Influence of the mobile phase and molecular structure parameters on the retention behavior of protonated basic solutes in chaotropic chromatography.

In this study, we present novel insights into the pH-dependent retention behavior of protonated basic solutes in chaotropic chromatography. To this end, two sets of experiments were performed to distinguish between mobile phase pH and ionic strength effects. In the first set, the ionic strength (I) was varied with the concentration of NaPF6 and additives that adjusted the mobile phase pH, while in the second set, I was kept constant by adding the appropriate amount of NaCl. In each set, the retention behavior of 13 analytes was qualitatively examined in 21 chromatographic systems, which were defined by the NaPF6 concentration in their aqueous phases (1-50mM) and the pH of their mobile phases (2, 3 or 4); the acetonitrile content was fixed at 40%. The addition of NaCl significantly reduced the differences among retention factors at studied pH values due to the effect of the Na+ ions on PF6-adsorption to the stationary phase and the magnitude of the consequential development of the surface potential. A quantitative description of the observed phenomenon was obtained by an extended thermodynamic approach. The contribution of ion-pair formation in the stationary phase to the retention of the solutes was confirmed across models at the studied pH values in the set with varying I. In the systems with a constant I, the shielding effect of the Na+ ions on the surface charge lowered the attractive surface potential and diminished the aforementioned interactions and hence the effect of the mobile phase pH on analyte retention. Eventually, we developed a readily interpretable empirical retention model that simultaneously takes into account analyte molecular structures and the most relevant chromatographic factors. Its coefficients have clear physical meaning, and owing to its good predictive capabilities, the model could be successfully used to clarify the contributions of analyte molecular structures and chromatographic factors to the specific processes underlying separation in chaotropic chromatography.

Investigation into the phenomena affecting the retention behavior of basic analytes in chaotropic chromatography: Joint effects of the most relevant chromatographic factors and analytes' molecular properties.

The aim of this study was to systematically investigate the phenomena affecting the retention behavior of structurally diverse basic drugs in ion-interaction chromatographic systems with chaotropic additives. To this end, the influence of three factors was studied: pH value of the aqueous phase, concentration of sodium hexafluorophosphate, and content of acetonitrile in the mobile phase. Mobile phase pH was found to affect the thermodynamic equilibria in the studied system beyond its effects on the analytes' ionization state. Specifically, increasing pH from 2 to 4 led to longer retention times, even with analytes which remain completely protonated. An explanation for this phenomenon was sought by studying the adsorption behavior of acetonitrile and chaotropic additive onto stationary phase. It was shown that the magnitude of the developed surface potential, which significantly affects retention - increases with pH, and that this can be attributed to the larger surface excess of acetonitrile. To study how analytes' structural properties influence their retention, quantitative structure-retention modeling was performed next. A support vector machine regression model was developed, relating mobile phase constituents and structural descriptors with retention data. While the ETA_EtaP_B_RC and XlogP can be considered as molecular descriptors which describe factors affecting retention in any RP-HPLC system, TDB9p and RDF45p are molecular descriptors which account for spatial arrangement of polarizable atoms and they can clearly relate to analytes' behavior on the stationary phase surface, where the electrostatic potential develops. Complementarity of analytes' structure with that of the electric double layer can be seen as a key factor influencing their retention behavior. Structural diversity of analytes and good predictive capabilities over a range of experimental conditions make the established model a useful tool in predicting retention behavior in the studied chromatographic system.

Development of liquid chromatographic method for the analysis of dabigatran etexilate mesilate and its ten impurities supported by quality-by-design methodology.

In this paper, the development of reversed-phase liquid chromatographic method for the analysis of dabigatran etexilate mesilate and its ten impurities supported by quality by design (QbD) approach is presented. The defined analytical target profile (ATP) was the efficient baseline separation and the accurate determination of the investigated analytes. The selected critical quality attributes (CQAs) were the separation criterions between the critical peak pairs because the mixture complexity imposed a gradient elution mode. The critical process parameters (CPPs) studied in this research were acetonitrile content at the beginning of gradient program, acetonitrile content at the end of gradient program and the gradient time. Plan of experiments was defined by Box-Behnken design. The experimental domains of the three selected factors x1--content of the acetonitrile at the start of linear gradient, x2--content of the acetonitrile at the end of linear gradient and x3--gradient time (tG) were [10%, 30%], [48%, 60%] and [8 min, 15 min], respectively. In order to define the design space (DS) as a zone where the desired quality criteria is met providing also the quality assurance, Monte Carlo simulations were performed. The uniform error distribution equal to the calculated standard error was added to the model coefficient estimates. Monte Carlo simulation included 5000 iterations in each of 3969 defined grid points and the region having the probability π ≥ 95% to achieve satisfactory values of all defined CQAs was computed. As a working point, following chromatographic conditions suited in the middle of the DS were chosen: 22% acetonitrile at the start of gradient program, 55.5% acetonitrile at the end of gradient program end and the gradient time of 11.5 min. The developed method was validated in order to prove its reliability.

