HPLC法测定阿法骨化醇中有关物质

目的:建立测定阿法骨化醇有关物质的HPLC梯度洗脱法,并对其中关键的有关物质D进行结构鉴定。方法:采用Waters XBridge C₁₈色谱柱(250 mm×4.6 mm,5μm),以乙腈-水-氨水(80∶20∶0.1)为流动相A,乙腈为流动相B,流速1.0 mL·min^-1,柱温为30℃,检测波长为265 nm;液质联用方法采用相同色谱柱和检测波长,以乙腈-水-氨水(80∶20∶0.1)为流动相,流速2.6 mL·min^-1,柱温30℃;采用APCI大气压化学离子源,正离子模式扫描,离子源电压为6.0 kV,毛细管温度为275℃,毛细管电压为35 V,Tube lens为125 V,雾化器温度为450℃。结果:阿法骨化醇与6种有关物质峰之间的分离良好;阿法骨化醇及各有关物质质量浓度在0.1~2.0μg·mL^-1范围内,与相应的峰面积呈良好的线性关系(R2>0.999);各有关物质的定量下限低于5 ng,检测下限低于1.5 ng。采用HPLC-MS/MS方法,有关物质D的结构鉴定为(1α,3β,5Z,7E)-9,10-开环胆甾-5,7,10(19)-三烯-1,3-二醇。结论:此HPLC梯度洗脱法专属性强,准确度高,优于现行标准方法,适用于阿法骨化醇产品的有关物质检查。鉴定了关键杂质D的结构,并将其订入质量标准,为现有药品标准的补充。

Determination of related substances in alfacalcidol by HPLC

Objective:To establish an HPLC gradient elution method for the determination of the related substances of alfacalcidol,and to identify the key related substance D.Methods:The HPLC method was carried out using a Waters XBridge C18(250 mm×4.6 mm,5μm);the mobile phase A was acetonitrile-water-ammonia(80∶20∶0.1),and the mobile phase B was acetonitrile;the flow rate was 1.0 mL·min^-1;the column temperature was 30℃and the detection wavelength was 265 nm.The column and detection wavelength of HPLC-MS/MS method were the same as those of HPLC method;the mobile phase was acetonitrile-water-ammonia(80∶20∶0.1);the flow rate was 2.6 mL·min^-1;the column temperature was 30℃.The HPLC-MS/MS system was equipped with an APCI source in positive ionization mode;the spray capillary voltage was 6.0 kV;capillary temperature was at 275℃;capillary voltage was 35 V;Tube lens was 125 V;atomizer temperature was at 450℃.Results:Alfacalcidol and its related substances were separated completely under the above chromatographic conditions.The standard curves of alfacalcidol and its related substances were linear within the range 0.1-2.0μg·mL^-1,with the correlation of 0.999.LOQs of related substances were all below 5 ng and LODs were below 1.5 ng.Impurity D was identified as(1α,3β,5 Z,7 E)-9,10-secocholesta-5,7,10(19)-triene-1,3-diol by means of HPLC-MS/MS analysis.Conclusion:The established method is specific,accurate and better than the existing standard method.Impurity D is identified and set into the quality standard.It is a supplement to the present standards.