Department of Chemistry, University of Nebraska-Lincoln, United States.
Department of Biological Systems Engineering, University of Nebraska-Lincoln, United States.
J Chromatogr A. 2023 Sep 27;1707:464307. doi: 10.1016/j.chroma.2023.464307. Epub 2023 Aug 18.
Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.
超快速亲和萃取 (UAE) 是一种微尺度亲和高效液相色谱法，可用于快速测量溶液中溶质结合剂相互作用的平衡常数。本研究利用色谱和平衡理论的通用图，考察了在 UAE 中获得此类相互作用的平衡常数的准确、精确和稳健测量所需的一般条件。预测结果与通过 UAE 在研究中获得的结果进行了比较，这些研究检查了各种药物与两种转运蛋白（人血清白蛋白和α-酸性糖蛋白）的结合情况。对于溶质平衡自由分数为中等值（F ≈ 0.20-0.80）的系统，这些结合研究的最精确和最稳健的条件。这些趋势与人血清白蛋白与华法林和格列齐特结合的 UAE 先前研究中观察到的趋势非常吻合。进一步确定了溶质的表观自由分数如何与溶质的离解速率、在 UAE 中允许溶质离解的时间以及溶质的平衡自由分数有关。这些结果也与华法林和格列齐特与人血清白蛋白结合的实验结果一致。最后一部分检查了由于溶质离解导致的表观自由分数的变化如何影响 UAE 测量的平衡常数的准确性。此外，还生成了理论图，以便在 UAE 中选择可在测量平衡常数时提供给定准确度的条件。为 UAE 创建的方程和确定的趋势是通用的，可以在未来的工作中扩展到其他溶质和结合剂。
