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| Simultaneous Determination of Vitamins A,D in Vitamin A and D Pharmaceutical by Online Two-dimensional Reverse-phase Liquid Chromatography |
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| DOI： |
| KeyWord:two-dimensional liquid chromatography（2D-LC） online separation vitamin D vitamin A pre-vitamin D vitamin A and D drops |
| Author | Institution |
| ZHANG Yan-hai，ZHANG Da-wei，CAO Ying，JIN Yan* |
1.赛默飞世尔科技（中国）有限公司;2.黑龙江农垦总医院;3.上海市兽药饲料检测所 |
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| Abstract: |
| A rapid method for the simultaneous determination of fat-soluble vitamins A and D in vitamin A and D pharmaceutical was developed using online two-dimensional liquid chromatography（2D-LC）.A type of polar embedded C18 was selected as the second dimension separating column according to system suitability testing requirement.C8 was chosen as first dimension separating column because fat-soluble substance was rich in matrix and a better orthogonality could meet with polar embedded C18 according to hydrophobic subtraction model.Sample solution after extracted with methanol without further purification was injected into liquid chromatography directly.The purification of vitamin D and quantification of vitamins A acetate were accomplished simultaneously in one dimensional separation using the right pump of Dual Gradient LC（DGLC） with methanol-acetonitrile-water as mobile phase.Vitamin D and pre-vitamin D were separated and quantified in two dimensional column by using the left pump of DGLC.The eluted target from the first dimensional column（1-D column） was collected by a 500 μL loop and then taken into the second dimensional column（2-D column） by the left pump of DGLC with high aqueous mobile phase.The cutting window could be confirmed by the retention time of target in one dimensional separation.Detection wavelengths of 325 nm and 264 nm were set at the diode-array detector.Vitamin A,vitamin D and pre vitamin D were quantified simultaneously and automatically in one injection by the external standard method.The results showed that the good linearities（r>0.999） for vitamin A and vitamin D could be obtained in the range of 0.1-250mg/L and 0.02-50.0mg/L,respectively.The recoveries of vitamin D ranged from 89.9% to 98.9%.The RSDs for precision and reproducibility are 0.48% and 1.2%,respectively.All the validating results indicated that vitamins A and D in pharmaceutical could be quantified rapidly and accurately by this method. |
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