Development of High-Performance Liquid Chromatographic Method for Vitamin D3 Analysis in Pharmaceutical Preparation

Enas Al-Qadi, Abdelkader Battah, Kamal Hadidi


In the present study, a rapid, simple and economical reversed phase-HPLC procedure has been developed for the determination of vitamin D3 and vitamin D2 in pharmaceutical preparations. The vitamins were separated isocratically on a Teknokroma C18 column (150×4.6mm, 3.5µm particle size) with a mobile phase consisting of isocratic acetonitrile and methanol (75:25, v/v) operated at 40ºC with retention times less than 12 min. The eluted vitamins were identified and monitored on a diode-array detector (DAD) at 265nm. The linearity of the method was excellent (r2 > 0.999), over the concentration range of 0-10µg/ml. The statistical evaluation of the method was examined performing intra-day and inter-day calibrations and were found to be satisfactory, with 2.600 ± 0.078% and 6.267 ± 0.050% accuracy and 0.001 ± 0.002% to 2.144 ± 0.050% precision results. Mean recovery of vitamin D3 was 98.2 ± 0.349% for different spiking levels. Detection limit was found to be 25ng/ml. No interference was found from other fat soluble vitamins (vitamin A, vitamin E and vitamin E acetate) that are commonly presents with vitamin D pharmaceutical preparation. The method was applied for the analysis of sixteen solid samples of pharmaceutical preparation (calcium with vitamin D). The results show only 31.25% of the tested samples give satisfactory agreement with the declared values.


Vit. D, HPLC, DAD and Pharmaceutical Preparation

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