Mammone Francesca Romana, Sadutto Daniele, Antoniella Eleonora, Pierini Marco, Cirilli Roberto
Centre for the Control and Evaluation of Medicines, Chemical Medicines Unit, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Department of Drug Chemistry and Technology, "Sapienza" University of Rome, 00185 Rome, Italy.
Molecules. 2025 Feb 28;30(5):1108. doi: 10.3390/molecules30051108.
Midazolam is a benzodiazepine that is utilized for the induction of anesthesia and the facilitation of procedural sedation. Despite the absence of stereogenic centers, the non-planar seven-membered ring devoid of reflection symmetry elements confers planar stereogenicity to the molecule. Due to the rapid conformational inversion of the and enantiomers, which occurs via a simple ring flip, high-performance liquid chromatography (HPLC) enantiomeric separation is restricted to sub-room temperature conditions. In this study, the energy barriers for the racemization of midazolam at five distinct temperatures and in acetonitrile/water mixtures were determined by monitoring the decay of the circular dichroism signal at a specific wavelength over time. The kinetic and thermodynamic data obtained were compared with those determined by dynamic enantioselective high-performance liquid chromatography using the Chiralpak IG-3 chiral stationary phase, which contains the amylose tris(3-chloro-5-methylphenylcarbamate) as the selector. The temperature-dependent dynamic HPLC of midazolam was carried out at the same temperatures and with the same aqueous mixtures used in parallel kinetic off-column experiments. To simulate dynamic chromatographic profiles, a lab-made computer program based on a stochastic model was utilized. The results indicated that the moderate influence of the stationary phase resulted in a slight increase in the activation barriers, which was more pronounced as the time spent in the column increased. This phenomenon was found to be mitigated when switching from a 250 mm × 4.6 mm, 3 µm, Chiralpak IG-3 column to a 50 mm × 4.6 mm, 1.6 µm, Chiralpak IG-U UHPLC column. The outcomes obtained under UHPLC conditions were found to be more closely aligned with those obtained through the ECD technique, with a discrepancy of only 0.1 kcal/mol or less, indicating a high degree of concordance between the two methods.
咪达唑仑是一种苯二氮䓬类药物，用于诱导麻醉和辅助程序镇静。尽管该分子没有手性中心，但缺乏镜像对称元素的非平面七元环赋予了分子平面手性。由于通过简单的环翻转发生的R和S对映体的快速构象反转，高效液相色谱（HPLC）对映体分离仅限于低于室温的条件。在本研究中，通过监测特定波长下圆二色性信号随时间的衰减，测定了咪达唑仑在五个不同温度下以及在乙腈/水混合物中的外消旋化能垒。将获得的动力学和热力学数据与使用Chiralpak IG-3手性固定相通过动态对映选择性高效液相色谱法测定的数据进行比较，该固定相包含直链淀粉三（3-氯-5-甲基苯基氨基甲酸酯）作为选择剂。在与平行动力学柱外实验相同的温度和相同的水性混合物条件下进行咪达唑仑的温度依赖性动态HPLC。为了模拟动态色谱图，使用了基于随机模型的自制计算机程序。结果表明，固定相的适度影响导致活化能垒略有增加，随着在柱中停留时间的增加，这种现象更加明显。当从250 mm×4.6 mm、3 µm的Chiralpak IG-3柱切换到50 mm×4.6 mm、1.6 µm的Chiralpak IG-U超高效液相色谱柱时，发现这种现象得到缓解。发现在超高效液相色谱条件下获得的结果与通过ECD技术获得的结果更接近，差异仅为0.1 kcal/mol或更小，表明两种方法具有高度一致性。
