Yoshii Tomoka, Sakama Akihiro, Kanamori Kazuyoshi, Nakanishi Kazuki, Imai Hiroaki, Citterio Daniel, Hiruta Yuki
Department of Applied Chemistry, Faculty of Science and Technology, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
J Chromatogr A. 2023 Jan 11;1688:463722. doi: 10.1016/j.chroma.2022.463722. Epub 2022 Dec 16.
Purification of basic drugs in reversed-phase mode is often difficult, mainly due to adsorption of positively charged compounds to the silica gel-based stationary phase. Since this adsorption can be suppressed under alkaline condition, columns with alkali-resistance are required. In addition, compounds with acid-sensitive structures are sometimes degraded during separation on silica gel-based columns which exhibit acidity due to their surface structure. We prepared an alkali-resistant reversed-phase packing material, Eggshell-PMAcO based on eggshells modified with an amphiphilic copolymer, poly(maleic acid-alt-1-octadecene) (PMAcO). The height equivalent to a theoretical plate (HETP) of the Eggshell-PMAcO column was improved by surface treatment with ammonium acetate buffer (900 mM, pH = 3.7), which is an inexpensive reagent, and the retention behavior for hydrophobic compounds was compared to a typical ODS column based on silica gel, resulting in sufficient selectivity of the eggshell-based column for hydrophobic compounds, as indicated by the ratio of retention factors of pentylbenzene and butylbenzene (Eggshell-PMAcO column: 1.55, ODS column: 1.65). Column temperature-dependent retention behavior of naphthalene was investigated in the temperature range from 25 °C to 45 °C, followed by the calculation of thermodynamic parameters. There was little difference in the standard molar enthalpy (Eggshell-PMAcO: -19.6 kJ/mol, ODS: -21.7 kJ/mol). The absolute value of the standard free Gibbs energy for the Eggshell-PMAcO column was much smaller than that of the ODS column (Eggshell-PMAcO: -0.284 kJ/mol, ODS: -13.0 kJ/mol), indicating that the Eggshell-PMAcO column had a weaker retention strength for naphthalene than the ODS column mainly due to the large difference in the standard molar entropy (Eggshell-PMAcO: -64.9 J/mol K, ODS column: -29.2 J/mol K). The retention capacities for imipramine under neutral (water/methanol) and alkaline (0.1% triethylamine water/methanol) conditions were 0.2 mg and 5 mg, respectively, based on injection mass-dependent HETP, retention factor and symmetry factor. Finally, the prepared column was applied to the purification of a building block for nucleic acid drugs. This study demonstrated that reversed-phase columns, which can be fabricated from eggshells and an amphiphilic copolymer in an inexpensive and eco-friendly way, have the ability to purify basic compounds and acid-sensitive compounds.
在反相模式下纯化碱性药物通常很困难，主要是由于带正电荷的化合物会吸附到硅胶基固定相上。由于这种吸附在碱性条件下可以被抑制，因此需要使用耐碱的色谱柱。此外，具有酸敏感结构的化合物有时会在基于硅胶的色谱柱上分离时发生降解，因为这些色谱柱由于其表面结构而呈现酸性。我们制备了一种耐碱反相填充材料，即基于用两亲共聚物聚（马来酸-alt-1-十八烯）（PMAcO）改性的蛋壳的蛋壳-PMAcO。用乙酸铵缓冲液（900 mM，pH = 3.7）进行表面处理可改善蛋壳-PMAcO色谱柱的理论塔板高度（HETP），乙酸铵缓冲液是一种廉价试剂，并且将其对疏水性化合物的保留行为与基于硅胶的典型ODS色谱柱进行了比较，结果表明基于蛋壳的色谱柱对疏水性化合物具有足够的选择性，以戊基苯和丁基苯的保留因子之比表示（蛋壳-PMAcO色谱柱：1.55，ODS色谱柱：1.65）。在25℃至45℃的温度范围内研究了萘的柱温依赖性保留行为，随后计算了热力学参数。标准摩尔焓几乎没有差异（蛋壳-PMAcO：-19.6 kJ/mol，ODS：-21.7 kJ/mol）。蛋壳-PMAcO色谱柱的标准自由吉布斯能的绝对值远小于ODS色谱柱（蛋壳-PMAcO：-0.284 kJ/mol，ODS：-13.0 kJ/mol），这表明蛋壳-PMAcO色谱柱对萘的保留强度比ODS色谱柱弱，主要是由于标准摩尔熵存在较大差异（蛋壳-PMAcO：-64.9 J/mol K，ODS色谱柱：-29.2 J/mol K）。基于进样质量相关的HETP、保留因子和对称因子，在中性（水/甲醇）和碱性（0.1%三乙胺水/甲醇）条件下，丙咪嗪的保留容量分别为0.2 mg和5 mg。最后，将制备的色谱柱应用于核酸药物的一个结构单元的纯化。这项研究表明，反相色谱柱可以以廉价且环保的方式由蛋壳和两亲共聚物制成，具有纯化碱性化合物和酸敏感化合物的能力。
