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,5 μm）,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0.1~2.0 μg·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。
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,5 μm）;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 0.1-2.0 μg·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,5Z,7E）-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.
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