HPLC Method Development and Validation for Simultaneous Quantification of Vitamins C and K3 in Hard Gelatin Capsules for their Potential Benefit in Postoperative Total Joint Arthroplasty
Yousif Rojeab, Mohamad Hassoun, Ellen
Hazelet, Deirdre Myers
Correspondence Address :
of Pharmaceutical and Biomedical Sciences
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Received on: August 21, 2017, Accepted on: September 18, 2017, Published on: October 06, 2017
Citation: Yousif Rojeab, Mohamad Hassoun, Ellen Hazelet, Deirdre Myers (2017). HPLC Method Development and Validation for Simultaneous Quantification of Vitamins C and K3 in Hard Gelatin Capsules for their Potential Benefit in Postoperative Total Joint Arthroplasty
Copyright: 2017 Yousif Rojeab, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Efficient, selective, rugged, simple and sensitive isocratic RP-HPLC method for simultaneous quantification of vitamins C and K3 was developed and validated. This method consisted of UV-detection at 254 nm and separation by a reversed-phase (RP)-C18 column. Mobile phase was composed of 50% methanol, 49% water and 1% glacial acetic acid, at flow rate of 1 mL/min. Injection volume (100 μL) consisted of varied concentrations of both vitamins (0.5 - 50 μg/mL) mixed with vitamin E (internal standard) in 1:1:2 volume ratio. Clear baseline resolution was achieved for all three compounds with retention times of 1.9, 3.3 and 4.3 min for vitamins C, K3 and E, respectively. The method exhibited excellent linearity over the entire concentration range for both vitamins with R2 of ≥ 0.9991. Intraday (n = 6) accuracy ranged from 92.2 - 102.0% and 99.4 - 106.7% for vitamins C and K3, respectively, while those for the inter-day assays (n = 12) ranged from 96.9 - 99.1% and 91.7 - 100.4%. This HPLC method was successfully implemented in quantification of vitamins C and K3 in compounded capsules containing the two ingredients to ensure content uniformity. For vitamin C, content was found to be 101 +/- 4% (mean +/- standard deviation) of the label claim of 500 mg. As for vitamin K3, content was found to be 87 +/- 6% of the label claim of 5 mg. These capsules were subsequently administered orally in a clinical trial aimed at evaluating any beneficial effect(s) of this vitamin mixture on postoperative total joint arthroplasty.
Keywords: UV-HPLC, Isocratic, Vitamin C, Vitamin K3, Simultaneous quantification