Zheng Xiwei, Matsuda Ryan, Hage David S
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
J Chromatogr A. 2014 Dec 5;1371:82-9. doi: 10.1016/j.chroma.2014.10.092. Epub 2014 Oct 31.
Ultrafast affinity extraction and a multi-dimensional affinity system were developed for measuring free drug fractions at therapeutic levels. This approach was used to compare the free fractions and global affinity constants of several sulfonylurea drugs in the presence of normal human serum albumin (HSA) or glycated forms of this protein, as are produced during diabetes. Affinity microcolumns containing immobilized HSA were first used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the HSA microcolumn, it was passed through a second HSA column for further separation and measurement. Items that were considered during the optimization of this approach included the column sizes and flow rates that were used, and the time at which the second column was placed on-line with the HSA microcolumn. This method required only 1.0 μL of a sample per injection and was able to measure free drug fractions as small as 0.09-2.58% with an absolute precision of ±0.02-0.5%. The results that were obtained indicated that glycation can affect the free fractions of sulfonylurea drugs at typical therapeutic levels and that the size of this effect varies with the level of HSA glycation. Global affinity constants that were estimated from these free drug fractions gave good agreement with those predicted from previous binding studies or determined through a reference method. The same approach could be utilized with other drugs and proteins or modified binding agents of clinical or pharmaceutical interest.
开发了超快速亲和萃取和多维亲和系统，用于测量治疗水平下的游离药物分数。该方法用于比较几种磺酰脲类药物在正常人血清白蛋白（HSA）或糖尿病期间产生的该蛋白糖化形式存在下的游离分数和整体亲和常数。首先使用含有固定化HSA的亲和微柱从注射的药物/蛋白质混合物中提取游离药物分数。当保留的药物从HSA微柱洗脱时，它通过第二个HSA柱进行进一步分离和测量。在优化该方法时考虑的因素包括所使用的柱尺寸和流速，以及第二个柱与HSA微柱联机的时间。该方法每次进样仅需1.0 μL样品，能够测量低至0.09 - 2.58%的游离药物分数，绝对精度为±0.02 - 0.5%。获得的结果表明，糖化会影响典型治疗水平下磺酰脲类药物的游离分数，且这种影响的大小随HSA糖化水平而变化。从这些游离药物分数估计的整体亲和常数与先前结合研究预测的或通过参考方法确定的结果高度一致。同样的方法可用于其他药物和蛋白质或具有临床或药学意义的修饰结合剂。
