A procedure is described for measuring pKa values in a short time, e.g., 4 min/assay. Samples, as 10 mM solutions, are prepared in DMSO in 96-well plates. A flowing pH gradient is produced by mixing two buffer solutions containing mixtures of weak acids and bases that do not absorb significantly in the UV above 250 nm. The sample solution is diluted with water and then injected directly into the flowing gradient, which then passes through a diode array spectrophotometer measuring in the UV wavelength range. The buffer has been formulated so that its acid-base titration curve is linear over a wide pH range, such that the pH of the gradient is a linear function of time. The solution pH in the measurement flow cell is therefore proportional to the time elapsed since the start of gradient generation. The sample's pKa values are calculated from the change in UV absorbance at multiple wavelengths as a function of pH. The pKa values of 71 drugs have been measured, and results compare well with values measured by pH-metric or traditional UV methods. Rules are suggested for the rapid inspection of data and the choice of method for the calculation of pKa from the data.

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites.

Solvation equations have been obtained for seven high performance liquid chromatographic ( HPLC) systems, generated in the reverse phase ( RP) mode with fast gradient elution. A training set of 40 compounds was used for each system. The seven equations were then used to calculate Abraham descriptors for a completely separate 40-compound test set. In this way the three descriptors dipolarity/polarizability S, hydrogen bond acidity A, and hydrogen bond basicity B were obtained. Five different procedures were used to calculate the descriptors, ( i) Microsoft 'Solver', (ii) a program that uses a set of three simultaneous equations, and which we denote as 'TripleX', (iii) a program similar to Solver that we denote as 'Descfit', (iv) a series of regression equations developed from compounds with known descriptors and ( v) a series of modified regression equations. We show that RP-HPLC data for a given compound in seven systems can be used to calculate the three Abraham descriptors reliably. We compare descriptors, and errors in the method, with those obtained from water-solvent partition systems.

The selectivity of Luna C18 Xterra C18 and Fluophase (perfluorinated C6) stationary phases has been investigated with aqueous acetonitrile, methanol and 2,2,2-trifluoroethanol mobile phases using linear solvation equations. The gradient retention times of a set of 60 compounds with known molecular descriptors have been determined. Linear solvation equations have been set up to describe the relationship between the gradient retention times and the molecular properties. The selectivity of the stationary phase/mobile phase systems was characterised by the regression coefficients of the molecular descriptors. The perfluorinated stationary phase showed very different selectivity using 2,2,2-trifluoroethanol (TFE) as co-solvent. Compounds with H-bond donor functionality were retained much less than in the other investigated high-performance liquid chromatography (HPLC) systems. This unique selectivity can be explained by the stronger adsorption of trifluoroethanol on the perfluorinated stationary phase surface, than on the hydrocarbon surface. It suggests the importance of the adsorbed organic modifiers in the separation mechanism during reversed-phase HPLC.

We propose a rapid method for the measurement of octanol/water partition coefficients (log P-oct) via fast gradient reversed phase retention and the calculation of the hydrogen bond acidity of the compounds. The cycle time of the generic gradient HPLC method is 5 minutes. The general solvation equation obtained for the log P-oct values and the fast gradient Chromatographic Hydrophobicity Indices with acetonitrile (CHIACN) and methanol (CHIMeOH) have been established. It has been revealed that the major difference between the log P-oct and CHIACN lipophilicity scales is their sensitivity towards the hydrogen bond acidity (Sigma alpha (H)(2)) of the compounds. The CHIACN values of the uncharged (neutral) compounds are measured, then the H-bond acidity term (Sigma alpha (H)(2)) is calculated from the structures. The log P-oct values are expressed using the following equation that was established from the data of 86 diverse compounds:Log P-oct = 0.054 CHIACN + 1.319 Sigma alpha (H)(2) - 1.877N=86 r=0.970 s=0.29 F=655A hydrogen bond count (HBC) is not as good as Sigma alpha (H)(2) as a measure of H-bond acidity but still gives an acceptable equation:Log P-oct = 0.047 CHIACN + 0.36 HBC - 1.10N=86 r=0.943 s=0.39 F=336In order to improve the correlation between the log P-oct and the CHIMeOH values several other terms, such as the H-bond basicity, polarisability and size term should also be considered. Therefore acetonitrile is suggested as the preferred organic modifier. This rapid method can be used as a high throughput lipophilicity screen for combinatorial libraries.

