高分辨采样二维液相色谱法测定维生素D滴剂中维生素D_(3)含量研究

Determination of vitamin D_(3)in vitamin D drops by high resolution sampling two-dimensional chromatography

目的:建立高分辨采样二维液相色谱法(HiRes 2D-LC)对维生素D滴剂中维生素D_(3)进行准确定量分析。方法:第一维液相色谱采用Thermo HYPERSIL Gold Silica(100 mm×2.1 mm,1.9μm)色谱柱,以正己烷-正戊醇(996∶4)为流动相,流速0.2 mL·min^(-1),检测波长265 nm,柱温40℃;第二维液相色谱采用ShimPack Velox Hilic(50 mm×2.1 mm,2.7μm)色谱柱,以正己烷-正戊醇-异丙醇(98∶1∶1)为流动相,流速0.5 mL·min^(-1),检测波长265 nm,柱温40℃。二维接口采用六位十四通阀,并配置2个多中心切割阀,对前维生素D_(3)峰和维生素D_(3)峰进行多次连续切割。结果:维生素D_(3)质量浓度在1.0184~5.092 mg·mL^(-1)范围内线性关系良好(r≥0.9998),前维生素D_(3)和维生素D_(3)精密度试验RSD分别为0.95%和0.40%,重复性试验RSD为0.41%,平均加标回收率为101.4%。供试品溶液在4℃和10℃放置12 h稳定,RSD分别为0.58%、0.66%。采用本法测得6批维生素D滴剂样品中维生素D_(3)的含量分别为100.4%、101.6%、100.9%、101.6%、102.7%、101.6%,与采用2020年版《中华人民共和国药典》四部通则0722第四法所测结果基本一致。结论:本方法具有优异的灵敏度、重复性和定量准确性,且省去烦琐的样品前处理步骤,缩短分析时间,改善长样品环带来的峰展宽等问题,显著提高了维生素D_(3)的含量测定效率,降低分析成本,为维生素D滴剂等主成分与辅料难分离制剂的准确定量提供新方法。

Objective:To establish an innovative analytical method based on high resolution sampling two-dimensional chromatography(HiRes 2D-LC)for determination of the content of vitamin D_(3)in vitamin D drops.Methods:Two-dimensional liquid chromatography was used.Thermo HYPERSIL Gold Silica(100 mm×2.1 mm,1.9μm)column was used in the first dimension with n-hexane-n-amyl alcohol(996∶4)as mobile phase.The flow rate was 0.2 mL·min^(-1).The samples were injected and tested at the wavelength of 265 nm.The column temperature was 40℃.In the second dimension liquid chromatography,ShimPack Velox Hilic(50 mm×2.1 mm,2.7μm)was used as the column with n-hexane-n-pentanol-isopropanol(98∶1∶1)as the mobile phase.The flow rate was 0.5 mL·min^(-1).The samples were injected and tested at the wavelength of 265 nm.The column temperature was 40℃.A six-position 14-way valve and was equipped with 2 multi-center cutting valves was equipped to make multiple consecutive cuts of the pre-vitamin D_(3)peak and vitamin D_(3)peak.Results:The calibration curves showed a good linearity at the range of 1.0184-5.092 mg·mL^(-1)(r≥0.9998).The precision test showed the RSDs of the peak area of pre-vitamin D_(3)and vitamin D_(3)were 0.95%and 0.40%,respectively.The repeatability test showed the RSD of vitamin D_(3)content was 0.41%.The average recovery rate(n=9)was 101.4%.The test solution was stable at 4℃and 10℃for 12 h,and the RSDs were 0.58%and 0.66%,respectively.The contents of vitamin D_(3)in the samples of vitamin D drops measured by this method were 100.4%,101.6%,100.9%,101.6%,102.7%and 101.6%,which was basically consistent with the results measured by the fourth method in General Chapter 0722 of ChP 2020 VolⅣ.Conclusion:This method has a good specificity and high sensitivity to accurately determine the content of vitamin D_(3)in vitamin D drops.