Ability of biomimetic chromatography and physicochemical systems to predict the skin permeation of neutral compounds. A comparison study.

Five in vitro physicochemical systems have been evaluated in terms of its ability to emulate the skin permeation of neutral compounds: the permeation in two different PAMPA membranes, the classical octanol-water partition coefficient, and two biomimetic chromatography systems, one based in cerasome electrokinetic chromatography and another based in reversed-phase liquid chromatography measurements. The coefficients of the solvation parameter model equation of the mentioned systems have been compared to the ones of the skin permeation process through different comparison parameters. Moreover, a method to predict whether a physicochemical system is able to emulate satisfactorily a biological one, just by the analysis of the equation coefficients has been developed. Results reveal that the two PAMPA systems are a good choice to emulate directly the skin permeation of neutral compounds. Instead, the other three systems need a volume correction term to provide a satisfactory emulation. However, after the correction, all the evaluated systems show a similar ability to emulate well skin permeation, as predicted.

Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns.

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k' < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Characterization of solute-solvent interactions in liquid chromatography systems: A fast method based on Abraham's linear solvation energy relationships.

The Abraham's solvation parameter model, based on linear solvation energy relationships (LSER), allows the accurate characterization of the selectivity of chromatographic systems according to solute-solvent interactions (polarizability, dipolarity, hydrogen bonding, and cavity formation). However, this method, based on multilinear regression analysis, requires the measurement of the retention factors of a considerably high number of compounds, turning it into a time-consuming low throughput method. Simpler methods such as Tanaka's scheme are preferred. In the present work, the Abraham's model is revisited to develop a fast and reliable method, similar to the one proposed by Tanaka, for the characterization of columns employed in reversed-phase liquid chromatography and particularly in hydrophilic interaction liquid chromatography. For this purpose, pairs of compounds are carefully selected in order to have in common all molecular descriptors except for a specific one (for instance, similar molecular volume, dipolarity, polarizability, and hydrogen bonding basicity features, but different hydrogen bonding acidity). Thus, the selectivity factor of a single pair of test compounds can provide information regarding the extent of the dissimilar solute-solvent interactions and their influence on chromatographic retention. The proposed characterization method includes the determination of the column hold-up volume and Abraham's cavity term by means of the injection of four alkyl ketone homologues. Therefore, five chromatographic runs in a reversed-phase column (four pairs of test solutes and a mixture of four homologues) are enough to characterize the selectivity of a chromatographic system. Tanaka's method is also analyzed from the LSER point of view.

Evaluation of the Ability of PAMPA Membranes to Emulate Biological Processes through the Abraham Solvation Parameter Model.

Two parallel artificial membrane permeability assay (PAMPA) systems intended for emulating skin permeability have been characterized through the solvation parameter model of Abraham using multilinear regression analysis. The coefficients of the obtained equations have been compared to the ones already established for other PAMPA membranes using statistical tools. The results indicate that both skin membranes are similar to each other in their physicochemical properties. However, they are different from other PAMPA membranes (e.g., intestinal absorption and blood-brain PAMPAs), mainly in terms of hydrophobicity and hydrogen bonding properties. Next, all PAMPA membranes have been compared to relevant biological processes also characterized through the solvation parameter model. The results highlight that skin-PAMPA membranes are a very good choice to emulate skin permeability.

Evaluation of Hold-Up Volume Determination Methods and Markers in Hydrophilic Interaction Liquid Chromatography.

Common methods for hold-up time and volume determination in Reversed-Phase Liquid Chromatography (RPLC) have been tested for Hydrophilic Interaction Liquid Chromatography (HILIC). A zwitterionic ZIC-HILIC column has been used for the testing. The pycnometric determination method, based on differences in column weight when filled with water or organic solvent, provides the overall volume of solvent inside the column. This includes the volume of eluent semi-sorbed on the packing of the column, which acts as the main stationary phase. The homologous series approach, based on the retention behavior of homologues in relation to their molecular volume, allows the determination of accurate hold-up volumes. However, the application of this method is time-consuming. In some cases, large neutral markers with poor dipolarity/polarizability and hydrogen bonding interactions can be used as hold-up volume markers. This is the case of dodecylbenzene and nonadecane-2-one in clearly HILIC behaving chromatographic systems, the use of decanophenone as a marker can be even extended to the boundary between HILIC and RPLC. The elution volume of the marker remains nearly unaffected by the concentration of ammonium acetate in the mobile phase up to 20 mM. The injection of pure solvents to produce minor base-line disturbance as hold-up markers is strongly discouraged, since solvent peaks are complex to interpret and depend on the ionic strength of the eluent.

