DataSheet1_Simultaneous effect of different chromatographic conditions on the chromatographic retention of pentapeptide derivatives (HGRFG and NPNPT).DOCX

Introduction: Oligopeptides exhibit great prospects for clinical application and its separation is of great importance in new drug development.Methods: To accurately predict the retention of pentapeptides with analogous structures in chromatography, the retention times of 57 pentapeptide derivatives in seven buffers at three temperatures and four mobile phase compositions were measured via reversed-phase high-performance liquid chromatography. The parameters (kHA, kA, and pKa) of the acid–base equilibrium were obtained by fitting the data corresponding to a sigmoidal function. We then studied the dependence of these parameters on the temperature (T), organic modifier composition (φ, methanol volume fraction), and polarity (PmN parameter). Finally, we proposed two six-parameter models with (1) pH and T and (2) pH and φ or PmN as the independent variables. These models were validated for their prediction capacities by linearly fitting the predicted retention factor k-value and the experimental k-value.Results: The results showed that logkHA and logkA exhibited linear relationships with 1/T, φ or PmN for all pentapeptides, especially for the acid pentapeptides. In the model of pH and T, the correlation coefficient (R2) of the acid pentapeptides was 0.8603, suggesting a certain prediction capability of chromatographic retention. Moreover, in the model of pH and φ or PmN, the R2 values of the acid and neutral pentapeptides were greater than 0.93, and the average root mean squared error was approximately 0.3, indicating that the k-values could be effectively predicted.Discussion: In summary, the two six-parameter models were appropriate to characterize the chromatographic retention of amphoteric compounds, especially the acid or neutral pentapeptides, and could predict the chromatographic retention of pentapeptide compounds.

引言：寡肽在临床应用方面展现出巨大的潜力，其在新药研发中的分离过程至关重要。方法：为准确预测具有相似结构的五肽在色谱中的保留时间，通过反相高效液相色谱法测量了57种五肽衍生物在七种缓冲液、三种温度和四种流动相组成下的保留时间。通过拟合S形函数对应的数据，获得了酸碱平衡参数（kHA、kA和pKa）。随后，我们研究了这些参数与温度（T）、有机改性剂组成（φ，甲醇体积分数）和极性（PmN参数）之间的关系。最终，我们提出了两个六参数模型，其中（1）pH和T以及（2）pH和φ或PmN作为自变量。通过线性拟合预测的保留因子k值和实验k值来验证这些模型在预测能力上的有效性。结果：结果表明，对所有五肽而言，logkHA和logkA与1/T、φ或PmN呈线性关系，尤其是在酸性五肽中。在pH和T的模型中，酸性五肽的相关系数（R2）为0.8603，表明色谱保留具有一定的预测能力。此外，在pH和φ或PmN的模型中，酸性和中性五肽的R2值均大于0.93，平均均方根误差约为0.3，表明k值可以有效地预测。讨论：总之，两个六参数模型适用于表征两性化合物的色谱保留，特别是酸或中性五肽，并能够预测五肽化合物的色谱保留。