Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
J Pharm Biomed Anal. 2013 Mar 5;75:112-7. doi: 10.1016/j.jpba.2012.11.018. Epub 2012 Nov 21.
The aim of this study is to further validate the use of ultrafiltration (UF) as a method for determining plasma protein binding (PPB) by demonstrating that non-specific binding (NSB) is not a limitation, even for highly lipophilic compounds, because NSB sites on the apparatus are passivated in the presence of plasma. Mass balance theory was used to calculate recovery of 20 commercial and seven investigational compounds during ultrafiltration in the presence and absence of plasma. PPB was also measured using this mass balance approach for comparison to PPB determined by rapid equilibrium dialysis (RED) and as found in the literature. Compound recovery during UF was dramatically different in the presence and absence of plasma for compounds with high NSB in PBS only. A comparison of PPB calculated by ultrafiltration with literature values or calculated by RED gave concordant results. Discrepancies could be explained by changes in pH, insufficient time to equilibrium, or compound instability during RED, problems which were circumvented by ultrafiltration. Therefore, NSB, as measured by the traditional incubation of compound in PBS, need not be an issue when choosing UF as a PPB assay method. It is more appropriate to calculate compound recovery from the device in plasma as measured by mass balance to determine the suitability of the method for an individual compound. The speed with which UF can be conducted additionally avoids changes in pH or compound loss that can occur with other methods. The mass balance approach to UF is thus a preferred method for rapid determination of PPB.
本研究旨在进一步验证超滤（UF）作为一种测定血浆蛋白结合率（PPB）的方法，方法是证明非特异性结合（NSB）不是限制因素，即使是对高度亲脂性化合物也是如此，因为在存在血浆的情况下，仪器上的 NSB 位点被钝化。使用质量平衡理论计算了 20 种商业化合物和 7 种研究化合物在存在和不存在血浆的情况下超滤过程中的回收率。还使用这种质量平衡方法测定了 PPB，并与通过快速平衡透析（RED）测定的 PPB 进行了比较，以及与文献中的值进行了比较。对于仅在 PBS 中具有高 NSB 的化合物，在存在和不存在血浆的情况下，UF 过程中的化合物回收率差异很大。超滤法计算的 PPB 与文献值或 RED 计算的 PPB 进行比较，结果一致。差异可以用 pH 值的变化、平衡时间不足或 RED 过程中化合物的不稳定性来解释，这些问题通过超滤得到了避免。因此，当选择 UF 作为 PPB 测定方法时，通过传统的在 PBS 中孵育化合物来测量的 NSB 不必成为问题。更适合的方法是根据质量平衡从血浆中测定设备中的化合物回收率，以确定该方法对单个化合物的适用性。UF 进行的速度还可以避免其他方法中可能发生的 pH 值变化或化合物损失。因此，质量平衡法 UF 是快速测定 PPB 的首选方法。
