制剂开发提供参考。

Objective To develop a method of determining the content of tetrodotoxin(TTX)and related substances in TTX bulk drugs by using the high-performance liquid chromatography-evaporative light scattering detector(HPLC-ELSD).Methods The determination was carried out by using a TSK Gel amide-80 column(150 mm×2 mm,5μm)at a flow rate of 0.2 mL/min,with a mobile phase comprising acetonitrile-0.1%formic acid solution containing 5 mmol/L ammonium acetate(70∶30,V/V),and a column temperature of 40℃.The ELSD evaporator temperature was set to 105℃,the nebulizer temperature was set to 80℃,and the gas flow rate was 1.80 L/min.Results TTX had a good linear relationship within the concentration range of 10-100μg/mL,with a correlation coefficient(R^(2))of 0.999,5.The detection limit(S/N=3)was 5 ng,and the lower limit of quantification(S/N=10)was 10 ng.The relative standard deviation in the accuracy test was 0.93%;the relative standard deviations for intra-day/inter-day precision tests were both less than 2%;the separation degree between degradation products and TTX in the degradation test was more than 2.0;and the relative standard deviations in the durability test were all less than 2%.Conclusion This method is applicable to determining TTX content and detecting related substances,which can provide a reference for the quality control of TTX bulk drugs and the development of its pharmaceutical preparation.