Orthogonal chromatographic descriptors for modelling Caco-2 drug permeability.

The use of chromatographic descriptors as alternative for Caco-2 permeability in drug absorption screening was evaluated. Therefore, retentions were measured on 17 Reversed-Phase Liquid Chromatographic systems, considered to be orthogonal or dissimilar, and an Immobilized Artificial Membrane (IAM) system. Retentions on a Micellar Liquid Chromatography system were taken from the literature. From this set of systems, those found dissimilar for the used data set were selected. The retention factors on these systems were then used as descriptors in QSAR modelling. Modelling was performed using Stepwise Multiple Linear Regression. This resulted in a model using only two chromatographic systems with good descriptive and acceptable predictive properties. A high qualitative model was obtained by combining both chromatographic systems selected in the previous model with a lipophilicity parameter (the squared Moriguchi n-octanol/water partition coefficient) and the molecular volume.

Development of a liquid chromatographic assay for an anti-HIV tablet containing lamivudine, zidovudine and TMC278.HCL.

A liquid chromatographic method was developed to analyse a tablet containing three anti-human immunodeficiency virus (HIV) compounds: lamivudine, zidovudine and a compound with the code name TMC278.HCl. Due to the presence of UV absorbing chromophores in the three active components, a single LC method with UV detection was developed. A Hypersil BDS C(18) column was used as stationary phase and the assay was performed with gradient elution using mobile phases containing acetonitrile, 0.2M potassium dihydrogen phosphate and water. The sample pretreatment is performed by treating the formulation with dimethyl sulfoxide-water (1:1) followed by filtration. After method development, the influence of the different chromatographic parameters on the separation, the interference of other active compounds and excipients, the repeatability and the linearity were investigated. The method was shown to be robust, selective, linear and repeatable. Finally, the content of the compounds in the tablet was determined.

Evaluation of orthogonal/dissimilar RP-HPLC systems sets for their suitability as method-development starting points for drug impurity profiles.

Orthogonal or dissimilar separation systems provide different selectivities and their application can facilitate the development of methods to identify and quantify impurities in a drug substance. Two sets of chromatographic systems potentially applicable for method development were evaluated using four drug/impurity profiles. The sets consist of orthogonal or dissimilar systems and systems with good overall separation properties, selected in earlier studies. The aim of this study is to evaluate these systems for selectivity differences in the impurity profiles. These differences should allow determining the number of compounds occurring in an impurity profile. Then, one or a very limited number of systems is to be proposed for further method development. To examine the selectivity changes and separation quality for each impurity profile, both the normalized retention times tau and the resolutions between pairs of consecutively eluting peaks were plotted on parallel axes, representing the systems. For each profile, several systems of the studied sets can serve as potential starting points for further method development. All impurities could be separated from the active substance and from each other on at least one system. However, for the different profiles, different systems were selected as best, which makes that each system in a given set has its importance, depending on the properties of the profile.

Dissimilar or orthogonal reversed-phase chromatographic systems: A comparison of selection techniques.

Developing an analytical separation procedure for an unknown mixture is a challenging issue. An important example is the separation and quantification of a new drug and its impurities. One approach to start method development is the screening of the mixture on dissimilar chromatographic systems, i.e. systems with large selectivity differences. After screening, the most suited system is retained for further method development. In a step prior to such strategy dissimilar chromatographic systems need to be selected. In this paper the performance of different chemometric selection approaches, described in the literature, was visually evaluated and compared. Additionally, orthogonal projection approach (OPA) was tested as another potential selection method. All techniques, including the OPA method, were able to select (a set of) dissimilar chromatographic systems and many similarities between the selections were observed. However, the Kennard and Stone algorithm performed best in selecting the most dissimilar systems in the earliest steps of the selection procedure. The generalized pairwise correlation method (GPCM) and the auto-associative multivariate regression trees (AAMRT) were also performing well. OPA and weighted pair group method using arithmetic averages (WPGMA) are less preferable.

Evaluation of chromatographic descriptors for the prediction of gastro-intestinal absorption of drugs.

The use of chromatographic descriptors in QSAR was evaluated. Therefore, retentions were measured on an immobilized artificial membrane system, 2 micellar liquid chromatography systems and 17 orthogonal or disimilar reversed-phase liquid chromatographic systems. It was investigated whether it was possible to model gastro-intestinal absorption as a function of chromatographic retentions applying two linear and one non-linear multivariate modeling technique. In a second step it was evaluated if models built with theoretical descriptors could be improved by adding the measured retention factors to the data set of descriptive variables. It was seen that gastro-intestinal absorption could be modelled in function of chromatographic retention using the non-linear modeling technique multivariate adaptive regression splines (MARS). The best models were obtained using a combination of theoretical and chromatographic descriptors with MARS as modeling technique.

Stationary phases in the screening of drug/impurity profiles and in their separation method development: identification of columns with different and similar selectivities.

