《Tab 1 Effect of mobile phase composition on various chromatographic parameters》

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《反相高效液相色谱(RP-HPLC)系统分析维生素D3软胶囊(油性基质)（英文）》

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RT:Retention time；α:Separation factor；k’:Capacity factor.

HPLC parameters of altering mobile phase were listed in Tab 1.Chromatograms were shown in Fig 4.As shown in Fig 4，the retention time gradually decreased as the proportion of ethanol or methanol to acetonitrile increased.Meanwhile，little addition of methanol and ethanol（1%）resulted in great resolution reduction between peak 2（pre-vitamin D3）and peak 3（tachysterol）.When proportion of either methanol or ethanol up to 10%，peak 2 and 3 merged together totally into one peak（peak 2+3）.Continuing up to 20%，peak 2，3 and 4 merged together into one peak（peak2+3+4）.Addition of water in acetonitrile greatly increased retention time.Meanwhile，it also resulted in resolution reduction between peak 2 and peak 3.Peak tailing in Fig 4C-a and peak fronting of peak 2+3 in Fig4C-b could be observed clearly.Continuing adding water until 15%，4 peaks were separated（Fig 4C-c）.The elution order changed from peak 1，2，3，4to peak 1，3，2，4.This revealed that the relative position of tachysterol changed gradually from the back to the front of pre-vitamin D3 as the proportion of water increased，further showed the apparent different elution rate change among different peaks especially between peak 2 and 3.Unfortunately，the retention time was so long that it was not available in analysis.Using 100%ACNas mobile phase achieved the best separation and appropriate retention time（Fig 2C）.