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 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.

Modeling Aquatic Toxicity through Chromatographic Systems.

Environmental risk assessment requires information about the toxicity of the growing number of chemical products coming from different origins that can contaminate water and become toxicants to aquatic species or other living beings via the trophic chain. Direct toxicity measurements using sensitive aquatic species can be carried out but they may become expensive and ethically questionable. Literature refers to the use of chromatographic measurements that correlate to the toxic effect of a compound over a specific aquatic species as an alternative to get toxicity information. In this work, we have studied the similarity in the response of the toxicity to different species and we have selected eight representative aquatic species (including tadpoles, fish, water fleas, protozoan, and bacteria) with known nonspecific toxicity to chemical substances. Next, we have selected four chromatographic systems offering good perspectives for surrogation of the eight selected aquatic systems, and thus prediction of toxicity from the chromatographic measurement. Then toxicity has been correlated to the chromatographic retention factor. Satisfactory correlation results have been obtained to emulate toxicity in five of the selected aquatic species through some of the chromatographic systems. Other aquatic species with similar characteristics to these five representative ones could also be emulated by using the same chromatographic systems. The final aim of this study is to model chemical products toxicity to aquatic species by means of chromatographic systems to reduce in vivo testing.

Microemulsion electrokinetic chromatography as a suitable tool for lipophilicity determination of acidic, neutral, and basic compounds.

In the present work, several MEEKC systems are studied to assess their suitability for lipophilicity determination of acidic, neutral, and basic compounds. Thus, several microemulsion compositions over a wide range of pH values (from 2.0 to 12.0), containing heptane, 1-butanol and different types and amounts of surfactant (SDS or sodium cholate: from 1.3 to 3.3%) are characterized using Abraham's solvation model. The addition of acetonitrile (up to 10%) is also studied, since it increases the resolution of the technique for the most lipophilic compounds. The system coefficients obtained are very similar to those of the 1-octanol/water, used as the reference lipophilicity index, allowing simple and linear correlations between the 1-octanol/water partition values (log Po/w ) and MEEKC mass distribution ratios (log kMEEKC ). Variations in the microemulsion composition (aqueous buffer, surfactant, concentration of ACN) did not significantly affect the similarity of the MEEKC systems to log Po/w partition.

Novel Instrument for Automated pK(a) Determination by Internal Standard Capillary Electrophoresis.

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

Unified pH values of liquid chromatography mobile phases.

This work introduces a conceptually new approach of measuring pH of mixed-solvent liquid chromatography (LC) mobile phases. Mobile phase pH is very important in LC, but its correct measurement is not straightforward, and all commonly used approaches have deficiencies. The new approach is based on the recently introduced unified pH (pH(abs)) scale, which enables direct comparison of acidities of solutions made in different solvents based on chemical potential of the proton in the solutions. This work represents the first experimental realization of the pH(abs) concept using differential potentiometric measurement for comparison of the chemical potentials of the proton in different solutions (connected by a salt bridge), together with earlier published reference points for obtaining the pH(abs) values (referenced to the gas phase) or pH(abs)(H2O) values (referenced to the aqueous solution). The liquid junction potentials were estimated in the framework of Izutsu's three-component method. pH(abs) values for a number of common LC and LC-MS mobile phases have been determined. The pH(abs) scale enables for the first time direct comparison of acidities of any LC mobile phases, with different organic additives, different buffer components, etc. A possible experimental protocol of putting this new approach into chromatographic practice has been envisaged and its applicability tested. It has been demonstrated that the ionization behavior of bases (cationic acids) in the mobile phases can be better predicted by using the pH(abs)(H2O) values and aqueous pKa values than by using the alternative means of expressing mobile phase acidity. Description of the ionization behavior of acids on the basis of pH(abs)(H2O) values is possible if the change of their pKa values with solvent composition change is taken into account.

Determination of acidity constants of sparingly soluble drugs in aqueous solution by the internal standard capillary electrophoresis method.

A set of 33 drugs with different solubilities, ranging from soluble to very insoluble, has been chosen in order to evaluate the performance of the internal standard CE method to determine acidity constants of compounds with limited solubility. The set of drugs tested in this work has been chosen as a function of their intrinsic solubility. For the most insoluble compounds, several analytical conditions to overcome the insolubility in aqueous buffers have been tested. This paper assesses the compound solubility limits for the IS-CE method in aqueous pKa determinations, and also compares the determined pKa s with the results from the literature data obtained by other methods. It is proved that IS-CE method determines acidity constants of sparingly soluble drugs in aqueous media (compounds with logS down to around -6), whereas other reference methods require the use of aqueous-organic solvent buffers and extrapolation procedures to obtain the aqueous pKa for the same compounds.

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).

Temperature variation effects on the determination of acidity constants through the internal standard-capillary electrophoresis method.

The internal standard (IS) CE (IS-CE) method is an interesting alternative to other methods for the determination of acidity constants of compounds. Although some of the advantages of this method have been already reported, the method has not been tested yet as regards to temperature effects. This has been the aim of this work, where it is demonstrated that the method can be applied successfully for the determination of pK(a) s at different temperatures, if the acidity constant of the IS at the desired temperature is known. The fact of obtaining the acidity constants at different temperatures allows the calculation of some thermodynamic quantities, such as the molar standard enthalpy and the molar standard entropy in a fast way. It is also demonstrated that if the IS and the test compound have similar standard enthalpy increment, the IS compensates uncontrolled possible temperature fluctuations (e.g., due to Joule heat) inside the capillary obtaining reliable acidity constant values at the desired temperature.

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.

Modeling nonspecific toxicity of organic compounds to the fathead minnow fish by means of chromatographic systems.

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.

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.

Estimation of biological properties by means of chromatographic systems: evaluation of the factors that contribute to the variance of biological-chromatographic correlations.

The performance of chromatographic systems to emulate biological systems is evaluated in terms of the precision that can be achieved. The variance obtained when biological parameters are correlated against physicochemical ones can be decomposed in three terms: the variance of the biological data, the variance of the physicochemical data, and the variance caused by the dissimilarity between the two correlated systems (biological and physicochemical). The three terms contribute to the overall variance observed when measurements in chromatographic systems are correlated with experimental biological properties. The Abraham linear free energy relationships (LFERs) provide a very good approach to characterize biological and physicochemical systems and thus the variance of the analyzed data and the similarity/dissimilarity between them. The contribution of the three variances to the precision of the biological parameter estimated in this way is evaluated from the characterization of the biological and chromatographic systems by means of the Abraham model. The proposed method is able to estimate the goodness of chromatographic systems to predict particular biological properties. In particular, this method is illustrated by comparison of toxicity data (-log LC(50)) for the fish fathead minnow with retention data (log k) in several micellar electrokinetic chromatography (MEKC) systems and also by correlations between retention data (log k) in the sodium taurocholate (STC) MEKC system and data of several biological systems.

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.

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 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.