# 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  
Chromotagraphic data obtained in IAM systems and with HSA phases can be used to estimate the distribution of substances in tissues. There are different proportions of phospholipids and proteins in the tissues, and the compositions of the cell membranes of the cells of different tissues differ from each other. Depending on the proportions of phospholipids and proteins in different tissues, the binding of drugs to tissues can be modelled by weighted sums of albumin and IAM binding. The binding of a substance in mucus can be modelled by determining the binding of the substance to a glycoprotein (AGP), since 90% of mucus is a glycoprotein. The permeability of a substance through the blood-brain barrier can be estimated by modelling the binding to brain tissue and the binding to plasma proteins. Of course, such studies only give us estimates, but with their help, it is possible to identify compounds that can penetrate the central nervous system. The distribution of drugs in the brain is determined by the properties of the molecule itself, such as its lipophilicity, its small size, its lack of binding to plasma proteins and its presence in a non-dissociated form. Any drug that is absorbed can remain in the blood--in which case, its duration of action is relatively short--or bind to the so-called transport proteins (serum albumin, \(\alpha\)- and \(\beta\)-globulins, and acidic \(\alpha\)-1-glycoprotein). A drug that is bound to a protein is not pharmacologically active--it is not transported to the tissues, is not metabolised, and is not excreted from the body. The binding of a drug by plasma proteins influences the concentration of the free form of the drug, which in turn influences the drug dose, the duration of the drug effect, and many other pharmacokinetic parameters. Different disease states cause changes in the amount of transport proteins, meaning that the drug dose may not be correct in some cases. Therefore, estimating the degree of binding of a drug, in addition to determining the volume of distribution, is of great importance, especially for drugs with a narrow therapeutic index (the effect of the drug may be ineffective or side effects may occur). The factors that influence the degree of binding of a drug to a protein are as follows:  
* The pH of the plasma--it can alter the ionisation of many compounds;
* The drug concentration--different saturation of the binding sites;
* The protein concentration--disease-related changes, age;
* The affinity of the drug molecules for protein-binding sites (physicochemical properties);  
Figure 5: Distribution of a substance in plasma and tissue. The presence of other substances in the body that bind to proteins at the same active sites;  
Exogenous (drugs, toxins), endogenous (urea, bilirubin, fatty acids, hormones). The degree of binding of a drug to proteins can be determined in systems with stationary phase HSA or BSA--basic transport proteins. It has been found that the retention times of drugs with protein phases are related to the percentage of drug binding by the following equation [60; 77]:  
\[\%bound=\frac{101\times 10^{logk}}{1+10^{logk}} \tag{6}\]  
Chromotographic measurements with gradient elution are frequently carried out. In systems with HSA columns, a phosphate buffer with a pH value of 7.4 is used as the mobile phase and 2-propanol (up to 35% by volume, as the proteins of the stationary phase are denatured above this concentration) is used as the organic modifier. Since the retention time of the substance in gradient elution depends on many factors, e.g., the flow rate of the mobile phase and the size of the column, it is necessary to calibrate the retention times in gradient elution with compounds whose percentage binding is known and which have been obtained using other methods, e.g., equilibrium dialysis, organic modifiers, ultrafiltration, capillary electrophoresis, etc., in order to standardise the results obtained so that the data can be compared between different laboratories.