Drug impurity profiling: Method optimization on dissimilar chromatographic systems: Part I: pH optimization of the aqueous phase.

The use of dissimilar chromatographic systems in drug impurity profiling can be very advantageous. Screening a new-drug impurity mixture on those systems not only enhances the chance that all impurities are revealed, but also allows choosing a suited system for further method development. In this paper several strategies were evaluated to predict the optimal pH (of the buffer used in the mobile phase) from the screening results. Four or five dissimilar stationary phases were screened at four pH values (between 2.5 and 9.4), in order to obtain maximal information about the composition of the sample and to select one column for the subsequent optimization. Different linear models (straight lines, 2nd and 3rd degree polynomials) based on these experiments were tested for their ability to predict the retention times (t(R)) of the impurities at intermediate pH values. The predicted t(R) values were then used to calculate minimal resolutions and eventually to select an optimal pH at which the highest minimal resolution is predicted. None of the applied models is accurate enough to predict correctly which peaks are worst separated at the indicated optimal pH. However, the best strategy (applying a second degree polynomial describing the t(R) measured at 3 consecutive screening pH values) did succeed in indicating an optimal pH at which a good separation of the impurities is obtained. Unfortunately, the resulting separation quality is not or only slightly better than the best separation obtained during screening. Therefore, it can be concluded that the most (time-) efficient approach to develop an impurity profile of a new drug is to screen it on four or five dissimilar columns at four different pH values and to retain the best screening conditions (without making predictions for intermediate conditions) for further optimization of the organic modifier composition of the mobile phase, and occasionally the temperature and the gradient. This is at least the case when the profiles have a complexity similar to those studied.

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.

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.

Orthogonality and similarity within silica-based reversed-phased chromatographic systems.

The starting point of this study was a current set of 32 chromatographic systems used to select initial conditions for method development to determine the impurity profile of a drug. The system exhibiting the best selectivity is then selected for further method development. In this current set eight silica-based phases are applied in conjunction with four mobile phases at different pH. In order to save time and resources, the possibilities for a meaningful subset selection were investigated. The most differing systems in terms of selectivity, in other words only the most orthogonal systems, need to be selected. Since the stationary phases are all silica-based, the selectivity differences are examined within a more homogeneous group than if, for instance, also zirconia- or polymer-based columns would be involved. To select the subset of systems also the best overall separation performances are taken into account. The selection is based both on the HPLC-DAD data of a generic set of 68 drugs, and on the LC-MS-DAD results for a mixture of 15 drugs, less different in structure. The orthogonality is evaluated using weighted-average-linkage dendrograms and color maps, both created from the Pearson-correlation coefficients r between normalized retention times r. The Derringer's desirability functions are applied to define the systems with the best overall separation performances. Proposals for different representative subsets of the initial 32 systems are made.

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.

Determination of diclazuril in animal feed by liquid chromatography.

A method is described for the determination of diclazuril (Janssen Research Compound R64433; trademark Clinacox) in chicken feed at the mg kg(-1) level. Compound R062646, a structure analogous to diclazuril, was used as the interna standard. The drug was extracted from food with acidified methanol. Diclazuril was then isolated by means of solid-phase extraction with a cartridge containing a C18 phase. The eluate was evaporated and the residue redissolved in dimethylformamide. An aliquot was injected onto a reversed-phase high-performance liquid chromatographic column and the drug substance quantified at 280 nm by an ultraviolet detector. Extraction (absolute) recoveries of 85% for both internal standard and diclazuril were obtained. The method is suitable for diclazuril concentrations ranging from 0.1 to 1.5 mg kg(-1). Method validation data are presented.