High-throughput log P determination by MEEKC coupled with UV and MS detections.

A high-throughput screening method using MEEKC was developed for the determination of 1-octanol-water partition coefficients (log P(oct)). Two approaches were carried out to decrease determination times to about 20 min per compound: (i) a dynamically coated capillary was used to increase the EOF at low pH, allowing the measurement of log P(oct) of acidic compounds and (ii) a short-end injection was performed to reduce the capillary effective length. The analytical conditions were optimized to determine the lipophilicity of neutral, basic, and acidic compounds with log P(oct) ranging from 0 to 5. The developed method was first applied to a well-balanced set of 35 reference compounds, and second to a set of 21 acidic and 29 basic pharmaceutical compounds. Finally, determinations were achieved with MS detection, allowing a 20-fold throughput increase thanks to sample pooling. An atmospheric pressure photoionization source was selected to advantageously replace ESI as it was less affected by the non-volatile BGE additives used in MEEKC.

Analytical tools for the physicochemical profiling of drug candidates to predict absorption/distribution.

The measurement of physicochemical properties at an early phase of drug discovery and development is crucial to reduce attrition rates due to poor biopharmaceutical properties. Among these properties, ionization, lipophilicity, solubility and permeability are mandatory to predict the pharmacokinetic behavior of NCEs (new chemical entities). Due to the high number of NCEs, the analytical tools used to measure these properties are automated and progressively adapted to high-throughput technologies. The present review is dedicated to experimental methods applied in the early drug discovery process for the determination of solubility, ionization constants, lipophilicity and permeability of small molecules. The principles and experimental conditions of the different methods are described, and important enhancements in terms of throughput are highlighted.

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections.

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 microm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P(oct) measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as beta-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-microm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application.

Lipophilicity determination of highly lipophilic compounds by liquid chromatography.

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P(oct) from 0 to 5. The best results in HPLC were obtained with the 20-mm Discovery RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30-mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10-mm Hypersil GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.

High-throughput log P determination by ultraperformance liquid chromatography: a convenient tool for medicinal chemists.

Accurate determinations of lipophilicity indices benefit from recent advances in chromatographic sciences such as the launch of ultraperformance liquid chromatography (UPLC). The fast strategy presented here emerges as a powerful method suitable for high-throughput log P measurements of therapeutic compounds in isocratic and gradient modes. Because UPLC columns are highly stable in basic pH conditions, this approach allows a direct lipophilicity estimation of basic compounds in their neutral forms.

A fast screening strategy for characterizing peptide delivery by transdermal iontophoresis.

Capillary zone electrophoresis (CZE) is a convenient experimental tool for mimicking the low-throughput in vitro skin model used to optimize the delivery of peptides by transdermal iontophoresis. This paper is devoted to the extraction of pertinent molecular parameters from CZE experiments at different pH values, the optimization of CZE experimental conditions, and the development of an in silico filter useful for drug design and development. The effective mobility (mu(eff)) of ten model dipeptides was measured by CZE at different pH values, enabling to determine their pK(a) values, charge and mu(eff) at any pH. The best linear correlation between the electromigration contribution to transdermal iontophoretic flux (J(EM)) measured across porcine skin with donor and acceptor compartments at pH 7.4 and charge/MW ratio was obtained at pH 6.5, which seems to be the most suitable pH to mimic the in vitro skin model. Therefore, the experimental strategy can be considerably shortened by using a single mu(eff) measurement at pH 6.5 as a predictor of J(EM). Additionally, pK(a) prediction software packages offer a fast access to charge/MW ratio using consensual molecular charges at pH 6.5, which suggests that this simple in silico filter can be used as a preliminary estimation of J(EM).

Rapid determination of pK (a) values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections.

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK (a)) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C(4)D) was used as an additional detection mode. The C(4)D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK (a) determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK (a) values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK (a) values were in accordance with the literature.

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure.

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.