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.

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.

The assay of a drug substance (DS) is one of the tests required to confirm the active pharmaceutical ingredient (API) quality at release. In the past, usually volumetric titration methods were performed, that were precise, but often non-specific. Nowadays specific chromatographic assay procedures are preferred. However, high performance liquid chromatographic (HPLC) methods, the way they are usually executed, tend to be less precise and have a larger total method variation compared to titration methods. The capabilities of fully validated titration and HPLC assay methods were determined and compared. It was studied which factors had the largest effects on the capability of chromatographic HPLC methods in order to improve their precision and precision-to-tolerance ratio. This was done using multiple Gage R&R (repeatability & reproducibility) studies and an experimental design approach. The investigations showed that it was feasible to define an HPLC method with a similar capability as the titration method. The most important factor determining the precision was demonstrated to be higher sample and reference material weights. When low weights are to be used, increasing the number of sample preparations and the number of reference solutions may enhance the method capability.

A set of 68 active pharmaceutical substances (mainly basic, but also some neutral and acidic) was earlier used to determine the orthogonality/similarity of chromatographic systems. The orthogonality of the systems was evaluated from correlation coefficients-based weighted-average-linkage (WAL) dendrograms and color maps. To increase the throughput in assessing systems, a representative subset of substances that leads to analogous conclusions about orthogonality/similarity was selected. Both the Kennard and Stone (K&S) algorithm applied on the autoscaled principal component analysis data from the Weighted Holistic Invariant Molecular (WHIM) descriptors of the test molecules, and WAL dendrograms on retention data were used for that purpose. A subset of 10 substances was found to give similar conclusions about orthogonality and similarity of the systems examined.

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.

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.

Application of the ICH procedure to the validation of a chiral capillary electrophoresis method for the quantification of the major stereoisomeric impurity of galantamine hydrobromide drug substance is discussed. Because the reproducibility of capillary electrophoresis as an analytical technique is often questioned, special emphasis was put on the robustness evaluation. The method was found to be accurate, precise and adequately robust, and consequently able to be transferred with confidence to other quality-control laboratories for the successful performance of the procedure.

Application of the ICH procedure to the validation of a chiral capillary electrophoresis method for the quantification of the major stereoisomeric impurity of galantamine hydrobromide drug substance is discussed. Because the reproducibility of capillary electrophoresis as an analytical technique is often questioned, special emphasis was put on the robustness evaluation. The method was found to be accurate, precise and adequately robust, and consequently able to be transferred with confidence to other quality-control laboratories for the successful performance of the procedure.

A robustness test was performed on a chromatographic method to identify and assay an active substance and two related compounds in film-coated tablet. For a number of responses the originally applied system suitability criteria were evaluated based on the results of the robustness test. Ambiguous situations can occur in situations where a method is found to be robust to assay the substances, as was the case here, but when system suitability criteria for some responses are violated. To avoid this, a proposal is made to define or re-define system suitability limits based on the results of the robustness test. From the effects found in the robustness test, the experimental conditions giving the worst result that still is acceptable and probable to occur are predicted and the system suitability limits are defined from replicated experiments in these conditions.

A Capillary Electrophoresis method was developed and applied successfully to test the quality of different drug formulations for release and stability studies. In the method an uncoated fused-silica capillary was employed containing a phosphoric acid buffer electrolyte which was brought to pH by triethylamine. The benzalkonium chlorides (BAC-C12 and BAC-C14) present in the standard were completely separated from each other and from the peaks of the main compound. Performance results of the method in terms of system repeatability, precision and accuracy are discussed.

The robustness of a method for the determination of vorozole in oral tablets was examined by applying a two-level, seven factor Plackett-Burman statistical experimental design. Five method variables that are sensitive to variation, especially during method transfer, were evaluated for their influence on the system suitability criteria set in the method procedure and on the analysis time. The method variables were investigated in a specified range above and below the nominal method conditions. They included the concentration of an ion-pairing agent, the percentage organic modifier at the start of the linear gradient, the mobile phase flow rate, the percentage organic modifier at the end of the linear gradient and the pH of the mobile phase. Two dummy factors were included in the design to estimate the experimental error. It was found that none of the five studied variables affected significantly (t-test, α=0.01) the capacity factor, the tailing factor or the analysis time. The resolution of the critical peak pair on the other hand, was significantly influenced by the factor pH. However, the responses for the resolution of all the experimental runs in the design were well above the system suitability limit stated in the normal assay procedure. Therefore, the method can overall be considered robust.

The separation of seventeen chlorophenol congeners and phenol was studied as a function of several variables. The pH and the concentration of sodium dodecylsulphate (SDS) were found to be important. During the implementation of a central composite design for the optimization of the separation it appeared that a part of the domain was not feasible as it resulted in very long migration times and extremely deformed peaks. Therefore, a D-optimal design was selected within the boundaries of the feasible region. The optimization of the selectivity did not result in selective regions for a simultaneous separation. It was, however, possible to find a region for the simultaneous separation of 15 compounds. Further optimization at these optimal conditions resulted in a separation where 17 peaks could be observed.

Method development and optimization procedures in capillary electrophoresis for small, mainly inorganic, ions are discussed. The selection of initial conditions and the optimization of selectivity are reviewed. The variables are grouped in three classes: variables related to the system, the sample, and the buffer electrolyte. The variables related to the buffer electrolyte are discussed in detail, as they are the ones primarily considered in selectivity adjustment. After dealing with the separation mechanisms for anions and cations, migration modelling procedures (both empirical and theoretical) are discussed. Examples of applications are provided.

In order to obtain a set of optimal experimental conditions for the separation of rare earth metal ions in capillary electrophoresis (CE), a multicriteria approach is applied. For this purpose, the Derringer's desirability function is proposed to determine conditions that will result in the most desirable combination of separation, sensitivity and analysis time. The CE separation method was optimized with the aid of a central composite design for two variables, i.e. the pH and the concentration of a complexing agent in the buffer electrolyte.

Nitrate and nitrite (and some other anions) were determined in vegetables by capillary electrophoresis (CE). The anions were extracted from the vegetables by mixing and diluting the samples with water at moderate temperature. The CE method is divided into two parts: a high-concentration-level method (for nitrate determination) and a low-concentration-level method (for nitrite determination). These CE methods were compared with a reference method (spectrophotometry after Jones reduction: official AOAC reference method for the determination of nitrates in foodstuffs). Parameters such as linearity, detection limit, quantification limit, precision and accuracy of the two techniques were investigated and compared. Both techniques resulted in acceptable linearity within their ranges. The detection limits of the CE methods were sufficiently low for the determination of the anions in vegetable samples. The precision and accuracy of the CE methods were comparable to those of the reference method. The precision was determined by evaluating the repeatability and the time-different intermediate precision, while the accuracy was investigated by comparing the slopes of the standard addition and external calibration lines and by evaluating the agreement between the results obtained with the CE and the reference spectrophotometric methods.

The separation of anions in capillary ion analysis below the critical micellar concentration of the modifier (cationic surfactant) is influenced by two chemical equilibria, the ionic dissociation which depends on the pH of the buffer electrolyte and the formation of ion association complexes in function of the concentration modifier. An expression of the mobility in function of the pH and concentration of the modifier is proposed and evaluated using experimental data. The experimental domain is examined with the aid of a central composite design. The model describes the mobility of anions in terms of physical and chemical constants of each ion (acid dissociation constant Ka, ion association constant Kia), the pH of the electrolyte, and the concentration of a modifier ([S]) in the buffer. The model is used to predict the mobility of each solute over a two-dimensional pH/[S] space.