## 3 Case Study: LC in Lipophilicity Assessment of Xanthone Derivatives  
To emphasize the dual role of LC in the determination of lipophilicity and its importance as a medicinal chemistry tool, we provide a case study describing the experience of our group with xanthone derivatives. Xanthone derivatives comprise an important class of oxygenated heterocyclic compounds in medicinal chemistry concerning not only its large range of biological and/or pharmacological activities [217; 218; 219], but also other applications, including as fluorescence probes [220; 221], and chiral stationary phases for LC [222; 223]. For the last several years, the search for new xanthone derivatives with potential bioactivity has remained one of the main areas of interest for our group [224]. Recently, the determination of lipophilicity of a small library of bioactive xanthone derivatives was performed in our group [225]. The chosen xanthone derivatives were previously synthesized and exhibited interesting biological activities, namely growth inhibitory effects on different tumour cell lines [226], nerve conduction blockade acting in a similar manner to local anaesthetics [227], and the inhibition of cyclooxygenase, the enzymes involved in inflammatory processes [228]. Among the different methods used for lipophilicity assessment, LC was applied for both indirect and direct determination of log P, specifically with RP-HPLC and with VALLME coupled with HPLC [225]. Regarding the RP-HPLC method, two hydrophobic RP silica-based columns with a C8 and a C18 containing hydrocarbon long-chain (same length, diameter, and particle size) were used to evaluate their influence on the experimental lipophilicity. Different percentages of methanol in the aqueous mobile phase were chosen with increments of 5% or 10%, ranging between 40 to 75%, in accordance with the OECD Guidelines. For each compound, the log k was determined (Equation (5)) at different proportions of methanol. At least five different percentages were used to obtain a linear relationship, and log k\({}_{\text{w}}\) was obtained by extrapolation to 100% of pure water. The different percentages of methanol in the mobile phase were chosen based on the theoretical log P values for each xanthone derivative [225]. As an example of the xanthone derivative XD-10 (Figure 5), the log k\({}_{\rm w}\) obtained in both columns (C8 and C18) was 3.71. Figure 6a shows the chromatograms of XD-10 on the C18 column and with different proportions of methanol in the mobile phase. As expected, the retention time increased with the decrease of the percentage of methanol in the mobile phase. Similar to other xanthone derivatives, for XD-10, a linear correlation between the chromatographic parameter log k and the proportion of methanol in the mobile phase was obtained, with R\({}^{2}\) of 0.9967 and 0.9982 for C18 and C8 columns, respectively. LC was also applied as an analytical method for direct quantification of the xanthone derivatives in the \(n\)-octanol phase after VALLME procedure [225]. For the analysis, a C18 hydrophobic silica-based column and a mixture of water/methanol (70:30 \(v/v\)) as a mobile phase were used. Once again, the theoretical log P values were the basis for the selection of the appropriate concentrations of the octanolic solutions for each xanthone derivative to fit the predictive concentrations in the \(n\)-octanol droplet into the linear zone of the calibration curve. The external calibration curves for xanthone derivatives demonstrated good linear correlations. As an example, for XD-10 (Figure 5), a R\({}^{2}\) of 0.9933 was achieved. Figure 6b shows the chromatograms for XD-10 present in \(n\)-octanol phase after VALLME procedure with different concentrations. As expected, the concentration of XD-10 found in the \(n\)-octanol phase increases with the augment of the concentration.