High-performance methods of evaluation of lipophilicity and acidity of drug candidates and other xenobiotics are highly requested in modern pharmaceutical and environmental research. Reversed-phase high-performance liquid chromatography (RP HPLC) might be particularly useful for the determination of both dissociation constant and the (pH-dependent) octanol-water partition coefficient related parameters as it would be applicable for convenient assays of multi-component mixtures. Our  general theory of combined pH/organic modifier gradient provides comprehensive equations relating gradient retention time and pH of the mobile phase. Based on this theory, a general method for the simultaneous determination of log kw (chromatographic parameter of hydrophobicity/lipophilicity) and dissociation constant (pKa) has been proposed^1-3. 

The purpose of this work was to facilitate our general approach by identification of individual components of the studied mixtures of analytes by means of RP HPLC coupled with Time-of-flight Mass Spectrometry with Electrospray Ionization Source (ESI-TOF-MS). At the same time, diverse experimental designs were tested to maximally speed up-the procedure. The accuracy of the proposed methodology was assessed by analyzing a set of known drugs (acids and bases). As the buffers, ammonium formate, ammonium acetate or ammonium bicarbonate, were used to control pH during individual chromatographic runs. The pKa and hydrophobicity parameters of the components of test mixtures were determined and the accuracy of the estimated values was assessed by comparing with the reference literature data. The gradient RP HPLC coupled with ESI-TOF-MS method allowed a fast determination of dissociation constant and hydrophobicity. The method was shown to be especially applicable for complex mixtures. The use of ESI-TOF-MS detection technique allowed to achieve the medium-throughput screening rate (100 compounds/day) without a need of using pure, isolated substances in substantial amounts.