Determination of the aqueous pKa of very insoluble drugs by capillary electrophoresis: Internal standards for methanol-water extrapolation.

A fast determination of acidity constants (pKa) of very insoluble drugs has become a necessity in drug discovery process because it often produces molecules that are highly lipophilic and sparingly soluble in water. In this work the high throughput internal standard capillary electrophoresis (IS-CE) method has been adapted to the determination of pKa of water insoluble compounds by measurement in methanol/aqueous buffer mixtures. For this purpose, the reference pKa values for a set of 46 acid-base compounds of varied structure (internal standards) have been established in methanol-water mixtures at several solvent composition levels (with a maximum of 40% methanol). The IS-CE method has been successfully applied to seven test drugs of different chemical nature with intrinsic solubilities lower than 10-6 M. pKa values have been determined at different methanol/aqueous buffer compositions and afterwards Yasuda-Shedlovsky extrapolation method has been applied to obtain the aqueous pKa. The obtained results have successfully been compared to literature ones obtained by other methods. It is concluded that the IS-CE method allows the determination of aqueous pKa values using low proportions of methanol, becoming then more accurate in the extrapolation procedure than other reference methods.

Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents.

Pycnometric and homologous series retention methods are used to determine the volume and mean composition of the water-rich layers partially adsorbed on the surface of several hydrophilic interaction liquid chromatography (HILIC) column fillings with acetonitrile-water and methanol-water as eluents. The findings obtained in this work confirm earlier studies using direct methods for measuring the stationary phase water content performed by Jandera's and Irgum's research groups. Water is preferentially adsorbed on the surface of the HILIC bonded phase in hydroorganic eluents containing more than 40% acetonitrile or 70% methanol, and a gradient of several water-rich transition layers between the polar bonded phase and the poorly polar bulk mobile phase is formed. These layers of reduced mobility act as HILIC stationary phases, retaining polar solutes. The volume of these layers and concentration of adsorbed water is much larger for acetonitrile-water than for methanol-water mobile phases. In hydroorganic eluents with less than 20-30% acetonitrile or 40% methanol the amount of preferentially adsorbed water is very small, and the observed retention behavior is close to the one in reversed-phase liquid chromatography (RPLC). In eluents with intermediate acetonitrile-water or methanol-water compositions a mixed HILIC-RPLC behavior is presented. Comparison of several HILIC columns shows that the highest water enrichment in the HILIC retention region for acetonitrile-water mobile phases is observed for zwitterionic and aminopropyl bonded phases, followed in minor grade for diol and polyvinyl alcohol functionalizations. Pentafluorophenyl bonded phase, usually considered a HILIC column, does not show significant water adsorption, nor HILIC retention.

Linear free energy relationship models for the retention of partially ionized acid-base compounds in reversed-phase liquid chromatography.

The LFER model of Abraham is applied to the retention of the neutral and ionic forms of 94 solutes in a C18 column and 40% v/v acetonitrile/water mobile phase. The results show that polarizability and cavity formation interactions increase retention, whereas dipole and hydrogen bonding interactions favours partition to the mobile phase and thus, they decrease retention. The coefficients of the ionic descriptors measure the effect of the electrostatic interactions and their contribution to partition of the cation or anion between the two mobile and stationary chromatographic phases. A new LFER model for application to the retention of partially dissociated acids and bases is derived averaging the descriptors of the neutral and ionic forms according to their degrees of ionization in the mobile phase. This new LFER model is satisfactorily compared to other literature modified Abraham models for a set of 498 retention data of partially dissociated acids and bases. All tested models require the calculation of the ionization degrees of the compounds at the measuring pH. Calculation of the ionization degrees in the chromatographic mobile phase (i.e. from pH and pKa in the eluent) give good correlations for all tested models. However, estimation of these ionization degrees from pH - pKa data in pure water gives biased estimations of the retention of the partially ionized solutes.