The classification or characterization of stationary phases based on chromatographic parameters, in general, requires different test solutes/mixtures and several mobile phases. To simplify the classification/characterization of reversed-phase liquid chromatographic columns, to be used in separating drug/impurity profiles, a new test procedure was proposed. It consists of injecting two mixtures of relatively similar active substances applying a standard gradient. The aim was to evaluate from this approach the selectivity differences and overall separation quality of newly tested columns compared to that in an earlier selected set of eight stationary phases. The selectivity differences of the columns were evaluated by correlation coefficient-based weighted-average-linkage dendrograms and color maps. Derringer's desirability functions were used to rank similar stationary phases according to their overall separation quality. Four columns of 27 examined were, for instance, considered different from the earlier selected eight and could be added to the selection. A number of tested stationary phases might be considered as alternatives for some from the initial set. For three columns the newly tested stationary phases did not contain alternatives.

Evaluation of chemometric techniques to select orthogonal chromatographic systems.

Several chemometric techniques were compared for their performance to determine the orthogonality and similarity between chromatographic systems. Pearson's correlation coefficient (r) based color maps earlier were used to indicate selectivity differences between systems. These maps, in which the systems were ranked according to decreasing or increasing dissimilarities observed in the weighted-average-linkage dendrogram, were now applied as reference method. A number of chemometric techniques were evaluated as potential alternative (visualization) methods for the same purpose. They include hierarchical clustering techniques (single, complete, unweighted-average-linkage, centroid and Ward's method), the Kennard and Stone algorithm, auto-associative multivariate regression trees (AAMRT), and the generalized pairwise correlation method (GPCM) with McNemar's statistical test. After all, the reference method remained our preferred technique to select orthogonal and identify similar systems.

Selection of orthogonal reversed-phase HPLC systems by univariate and auto-associative multivariate regression trees.

In order to select chromatographic starting conditions to be optimized during further method development of the separation of a given mixture, so-called generic orthogonal chromatographic systems could be explored in parallel. In this paper the use of univariate and multivariate regression trees (MRT) was studied to define the most orthogonal subset from a given set of chromatographic systems. Two data sets were considered, which contain the retention data of 68 structurally diversive drugs on sets of 32 and 38 chromatographic systems, respectively. For both the univariate and multivariate approaches no other data but the measured retention factors are needed to build the decision trees. Since multivariate regression trees are used in an unsupervised way, they are called auto-associative multivariate regression trees (AAMRT). For all decision trees used, a variable importance list of the predictor variables can be derived. It was concluded that based on these ranked lists, both for univariate and multivariate regression trees, a selection of the most orthogonal systems from a given set of systems can be obtained in a user-friendly and fast way.

Selection of reversed-phase liquid chromatographic columns with diverse selectivity towards the potential separation of impurities in drugs.

To select appropriate stationary phases from the continuously expanding supply of potentially suitable HPLC columns, the properties of 28 frequently applied stationary phases were determined by measuring several chromatographic parameters. From these results, based on chromatographic expertise, eight stationary phases with different properties and selectivities were selected. The aim of this study is to apply chemometric tools to evaluate the initially selected set of columns, i.e. a more systematic approach for making such a selection is examined. Starting from the information obtained on the 28 stationary phases, the re-evaluation was performed independently based on the chemometric techniques Pareto-optimality, principal component analysis (PCA), and Derringer's desirability functions. The aim was to select a set of efficient columns exhibiting large selectivity differences. The chemometrically selected stationary phases were divided in groups based on hydrophobicity, a critical retention-determining property in reversed-phase chromatography. This allowed to further reducing the selection to three columns. It is demonstrated that the selection by the chemometric approaches in general is fairly comparable with the initial selection.

Determining orthogonal and similar chromatographic systems from the injection of mixtures in liquid chromatography-diode array detection and the interpretation of correlation coefficients color maps.

Generic orthogonal chromatographic systems might be helpful tools as potential starting points in the development of methods to separate impurities and the active substance in drugs with unknown impurity profiles. The orthogonality of 38 chromatographic systems was evaluated from weighted-average-linkage dendrograms and color maps, both based on the correlation coefficients between the retention factors on the different systems. On each chromatographic system, 68 drug substances were injected as mixtures of three or four components to increase the throughput. The (overlapping) peaks were identified and resolved with a peak purity algorithm, orthogonal projection approach (OPA). The visualization techniques applied allowed a simple evaluation of orthogonal and (groups of) similar systems.

Determining orthogonal chromatographic systems prior to the development of methods to characterise impurities in drug substances.

To define starting conditions for the development of methods to separate impurities from the active substance and from each other in drugs with an unknown impurity profile, the parallel application of generic orthogonal chromatographic systems could be useful. The possibilities to define orthogonal chromatographic systems were examined by calculation of the correlation coefficients between retention factors k for a set of 68 drugs on 11 systems, by visual evaluation of the selectivity differences, by using principal component analysis, by drawing color maps and evaluating dendrograms. A zirconia-based stationary phase coated with a polybutadiene (PBD) polymer and three silica-based phases (base-deactivated, polar-embedded and monolithic) were used. Besides the stationary phase, the influence of pH and of organic modifier, on the selectivity of a system were evaluated. The dendrograms of hierarchical clusters were found good aids to assess orthogonality of chromatographic systems. The PBD-zirconia phase/methanol/pH 2.5 system is found most orthogonal towards several silica-based systems, e.g. a base-deactivated C16 -amide silica/methanol/pH 2.5 system. The orthogonality was validated using cross-validation, and two other validation sets, i.e. a set of non-ionizable solutes and a mixture of a drug and its impurities.