目的：制备骨化三醇前体对照品,进行方法研究,并建立高效液相色谱法测定骨化三醇软胶囊中骨化三醇前体的含量。方法：采用 C8 色谱柱,以乙腈 -Tris 缓冲液（配制 1 g·L-1的 3-羟甲基氨基甲烷溶液, 用磷酸调节 pH为 7.0~7.5）为流动相,梯度洗脱,流速 1 mL·min-1,检测波长 265 nm,柱温 40 ℃,进样体积 20 μL。结果：骨化三醇前体定量测定方法专属性良好,骨化三醇质量浓度在 1.275~255.0 μg·mL-1 范围内线性良好,骨化三醇前体质量浓度在 0.142~2.126 μg·mL-1浓度范围内线性良好,骨化三醇和前体的检测下限分别为 0.006 4 μg·mL-1 和 0.035 μg·mL-1,定量下限分别为 0.032 μg·mL-1 和 0.12 μg·mL-1,精密度良好, 平均加样回收率 100.0%,选择加校正因子的面积归一化法进行计算,对不同批次的骨化三醇软胶囊进行测定,前体平均含量分别为 6.06%、5.08%、6.12%。结论：本方法操作简便,结果准确,可用于骨化三醇软胶囊中骨化三醇前体的测定,可为具有相似结构的骨化醇类药品控制前体限度提供参考。
Objective：To study the preparation method of pre-calcitriol reference substance,and establish an high performance liquid chromatography method to determine the content of pre-calcitriol in calcitriol soft capsules. Methods：The determination was performed on a C8 column with acetonitrile-Tris buffer as mobile phase,wherein a Tris buffer solution was obtained by preparing 1 g·L-1 of 3-（hydroxymethyl）aminomethane solution and then adjusting pH to 7.0-7.5 with phosphoric acid. The flow rate was set at 1 mL·min-1 and the detection wavelength was set at 265 nm. The column temperature was set at 40 ℃ and the injection volume was 20 μL. Results：The specificity of this quantitative method was good,the linearity of calcitriol was within the concentration range of 1.275-255.0 μg·mL-1,and the linearity of pre-calcitriol was within the concentration range of 0.142-2.126 μg·mL-1. The limit of detection of calcitriol and pre-calcitriol were 0.006 4 μg·mL-1 and 0.035 μg·mL-1 respectively,and the quantitative limits were 0.032 μg·mL-1 and 0.12 μg·mL-1 respectively. The precision was good,and the average recovery rate of added samples was 100.0%. An area normalization method with correction factors was selected for calculation,calcitriol soft capsules of different batches were determined, and pre-calcitriol contents of the calcitriol soft capsules were 6.06%,5.08% and 6.12% respectively. Conclusion：This method is simple and accurate,and therefore can be used for determining pre-calcitriol in calcitriol soft capsules. It can be used as a reference for the control of precursor limits of calcitriol drugs with similar structure.
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