HILIC characterization: Estimation of phase volumes and composition for a zwitterionic column.

A methodology for the estimation of the different phase volumes in HILIC is presented. For a ZIC-HILIC column the mobile phase volume (hold-up volume) is determined in several acetonitrile- and methanol-water compositions by a Linear Free Energy Relationships (LFER) homologous series approach involving n-alkyl-benzenes, -phenones, and -ketones. We demonstrate that the column works as a HILIC column when the mobile phase contains high and medium proportions of methanol or acetonitrile. However, for acetonitrile contents below 20%, or 40% for methanol, same column works in RPLC. In between, a mixed HILIC-RPLC behavior is observed, and solutes of low molecular volume are retained as in HILIC mode, but the largest ones show RPLC retention. From the homologous series retention data and pycnometric measurements involving the pure organic solvents and their mixtures with water, the mean solvent composition of the water-rich transition layers between column functionalization and the bulk mobile phase, which act as stationary phase, is estimated. Finally, the phase ratio between stationary and mobile phases is also estimated for each eluent composition, allowing the calculation of the corresponding stationary phase volumes. All volumes are strongly dependent on the water content in the eluent, especially when acetonitrile is selected as mobile phase constituent. In HILIC mode, when the water content in the hydroorganic mobile phase increases, the volumes of mobile phase decrease, but the volumes of stationary phase (mainly the water layer adsorbed onto the bonded-phase and the water-enriched interface) increase. However, at high water concentrations, where the column works in RPLC mode, the mobile phase volume increases and the stationary phase (which is now the bonded zwitterion) volume decreases when increasing the water percentage in the mobile phase.

Determination of acidity constants at 37 °C through the internal standard capillary electrophoresis (IS-CE) method: internal standards and application to polyprotic drugs.

This work provides the pKa at the biorelevant temperature of 37 °C for a set of compounds proposed as internal standards for the internal standard capillary electrophoresis (IS-CE) method. This is a high throughput method that allows the determination of the acidity constants of compounds in a short time, avoiding the exact measurement of the pH of the buffers used. pH electrode calibration at 37 °C can be avoided too. In order to anchor the pKa values obtained through the IS-CE method in the pH scale, the acidity constant at 37 °C of some of the standards has been determined also by the reference potentiometric method. In general, a decrease in the pKa value is observed when changing the temperature from 25 to 37 °C, and the magnitude of the change depends on the nature of the compounds. Once the pKa values at 37 °C of the internal standards have been established, the method is applied to the determination of the acidity constants of seven polyprotic (5 diprotic and 2 triprotic) drugs. The obtained mobility-pH profiles show well-defined curves, and the fits provide precise pKa values. Due to the lack of reference data at 37 °C only the pKa values of labetalol can be compared to values from the literature, and a very good agreement is observed.

Optimization of experimental conditions for skin-PAMPA measurements.

In recent years, the parallel artificial membrane permeability assay (PAMPA) has been extended for prediction of skin permeation by developing an artificial membrane which mimics the stratum corneum structure, skin-PAMPA. In the present work, the different parameters affecting skin-PAMPA permeability, such as incubation time and stirring, have been studied to establish ideal assay conditions to generate quality data for a screening of active pharmaceutical ingredients (API) in early stage drug discovery. Another important parameter is membrane retention, which shows dependence on lipophilicity when compounds are in their neutral form. Furthermore, the stability of the membrane has been investigated at different pH values, especially at basic pHs. Finally, a good correlation between human skin permeability and skin-PAMPA permeability, with a large dataset (n = 46), has been established. The optimized assay conditions were an incubation time of 4 hours with stirring in a pH below 8. With all these considerations the thickness of the aqueous boundary layer is decreased as much as possible and the membrane stability is guaranteed.

