Dobó Máté, Dombi Gergely, Köteles István, Fiser Béla, Kis Csenge, Szabó Zoltán-István, Tóth Gergő
Department of Pharmaceutical Chemistry, Semmelweis University, Hogyes 9, H-1092 Budapest, Hungary.
Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 19, 41390 Göteborg, Sweden.
Int J Mol Sci. 2024 Feb 26;25(5):2697. doi: 10.3390/ijms25052697.
A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (//) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors' knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.
建立了一种反相高效液相色谱（HPLC）法，用于同时测定右酮洛芬的潜在杂质，包括异构体R-酮洛芬。在筛选了四种多糖柱（Lux直链淀粉-1、Lux直链淀粉-2、Lux纤维素-1和Lux纤维素-2）在极性有机模式和反相模式下的分离能力后，仅在Lux直链淀粉-2柱上，于酸化的乙腈/水混合物中观察到了适当的对映体分离。对流动相组成和温度对对映选择性和化学选择性进行的详细研究显示了许多意外发现。结果表明，通过改变温度和流动相组成，可以微调分离度和对映体洗脱顺序。此外，在直链淀粉型柱上使用甲醇/水混合物的反相模式下，还观察到了保留时间和对映选择性的滞后现象。这可能表明直链淀粉柱的三维结构会因从极性有机模式转变为反相模式而发生变化，从而影响对映体分离过程。在反相模式下还观察到了温度依赖性对映体洗脱顺序以及在操作温度范围内罕见的焓/熵控制对映体分离。为了找到测定右酮洛芬杂质的最佳方法条件，进行了全因子优化设计。使用优化参数（Lux直链淀粉-2柱，水/乙腈/乙酸为50/50/0.1（v/v/v），流速为1 mL/min，温度为20℃），在15分钟内实现了所有化合物之间的基线分离。我们新开发的HPLC方法按照现行指南进行了验证，并在市售药物制剂上进行了应用测试。据作者所知，这是首次报道多糖柱在反相模式下的滞后行为的研究。
