目的:建立固相萃取-高效液相色谱法(SPE-HPLC法)快速检测维生素D滴剂(软胶囊)中维生素D3有关物质反式维生素D3(杂质A)、7-去氢胆固醇(杂质B)、光甾醇3(杂质C)、异速甾醇3(杂质D)。方法:取内容物约1.2 g(约相当于维生素D3 1 548 IU),精密称定,置于试管中,加入异辛烷4 mL,涡旋混匀,采用固相萃取小柱进行净化,以正己烷-乙酸乙酯(85∶15)为洗脱剂,无水乙醇为解吸剂;采用Luna Silica(2)100 Å硅胶柱(150 mm×4.6 mm,3 μm),以正己烷-正戊醇(996.5∶3.5)为流动相,采用加校正因子主成分自身对照法计算维生素D3各杂质的含量。结果:前维生素D3与植物油分离度＞1.5。维生素D3的线性范围为0.02~0.80 μg·mL-1,r=0.999 5;杂质A的线性范围为0.02~0.80 μg·mL-1,r=0.999 9;杂质B的线性范围为0.02~0.80 μg·mL-1,r=0.999 9;杂质C的线性范围为0.02~0.80 μg·mL-1,r=0.999 9;杂质D的线性范围为0.02~0.80 μg·mL-1,r=0.999 9。维生素D3杂质A~D的平均回收率(n=9)为93.2%~102.9%。对3批维生素D滴剂(软胶囊)样品进行检测,结果已知杂质及其他最大单个杂质含量均≤0.5%,杂质总含量≤1.0%。结论:本方法经系统方法学验证,适用于维生素D滴剂(软胶囊)中维生素D3有关物质的检测,且具有操作简便、快速、准确的优势,为脂溶性维生素D3制剂的质量控制提供了技术保证。
Objective: To establish a solid-phase extraction-high performance liquid chromatography(SPE-HPLC) method for the rapid detection of related substances of vitamin D3, trans vitamin D3 (impurity A), 7-dehydrocholesterol (impurity B), lumisterol 3 (impurity C), and isotachysterol 3 (impurity D), in vitamin D drops (soft capsules). Methods: The content, which was approximately equivalent to vitamin D3 1 548 IU, was taken in about 1.2 g, weighed accurately, put into a test tube, 4 mL of isooctane was added to it, and it was swirled and mixed well. Purification was performed using solid phase extraction columns, with n-hexane-ethyl acetate(85∶15) as the elution agent and anhydrous ethanol as the desorption agent. A Luna Silica(2) 100 Å silica gel column(150 mm×4.6 mm, 3 μm) was used, with n-hexane-n-pentanol (996.5∶3.5) as the mobile phase. The content of vitamin D3 impurities was calculated using the adding standard calibration factors principal component self-control method. Results: The separation degree between pre-vitamin D3 and vegetable oil was greater than 1.5. The linear range of vitamin D3 was 0.02-0.80 μg·mL-1, with r=0.999 5. The linear range of impurity A was 0.02 -0.80 μg·mL-1, with r=0.999 9. The linear range of impurity B was 0.02-0.80 μg·mL-1, with r=0.999 9. The linear range of impurity C was 0.02-0.80 μg·mL-1, with r=0.999 9. The linear range of impurity D was 0.02-0.80 μg·mL-1, with r=0.999 9. The average recovery rate of impurities A-D (n=9) was 93.2%-102.9%. The detection results of 3 batches of vitamin D drops (soft capsules) showed that the content of known impurities and other maximum single impurities were less than 0.5%, and the total content of impurities was less than 1.0%. Conclusion: The results indicate that the established method is suitable for the detection of vitamin D3 related substances in vitamin D drops (soft capsules). It has the advantages of being easy to operate, providing rapid and accurate results, and providing technical assurance for the quality control of fat-soluble vitamin D3 preparations.
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