Estimation of the octanol-water distribution coefficient of basic compounds by a cationic microemulsion electrokinetic chromatography system.

The octanol-water partition coefficient (Po/w), or the octanol-water distribution coefficient (Do/w) for ionized compounds, is a key parameter in the drug development process. In a previous work, this parameter was estimated through the retention factor measurements in a sodium dodecyl sulfate (SDS) - microemulsion electrokinetic chromatography (MEEKC) system for acidic compounds. Nonetheless, when ionized basic compounds were analyzed, undesirable ion pairs were formed with the anionic surfactant and avoided a good estimation of log Do/w. For this reason, an alternative MEEKC system based on a cationic surfactant has been evaluated to estimate Po/w or Do/w of neutral compounds and ionized bases. To this end, it has been characterized through the solvation parameter model (SPM) and compared to the octanol-water partition system. Results pointed out that both systems show a similar partition behavior. Hence, the log Po/w of a set of neutral compounds has been successfully correlated against the logarithm of the retention factor (log k) determined in this MEEKC system. Then, the log Do/w of 6 model bases have been estimated at different pH values and they have been compared to data from the literature, determined by the reference shake-flask and potentiometric methods. Good agreement has been observed between the literature and the estimated values when the base is neutral or partially ionized (up to 99% of ionization).

Estimation of the octanol-water distribution coefficient of acidic compounds by microemulsion electrokinetic chromatography.

The feasibility of extending the determination of the lipophilicity of partially ionized acids (log Do/w) by microemulsion electrokinetic chromatography (MEEKC) is tested. Theoretical considerations predict that a linear log Do/w vs. log k correlation can be obtained only when the neutral and ionic forms of an acid follow the same correlation equation and the slope of the correlation is unity. In practice, since the lipophilicity of the neutral acid is much higher than that of the ionic form and the correlation slope is not very different from 1, the general linear correlation for neutral compounds can be applied across most of the ionization range of the acid. The linear correlation between log Po/w and log k of 20 neutral solutes (calibration curve) has been established and extended to 6 acids used as models, tested across their full ionization range. log Do/w-pH, and log k-pH profiles have been obtained for these 6 acids, and plotted log Do/w against log k for any acid at any degree of ionization. Furthermore, the log Do/w of the acids has been estimated from the calibration curve and log k-pH profile, and compared to values in the literature determined using reference methods such as the shake-flask one. Accurate values have been obtained using the MEEKC method when the acids are in their neutral form or partially ionized (ionization degree, α < 0.995). However, this parameter is overestimated when the acids are highly or fully ionized (α ≈ 1). Finally, in order to test the applicability of this method, we have applied the same procedure to estimate log Do/w at pH = 7.4 (blood physiological pH) of a set of 30 additional compounds (including partially and fully ionized acids). The results at this pH follow the same trend observed in the 6 model acids, and validate the application of the method for Do/w determination, except when α is very close to 1.

Lecithin liposomes and microemulsions as new chromatographic phases.

Lecithins are phospholipidic mixtures that can be part of microemulsions and liposomes. In this work, ready-to-use preparations of lecithin have been tested as pseudostationary and mobile phases in EKC and LC, respectively. The selectivity of two EKC systems, one based on lecithin microemulsions (LMEEKC) and another on liposomes (LLEKC), and of a LC system based on lecithin microemulsions (MELC) has been evaluated through the solvation parameter model. In all cases, solute volume and hydrogen-bond basicity are the main descriptors that drive the partition process. While solute volume favors the retention of solutes, hydrogen-bond basicity has the contrary effect. In lecithin-based EKC systems the hydrogen-bond acidity of the solute leads to a higher retention while in the lecithin-based LC system a minor retention is produced. The three lecithin systems have been compared through the solvation parameter model to other chromatographic systems, most of them containing phospholipids. Principal component analysis reveals that lecithin systems cluster together with the other EKC systems based on phospholipids, with an immobilized artificial membrane (IAM) LC system, with the octanol/water reference partition system, and with a SDS-based microemulsion. Thus, they all show similar selectivity. However, the great advantage of using the ready-to use lecithin systems is that the laborious liposome preparation is avoided, and that their commercial availability makes them more affordable than IAM LC columns. Finally, taking into account that lecithin has a high semblance to the mammalian cell membranes composition, the ability of the three lecithin systems to mimic the pass of the solutes through the membranes has been evaluated. Experimental determinations have demonstrated that the skin partition of neutral solutes can be easily emulated, especially using the lecithin-microemulsion EKC method. The model is robust and shows good prediction ability.

