Detection of related substances in vitamin D drops(soft capsules)by SPE-HPLC
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-de-hydrocholesterol(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 compo-nent 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 im-purity A was 0.02-0.80 μg·mL-1,with r=0.999 9.The linear range of impurity B was 0.02-0.80μg·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 advanta-ges of being easy to operate,providing rapid and accurate results,and providing technical assurance for the quali-ty control of fat-soluble vitamin D3 preparations.
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