Department of Pharmaceutics, Kobe Pharmaceutical University.
Chem Pharm Bull (Tokyo). 2020;68(12):1233-1237. doi: 10.1248/cpb.c20-00448.
The aim of this study was to investigate appropriate analytical conditions for hydrophilic nucleosides and nucleotides (monophosphates and triphosphates) by HPLC methods using a mixed-mode AX-C18 column with anion-exchange and hydrophobic interactions by quaternary ammonium and C18, respectively, and a reversed-phase pentabromobenzyl (PBr) column with dispersion force and hydrophobic interactions by PBr group. The higher compound polarity led to stronger retention on AX-C18 (triphosphates > monophosphates > nucleosides). AX-C18 demonstrated feasible retention of nucleotides via anion-exchange interaction by increasing the salt and methanol concentrations. In contrast, on PBr, the lower compound polarity led to stronger retention. On PBr, feasible retention of both nucleosides and nucleotides was obtained via dispersion interactions with purine and pyrimidine rings by increasing the methanol concentration. Regarding the pH of phosphate buffer used as the mobile phase, pH 7.0 should be used in measuring nucleoside triphosphates on AX-C18, whereas pH 2.5 is better suited for measuring nucleotides on PBr. In terms of selectivity to highly hydrophilic nucleotides, the mixed-mode AX-C18 column had an advantage over the reverse-phase PBr column. In contrast, PBr column was more versatile than the AX-C18 column. Taken together, HPLC analyses of nucleosides and nucleotides should be carried out by optimizing the interactions between the stationary phase and nucleic acids.
本研究旨在通过 HPLC 方法，使用混合模式 AX-C18 柱，通过季铵和 C18 分别实现阴离子交换和疏水相互作用，以及使用反相 pentabromobenzyl（PBr）柱，通过 PBr 基团实现分散力和疏水相互作用，来研究亲水性核苷和核苷酸（单磷酸和三磷酸）的合适分析条件。化合物的极性越高，在 AX-C18 上的保留越强（三磷酸 > 单磷酸 > 核苷）。通过增加盐和甲醇浓度，AX-C18 可以通过阴离子交换相互作用实现对核苷酸的可行保留。相比之下，在 PBr 上，化合物的极性越低，保留越强。通过增加甲醇浓度，在 PBr 上可以通过嘌呤和嘧啶环与分散相互作用实现对核苷和核苷酸的可行保留。关于用作流动相的磷酸盐缓冲液的 pH 值，AX-C18 上测量核苷三磷酸时应使用 pH 7.0，而 PBr 上测量核苷酸时 pH 2.5 更合适。就高度亲水核苷酸的选择性而言，混合模式 AX-C18 柱优于反相 PBr 柱。相比之下，PBr 柱比 AX-C18 柱更通用。综上所述，应对固定相和核酸之间的相互作用进行优化，以进行核苷和核苷酸的 HPLC 分析。