Characterization of hydrophilic interaction liquid chromatography retention by a linear free energy relationship. Comparison to reversed- and normal-phase retentions.

The Abraham solvation parameter model, a linear free energy relationship (LFER) approach, has been used to characterize a polymeric zwitterionic (sulfobetaine) column in HILIC mode. When acetonitrile (MeCN) is used in the preparation of mobile phases the main solute characteristics affecting the chromatographic behavior of analytes are the molecular size and the hydrogen-bonding (both acidity and basicity) interactions. The former property is more favorable in the acetonitrile-rich mobile phase, reducing thus the retention, but the latter reveals a higher affinity for the water layer adsorbed on the stationary phase, enhancing retention. However, if the aprotic acetonitrile is replaced by methanol, a hydrogen-bond acidic solvent, solute hydrogen-bond basicity does not contribute any more to retention, quite the opposite. Thus, a slightly different selectivity is observed in methanol/water than in acetonitrile/water. Normal-phase mode and HILIC-MeCN share the same main factors affecting retention. For reversed-phase and immobilized artificial membrane (IAM) chromatography, the solute molecular size increase retention because of the lower amount of energy required in the formation of a cavity in the solvated stationary phase. On the contrary, the analyte hydrogen-bond basicity favors interactions with the hydroorganic mobile phase and reduces retention. The determined parameters justify the reversed selectivity commonly observed in HILIC in reference to reversed-phase. In most instances, the least retained solutes in reversed-phase are the most retained in HILIC.

Influence of the acid-base ionization of drugs in their retention in reversed-phase liquid chromatography.

The effect of the ionization in the RP-HPLC retention of 66 acid-base compounds, most of them drugs of pharmaceutical interest, is studied. The retention time of the compounds can be related to the pH measured in the mobile phase (pwsH) through the sigmoidal equations derived from distribution of the neutral and ionic forms of the drug into the stationary and mobile phases. Fitting of the obtained retention vs. pH profiles provides the retention times of the ionic and neutral forms and the pKa values of the drugs in the mobile phase (pwsKa). The obtained pwsKa values are linearly correlated to the pKa values in water (pwwKa) with two different correlations, one for neutral acids and another for neutral bases that reflect the different influence of the dielectric constant of the medium in ionization of acids and bases. The retention of the neutral species is well correlated to the octanol-water partition coefficient of the drugs as measure of the lipophilicity of the drug, which affects chromatographic retention. Also, the retention time of the ionized forms is related to the retention time of the neutral forms by two different linear correlations, one for anions and the other for cations. These last correlations point out the different retention behaviour of anions and cations: anions are less retained than cations of the same lipophilicity, as measured by the octanol-water partition coefficient of the neutral form. The different retention behaviour of anionic, cationic and neutral forms is confirmed by the hold-up times obtained from different approaches: pycnometry and retention times of anionic (KBr and KI) and neutral (DMSO) markers. Hold-up times obtained by pycnometric measurements agree with those obtained by retention of neutral markers (0.83-0.85 min), whereas hold-up time for anions is mobile phase pH dependent. At acidic pH it is similar to the hold-up time for neutral markers (0.83 min), but then it decreases with the increase of mobile phase pH to 0.65 min at pH 11. The decrease can be explained by the ionization of the silanols of the column and exclusion of anions by charge repulsion. Although not directly measured, the obtained retention data and correlations indicate hold-up time for cations are similar or slightly lower than hold-up time for neutral compounds (0.77-0.83 min). The model proposed and the correlations obtained can be very useful for its implementation in retention prediction algorithms for optimization of separation purposes.

