Department of Chemistry, University of Nebraska-Lincoln, NE 68588-0304, USA.
Department of Chemistry, University of Nebraska-Lincoln, NE 68588-0304, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Dec 15;1187:123047. doi: 10.1016/j.jchromb.2021.123047. Epub 2021 Nov 17.
Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18-22% over 100-150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.
超快速亲和萃取 (UAE) 最近已被开发并用于测量具有可溶性结合剂（如血清蛋白）的药物和其他靶标中的非结合（或游离）分数和结合或速率常数。本研究考察了在低和高流速（例如，0.5 和 3.5 mL/min）下使用 10 mm×2.1 mm i.d. 亲和微柱进行 UAE 时，微柱的长期稳定性，在一系列延长的注射中进行了这项稳定性研究。该稳定性使用固定化人血清白蛋白 (HSA) 和含有药物华法林的样品进行了研究，这些样品含有或不含有可溶性 HSA 作为模型系统。在存在可溶性 HSA 的情况下，在 0.5 mL/min 下测量的游离华法林分数在 150 次注射内稳定，在 3.5 mL/min 下变化 <10%。通过 3.5 mL/min 的 UAE 估算的华法林与 HSA 的缔合平衡常数在 50 次注射内没有明显变化，在 100-150 次注射内仅变化约 18-22%。通过结合 0.5 和 3.5 mL/min 的 UAE 结果并采用新的两点法，发现了华法林从 HSA 上的离解速率常数，即使在 200 次注射后，该值也没有明显变化。还考虑了延长微柱使用对华法林的保留时间、峰宽和峰不对称性以及微柱的背压的影响。这些结果表明，UAE 和 HSA 微柱可用于在大量注射中提供游离溶质分数、结合常数和速率常数的一致值。这些结果将有助于未来的工作，为评估、进一步开发和使用 UAE 来表征基于溶液的样品中涉及其他药物和结合剂的相互作用提供指导。
