二维液相色谱法测定维生素D2软胶囊中速甾醇的含量

Determination of the Content of Tachysterol in Vitamin D Soft Capsules by Two-dimensional Liquid Chromatography

目的建立二维液相色谱法检测维生素D2软胶囊中速甾醇含量的方法。方法检测波长281 nm;流速0.5 mL·min-1;柱温40℃;一维色谱条件:采用Thermo Acclaim HILIC丙基酰胺键合硅胶柱(2.1 mm×150 mm,3μm),以正己烷-正戊醇-异丙醇(998∶0.5∶1.5)为流动相;二维色谱条件:采用Thermo硅胶柱(3 mm×100 mm,1.8μm),以正己烷(A)-正己烷-正戊醇-异丙醇(96∶2∶2)(B)为流动相,梯度洗脱。结果速甾醇在0.032 5~0.325μg·mL^-1内与峰面积的线性关系良好,r=0.999 9;回收率为99.35%(RSD=1.4%,n=9),速甾醇的相对校正因子为0.047 6,检测限为1.6ng·mL^-1,定量限为4.8 ng·mL^-1。结论建立的方法符合杂质测定的要求,可以准确测定维生素D2软胶囊中速甾醇的含量。

OBJECTIVE To establish a method for the determination of tachysterol in vitamin D2 soft capsules using two-dimensional liquid chromatography. METHODS Test wavelength: 281 nm;velocity of flow 0.5 mL·min-1;column temperature: 40 ℃. One-dimensional chromatographic condition: Thermo Acclaim HILIC propylamide bonded silica gel column(2.1 mm×150 mm, 3 μm) was used, hexane-pentyl alcohol-isopropanol(998∶0.5∶1.5) as mobile phase. Two-dimensional chromatographic condition: Thermo silica column(3 mm×100 mm, 1.8 μm) was used, hexane(A)-hexane-pentyl alcoholisopropanol(96∶2∶2)(B) as mobile phase, gradient elution. RESULTS In the range of 0.032 5-0.325 μg·m L^-1, tachysterol could obtain a good linear relationship(r=0.999 9), the recovery of tachysterol was 99.35%(RSD=1.4%, n=9). The relation correction factor of tachysterol was 0.047 6, the LOD was 1.6 ng·mL^-1, the LOQ was 4.8 ng·mL^-1. CONCLUSION The method conform to the requirement of impurity determination, and the content of the tachysterol in vitamin D2 soft capsules can be accurately measured.