# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 4 Chromatography in the Determination of Pharmacokine Parameters of Substances  
The pharmacokinetic parameters of a drug enable the determination of the drug dosage and the prediction of the expected drug efficacy. Compounds with optimal lipophilicity often have suitable pharmacokinetic parameters so that the determination of the lipophilic properties of compounds can be used to determine important pharmacokinetic parameters. If the number of aromatic rings of a substance is combined with the Chrom log\(D\) value, the so-called _PFI_ values (_Property Forecast Index_) are obtained [72; 73; 74]. This parameter indicates which properties of a compound influence the LADME process. The _PFI_ value is calculated on the basis of the following relationship:  
\[\textit{PFI}=\textit{Chrom}\ \text{log}D+n \tag{4}\]  
where \(n\) is the number of aromatic rings in the molecule and _Chrom_ log\(D\) is determined based on chromatographic measurements. Based on the analysis of _PFI_ values for more than 40,000 substances, compounds with _PFI_ values between three and six were found to have good pharmacokinetic parameters. The distribution of a drug is another important process that influences the pharmacological effect of the preparation. The pharmacokinetic parameters that characterise the distribution of a drug in the body are the distribution constant, the volume of distribution, and the degree of binding of the drug to blood proteins. The volume of distribution can be defined as the ratio between the drug dose and the plasma concentration at steady state:  
\[V_{d}=\frac{X}{C} \tag{5}\]  
where \(V_{d}\)--the volume of distribution [dm\({}^{3}\)], \(X\)--the drug dose (total content of the drug in the body), \(C\)--the measured concentration of the drug in the blood (serum). This quantity (volume of distribution), which helps to describe the quantitative distribution of the drug in the body, is proportional to the distribution of the drug between the plasma and the tissue compartment. * If \(V_{d}<5\) dm\({}^{3}\), then the drug is in the blood and it is distributed only in the circulatory system. * If 10 dm\({}^{3}<V_{d}<\) 20 dm\({}^{3}\), this means that the drug penetrates the extracellular fluid;
* If 25 dm\({}^{3}<V_{d}<\) 30 dm\({}^{3}\), the drug is in the intracellular fluid (_ICFV_);
* If it is approx. 40 dm\({}^{3}\), the drug is distributed in all the bodily fluids;
* If it is approx. 100 dm\({}^{3}\), this means that the drug accumulates in tissues and organs. Drugs of lipophilic nature (e.g., diazepam) easily penetrate cell membranes and have a large volume of distribution, as their plasma concentration is low. Drugs that bind to plasma proteins have a small volume of distribution (up to 10 dm\({}^{3}\)), drugs that have an affinity for binding to peripheral tissue have a very high volume of distribution (e.g., digoxin--about 500 dm\({}^{3}\)), whereas azithromycin, which accumulates in intracellular lysosomes, has a volume of distribution of about 2000 dm\({}^{3}\). The volume of distribution depends on the affinity of a substance for phospholipids and plasma proteins (Figure 5). It has been observed that positively charged compounds bind more strongly to phospholipids and negatively charged cell membranes, indicating a high accumulation of drugs in tissues [75]. Although tissues contain large amounts of proteins (including albumin), they have many barriers, many biological membranes, and other lipid components, so the accumulation of drugs in tissues is mainly determined by the interactions of molecules with lipids. Negatively charged compounds bind more strongly to albumin and remain present in the blood plasma. In summary, compounds that bind more strongly to phospholipids show greater accumulation in tissues than compounds that bind strongly to plasma proteins, which mostly remain in the blood. Until recently, the value of the volume of distribution could only be determined by in vivo experiments.