# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 4 Chromatography in the Determination of Pharmacokine Parameters of Substances
### Determination of the Distribution of Substances in Tissues
#### 4.1.3 Determination of \(K_{d}\) Values against a Standard  
Another method for determining the \(K_{D}\) value is to determine the receptor dosage constant based on the retention time of the substance. The method for determining the \(K_{D}\) value in relation to a standard is to determine the value of the retention factor for an \(S\) standard with a known \(K_{DS}\):  
\[\frac{k_{x}}{k_{s}}=\frac{K_{Dx}}{K_{Ds}}\quad K_{Dx}=\frac{k_{x}}{k_{s}}K_{Ds} \tag{9}\]  
where \(k_{x}\) and \(k_{s}\) represent the retention coefficient of the investigated substance and standard, respectively; \(K_{DX}\) and \(K_{DS}\) represent the \(K_{D}\) value of the investigated substance and the standard, respectively.  
This is a practical method that requires much smaller substitution amounts than the frontal analysis method so that \(K_{D}\) values for several substances can be determined from a single standard. [PERSON] et al. investigated the affinity of different ligands for \(\alpha\)1-AR and three subtypes of this receptor in stationary-phase CMC systems with cell membranes with high expression of \(\alpha\)1A-adrenooreceptor (AR), \(\alpha\)1B-AR, and \(\alpha\)1D-AR. They ranked the test substances according to increasing affinity for the receptors studied and obtained results that were consistent with those found in vivo in the literature [81]. On the other hand, in stationary phase, [PERSON] et al. showed CMC systems based on guinea pig cardiac muscle and jejunum cells as well as cardiac muscle, jejunum, and rat brain, and the results obtained were in agreement with those of the frontal analysis.  
1.4 Chromatography as an Analytical Method for the Determination of Interactions of Substances with Protein  
Another application of chromatographic methods to determine the bound form of a drug is their use as methods of quantitative analysis. The percentage of the bound  
Figure 6: Determination of \(K_{D}\) values from Equation (8).  
form is determined in a sample of buffer solution (pH = 7.4), test substance (at a known concentration), and test protein (at a known concentration). In such a solution, the test substance is adsorbed onto the protein molecules. The protein must then be removed from the solution, usually by adsorption onto suitable adsorbents. The deproteinised sample prepared in this way is subjected to quantitative analysis in a chromatographic system that is adapted to the type of drug.  
Another way to determine the percentage of the bound form, which allows the tedious step of deproteinising the sample to be avoided, is to use micellar chromatography to quantify the percentage of the bound drug. In micelle chromatography, proteins can dissolve in micelles so that a protein-containing sample can be injected into the chromatography column [18; 19; 49; 54; 82].  
In order to determine the free form of drugs with a micellar system, it is necessary to use a surfactant whose surface charge of the micelles is opposite to that of the protein--they then become binding analytes. In mimetic MLC systems, the pH value of the mobile phase is 7.4 (physiological plasma pH value). The pI value of albumin is 5.8. Under the conditions of the experiment, the albumin molecules are therefore negatively charged at pH = 7.4. Thus, it is important to use a cationic surfactant (e.g., CTAB), which forms positively charged micelles, and not an anionic one (e.g., SDS).