Retention data for a set of 69 compounds using rapid gradient elution are obtained on a wide range of reversed-phase stationary phases and organic modifiers. The chromatographic stationary phases studied are Inertsil (IN)-ODS, pentafluorophenyl, fluoro-octyl, n-propylcyano, Polymer (PLRP-S 100), and hexylphenyl. The organic solvent modifiers are 2,2,2-trifluoroethanol (TFE); 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP); isopropanol; methanol (MeOH); acetonitrile (AcN); tetrahydrofuran; 1,4-dioxane; N,N-dimethylformamide; and mixed solvents of dimethylsulfoxide (DMSO) with AcN and DMSO with MeOH (1:1). A total of 25 chromatographic systems are analyzed using a solvation equation. In general, most of the systems give reasonable statistics. The selectivity of the reversed phase-high-performance liquid chromatographic (HPLC) systems with respect to the solute's dipolarity-polarity, hydrogen-bond acidity, and basicity are reflected in correspondingly large coefficients in the solvation equation. We wanted to find the most orthogonal HPLC systems, showing the highest possible selectivity difference in order to derive molecular descriptors using the gradient retention times of a compound. We selected eight chromatographic systems that have a large range of coefficients of interest (s, a, and b) similar to those found in water-solvent partitions used previously to derive molecular descriptors. The systems selected are IN-ODS phases with AcN, MeOH, TFE, and HFIP as mobile phase, PLRP-S 100 phase with AcN, propylcyano phase with AcN and MeOH, and fluorooctyl phase with TFE. Using the retention data obtained for a compound in the selected chromatographic systems, we can estimate the molecular descriptors with the faster and simpler gradient elution method.

The solvation equation [Eq. (1)] can be applied to reversed-phase HPLC capacity factors (i.e. logSP=logk).(1)logSP=c+rR2+sπH2+aΣαH2+bΣβH2+vVxS.SP is a solute property (e.g., solubility or partition coefficient) and the explanatory variables are solute descriptors as follows: R2 is an excess molar refraction, π2H is the solute dipolarity/dipolarisability, Σα2H and Σβ2H are the solute overall or effective hydrogen-bond acidity and basicity, and Vx is the McGowan characteristic volume and c, r, s, a, b and v are constants that are characteristic of the particular mobile/stationary phase combination. Only a limited hydrophobicity range can be covered using logk values obtained by isocratic chromatography at a fixed mobile phase composition. Gradient elution is more versatile and 29 compounds were examined under 20 different reversed-phase HPLC conditions using automated fast gradient methods. Using Eq. (1)with gradient retention time (tRg) in place of logSP an excellent correlation was obtained with the solute descriptors. This provides an experimental demonstration that gradient retention times (tRg) can be treated as linear free-energy related parameters just like logk or logP values. Because gradient retention times cannot be used for inter-laboratory comparisons, they were scaled by conversion to chromatographic hydrophobicity index (CHI) values by correlation with data from a calibration set of compounds with known CHI values. By comparing the coefficients r, s, a, b and v of Eq. (1)as determined from different chromatographic systems, the relative effects of different solute properties can be revealed and thus the selectivity of HPLC columns can be predicted. It is also possible to compare chromatographic behaviour with that of other partition systems, such as octanol–water, cyclohexane–water and blood–brain barrier distributions.

The chromatographic hydrophobicity index (CE-II) obtained from high-throughput gradient elution reversed-phase HPLC with ODS column and acetonitrile mobile phase has been shown to be well correlated with log k values obtained by isocratic elution in the same system; between CHI and log k(50), the correlation coefficient was 0.99 for a very diverse set of 55 compounds. CBI and log k(50) are moderately correlated with log P (water/octanol), and both can be used as alternative measures of lipophilicity. Analyses using the general solvation equation of Abraham shows that the solute factors that influence CHI and log k(50) are not entirely the same as those that influence log P, so that neither CHI nor log k(50) can be used as a direct measure of log P and vice versa, However, the factors that influence CHI are qualitatively and quantitatively the same as those that influence log k(50), so that the rapidly determined CHI indexes encode exactly the same information as do isocratic log values.

A new chromatographic hydrophobicity index (CHI) is described which can be used as part of a protocol for high-throughput (50-100 compounds/day) physicochemical property profiling for rational drug design. The index is derived from retention times (t(R)) observed in a fast gradient reversed-phase HPLC method. The isocratic retention factors (log k') were measured for a series of 76 structurally unrelated compounds by using various concentrations of acetonitrile in the mobile phase. By plotting the log k' as a function of the acetonitrile concentration, the slope (S) and the intercept (log k'(w)) values were calculated. The previously validated index of hydrophobicity φ(0) was calculated as -log k'(w)/S. A good linear correlation was obtained between the gradient retention time values, t(R) and the isocratically determined φ(0) values for the 76 compounds. The constants of this linear correlation can be used to calculate CHI. For most compounds, CHI is between 0 and 100 and in this range it approximates to the percentage (by volume) of acetonitrile required to achieve an equal distribution of compound between the mobile and the stationary phases. CHI values can be measured using acidic, neutral, or slightly basic eluents. Values corresponding to the neutral form of molecules could be measured for 52 of the compounds and showed good correlation (r = 0.851) to the calculated octanol/water partition coefficient (c log P) values.