Determination of the retention factor of ionizable compounds in microemulsion electrokinetic chromatography.

Determination of the retention factor of ionized compounds in microemulsion electrokinetic chromatography requires two mobility measurements at the same pH: one in the presence of the microemulsion and another in plain buffer. However, it has been observed that in some cases subtracting one mobility from another determined in a different medium leads to negative retention factors, which makes no sense from a chemical point of view. This indicates that there is some error in the process which has a direct impact when retention factors are used for further applications. Here, we evaluate how the components of the microemulsion confer different properties to the buffer medium, particularly varying the viscosity parameter (which is inversely related to mobility). Whereas sodium dodecyl sulfate, the surfactant used in the microemulsion, has little effect on the medium viscosity (only an increase of 5%-6%), the presence of 1-butanol, used as a stabilizer, increases it by around 30%. Meanwhile, heptane, which is used as an oil, provokes a slight decrease. Consequently, the mobilities obtained in the microemulsion system are shifted to higher values (less negative mobilities) compared to mobilities obtained in the aqueous buffer, and so one cannot be directly subtracted from the other. Since the microemulsion-buffer medium cannot be directly reproduced, we propose a correction that takes into account the variation of viscosities. This is determined from the electrophoretic mobility of the benzoate ion. As this ion does not interact with the microemulsion, the ratio of its mobilities (measured in plain buffer and microemulsion) is equivalent to the ratio of viscosities, and can be used as the correction factor for other measurements. Thus, mobilities in buffer and microemulsion media are placed on the same scale, overcoming the errors in retention factor determination.

Retention-pH profiles of acids and bases in hydrophilic interaction liquid chromatography.

The high proportion of acetonitrile used in many HILIC mobile phases significantly changes the acid-base properties of pH buffers and analytes foreseen from available data in water. In this paper, the recommended stability pH range for chromatographic columns is examined with various acetonitrile/water mixtures, resulting in a significant broadening in the operational pH window with the content of organic solvent. Additionally, the challenge of buffer selection in HILIC is also addressed. Commonly used ammonium acetate shrinks its pH buffering range in acetonitrile-rich mobile phases due to variations in the dissociation constants of the buffer constituents (acetic acid and ammonium). Thus, other organic acids such as formic acid, TFA, and succinimide have been studied as buffers in order to fully cover the pH range of use of the column. Also the retention-pH profiles of several acids and bases have been studied in 80% and 90% acetonitrile using the proposed buffers and their behavior compared to that obtained with buffers prepared from oxalic acid, pyrrolidine, and triethylamine. The latter two show additional interactions in 80% acetonitrile that distort the expected retention-pH profiles of acid analytes, but not the ones of bases. In 90% acetonitrile the profiles are affected by significant additional solute-buffer interactions that might be caused by ion pairing, homo- and heteroassociation in this low ion solvating medium.

Chasing the elusive hold-up time from an LFER approach.

A homologous series approach derived from the Abraham's solvation model was developed for the determination of hold-up times. Firstly, it was tested from reversed-phase liquid chromatography data obtained in the literature involving several series of homologues, followed by its application in a polymeric zwitterionic HILIC column using two different homologous series (n-alkyl benzenes and n-alkyl phenones). Acetonitrile and methanol were selected as organic modifiers in a composition range between 80% and 100% in volume. Results obtained for both series were consistent, and hold-up times were found to be strongly dependent on the water content and the organic modifier nature of the mobile phase.

Critical comparison of shake-flask, potentiometric and chromatographic methods for lipophilicity evaluation (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs.

In the present study three different procedures have been compared for the determination of the lipophilicity of the unionized species (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs. Shake-flask, potentiometric and chromatographic approaches have been assayed in a set of 66 representative compounds in different phases of advanced development. An excellent equivalence has been found between log Po/w values obtained by shake-flask and potentiometry, while the chromatographic approach is less accurate but very convenient for screening purposes when a high-throughput is required. In the case of zwitterionic and amphoteric compounds, either for shake-flask and chromatographic methods, the pH has to be accurately selected in order to ensure the compound to be in its neutral form.