Figure 8.11 Schematic representation of the Hummel-Dreyer capillary zone electro-phoretic method. Zone identification: 1, drug; 2, bovine serum albumin; 3, buffer; 4, BSA-drug complex. (From Kraak et al., 1992, with permission.)
absorb drug molecules again. Reabsorption continues to equilibrium (i.e., the drug is reconcentrated to its original level). From this moment, a steady state is reached and two negative bands appear on the electropherogram; the first corresponds to the bound drug, the second represents the free drug zone (Fig. 8.12).
The last approach, also applicable to CZE, is analogous to frontal analysis. Here the capillary at the beginning of the experiment is filled with the background electrolyte only, and a very large sample drug containing buffer, protein, and the drug is injected into the capillary. Because of the differences in electrophoretic mobility between the drug and the protein, the free drug starts to leak out at the rear edge of the plug. A plateau corresponding to the free drug concentration is formed. At the front edge of the plug another plateau is created, representing the free protein. Thus, the result of this analysis is a three-step profile in which the free protein plateau is followed by the zone of protein-drug complex and the profile is terminated by a plateau corresponding to free drug. The height of the drug plateau is taken as a measure of free drug concentration. As stressed by Kraak et al. (1992), depending on the detection wavelength used, the first plateau is frequently not seen by the detector (Fig. 8.13).
Figure 8.12 Schematic representation of the vacancy peak capillary zone electrophoresis method. Zone identification as in Figure 8.11. (From Kraak et al., 1992, with permission.)
Thus the three approaches to drug binding assays are represented according to the schemes in Figures 8.11 to 8.13. Kraak et al. (1992), testing binding of warfarin by bovine serum albumin, used 0.067 potassium phosphate buffer at pH 7.4 as background electrolyte. For the practical evaluation of data by the Hummel-Dreyer method, the simplified approach presented by Pinkerton and Koeplinger (1990) was applied. In this approach two injections at a given warfarin concentration were applied: the sample consisting of buffer and protein and the blank buffer. From the peak areas the bound drug [Db] concentration can be calculated.
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