RP-HPLC法测定维生素D3制剂中的有关物质
Determination of related substances of vitamin D3 in its preparations by RP-HPLC
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摘要: 建立维生素D3制剂中有关物质检查的反相HPLC方法。以十八烷基硅烷键合硅胶为填充剂；乙腈为流动相；柱温20 ℃；检测波长为265 nm；流速1.0 mL/min。建立的方法可以使维生素D3与相邻杂质有效分离。通过光热反应特性和LC-MS-DAD对相邻杂质进行了定性。维生素D3与其主要杂质前维生素D3、反式维生素D3和速甾醇D3互为同分异构体，在LC-MS上具有相同的特征离子峰m/z 385.27，四者的最大紫外吸收波长分别为265，265，275，279 nm。该法专属性、耐用性较好，检测限和定量限分别为0.1μg/mL和0.3 μg/mL。供试品溶液在室温下放置24 h稳定。应用本法对3种维生素D3制剂进行有关物质检查，并与正相HPLC法比较，两种方法结果一致，表明本法适用于维生素D3制剂中有关物质检查。Abstract: An isocratic RP-HPLC method for the determination of the related substances of vitamin D3 in its preparations was established.The chromatographic separation was achieved on amethyst C18-H column (150 mm×4.6 mm，5 μm），using acetonitrile as the mobile phase.The detection was performed at 265 nm，and the column temperature was 20 °C.A good resolution with baseline separation was obtained.Impurities were identified using photochemical and thermal experiments and LC-MS-DAD.The APCI-MS spectra gave the same [M+H]+ peak at m/z 385.27 as their base peaks for vitamin D3，pre-vitamin D3，trans-vitamin D3，and tachysterol 3.The UV λmax of vitamin D3,pre-vitamin D3,trans-vitamin D3,and tachysterol3 were 265 nm，265 nm，275 nm and 279 nm，respectively.The method was specific and robust.The limit of detection was 0.1 μg/mL，and the limit of quantification was 0.3 μg/mL.Test solution was stable for 24 h at room temperature.Three kinds of vitamin D3 preparations were tested using the developed method and the current official method，the results showed no differences.The developed method was suitable for the determination of the related substances of vitamin D3 in its preparations.
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Keywords:
- RP-HPLC /
- vitamin D3 /
- related substances /
- content determination
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