Fayed Ahmed Salah, Hegazy Maha Abdel-Monem, Wahab Nada Sayed Abdel
Cairo University Faculty of Pharmacy, Analytical Chemistry Department, Kasr El-Aini Street, Cairo, Egypt 11562.
Beni Suef University, Faculty of Pharmacy, Analytical Chemistry Department, Alshahed Shehata Ahmed Hegazy St, Beni Suef, Egypt 62514.
J AOAC Int. 2016 Nov 1;99(6):1513-1521. doi: 10.5740/jaoacint.16-0023. Epub 2016 Oct 1.
New, simple, highly sensitive, precise, and accurate gradient reversed-phase chromatographic methods were developed using HPLC and ultra-HPLC (UPLC) systems for the determination of five components, namely thiamine, pyridoxine, cyanocobalamin, benfotiamine, and diclofenac in tablets and capsules. The methods were compared for their efficiency in the separation and determination of these five compounds using two different C18 columns (250 × 4.6 mm, 5 μm; and 100 × 4.6 mm, 2.6 μm) for HPLC and UPLC, respectively. Chromatographic separation was performed with a mobile phase containing acetonitrile and 0.025 M phosphate buffer (pH 3.5), with a gradient program and a flow rate of 1.5 and 1.0 mL/min for both methods, respectively. The methods were validated according to International Conference on Harmonization guidelines. Linearity was achieved in the range of 5.00 to 150.00 μg/mL for each of the five compounds. Ruggedness and intermediate precision were confirmed by different analysts on different columns on different days. Moreover, the components were subjected to an accelerated stability study under acidic, alkaline, and oxidative stress conditions and no interfering peaks were observed. The five compounds were efficiently separated in <20 min by HPLC, whereas for UPLC, separation was achieved in <8 min, which dramatically decreased the consumption of organic solvents.
采用高效液相色谱（HPLC）和超高效液相色谱（UPLC）系统，开发了新的、简单的、高灵敏度、精确且准确的梯度反相色谱方法，用于测定片剂和胶囊中的五种成分，即硫胺素、吡哆醇、氰钴胺、苯磷硫胺和双氯芬酸。使用两根不同的C18色谱柱（分别为250×4.6 mm，5μm；和100×4.6 mm，2.6μm），分别用于HPLC和UPLC，比较了这两种方法在分离和测定这五种化合物方面的效率。色谱分离采用含有乙腈和0.025 M磷酸盐缓冲液（pH 3.5）的流动相，两种方法均采用梯度程序，流速分别为1.5和1.0 mL/min。这些方法根据国际协调会议指南进行了验证。五种化合物在5.00至150.00μg/mL范围内均实现了线性关系。不同分析人员在不同日期使用不同色谱柱确认了耐用性和中间精密度。此外，对这些成分在酸性、碱性和氧化应激条件下进行了加速稳定性研究，未观察到干扰峰。通过HPLC可在<20分钟内有效分离这五种化合物，而对于UPLC，在<8分钟内即可实现分离，这大大减少了有机溶剂的消耗。