The influence of salt chaotropicity, column hydrophobicity and analytes' molecular properties on the retention of pramipexole and its impurities.

The aim of this study was to examine the interaction of the chaotropic salts of different position in Hofmeister series (CF3COONa, NaClO4, NaPF6) added to the mobile phase with the stationary phases of different hydrophobicity (C8 and C18 XTerra(®) columns), as well as their common influence on the retention behavior of pramipexole and its structurally related impurities. The extended thermodynamic approach enabled the understanding of the underlying separation mechanism. Comparing six different column-salt systems it was observed that general system hydrophobicity presented by salt chaotropicity and column hydrophobicity favors stationary phase ion-pairing over the ion-pair formation in the eluent. Further, an attempt was made to describe the influence of analytes' nature on their retention behavior in such chromatographic systems. An analysis is performed in order to select and elucidate the molecular descriptors (electrostatical, quantum-chemical, geometrical, topological, and constitutional) that best explain the experimental evidence and findings obtained by the thermodynamic approach. The results of this analysis suggest that analytes' charge distribution and its complementarity to the structure of the electric double layer formed on the surface of the stationary phase upon the addition of chaotropic additives can be useful for understanding the differences in retention of structurally related analytes. These findings provide a novel understanding of the interactions between all the components of the chromatographic system containing chaotropic additive and a good basis for further investigations suggesting the development of generally applicable predictors in structure-retention relationship studies in related chromatographic systems.

Chaotropic salts in liquid chromatographic method development for the determination of pramipexole and its impurities following quality-by-design principles.

The aim of this paper is to present a development of liquid chromatographic method when chaotropic salts are used as mobile phase additives following the QbD principles. The effect of critical process parameters (column chemistry, salt nature and concentration, acetonitrile content and column temperature) on the critical quality attributes (retention of the first and last eluting peak and separation of the critical peak pairs) was studied applying the design of experiments-design space methodology (DoE-DS). D-optimal design is chosen in order to simultaneously examine both categorical and numerical factors in minimal number of experiments. Two ways for the achievement of quality assurance were performed and compared. Namely, the uncertainty originating from the models was assessed by Monte Carlo simulations propagating the error equal to the variance of the model residuals and propagating the error originating from the model coefficients' calculation. The baseline separation of pramipexole and its five impurities is achieved fulfilling all the required criteria while the method validation proved its reliability.

Testing the capability of a polynomial-modified gaussian model in the description and simulation of chromatographic peaks of amlodipine and its impurity in ion-interaction chromatography.

In this paper, the capability of a polynomial-modified Gaussian model to relate the peak shape of basic analytes, amlodipine, and its impurity A, with the change of chromatographic conditions was tested. For the accurate simulation of real chromatographic peaks the authors proposed the three-step procedure based on indirect modeling of peak width at 10% of peak height (W0.1), individual values of left-half width (A) and right-half width (B), number of theoretical plates (N), and tailing factor (Tf). The values of retention factors corresponding to the peak beginning (k(B)), peak apex (k(A)), peak ending (k(E)), and peak heights (H0) of the analytes were directly modeled. Then, the investigated experimental domain was divided to acquire a grid of appropriate density, which allowed the subsequent calculation of W0.1, A, B, N, and Tf. On the basis of the predicted results for Tf and N, as well as the defined criteria for the simulation the following conditions were selected: 33% acetonitrile/67% aqueous phase (55 mM perchloric acid, pH 2.2) at 40°C column temperature. Perfect agreement between predicted and experimental values was obtained confirming the ability of polynomial modified Gaussian model and three-step procedure to successfully simulate the real chromatograms in ion-interaction chromatography.

The influence of inorganic salts with chaotropic properties on the chromatographic behavior of ropinirole and its two impurities.

Chaotropic agents recently gained popularity as interesting and useful mobile phase additives in liquid chromatography due to their effect on analytes retention, peak symmetry and separation efficiency. They mimic the role of classical ion-pairing agents, but with less drawbacks, so their use becomes attractive in the field of pharmaceutical analysis. In this paper, the influence of sodium trifluoroacetate and sodium perchlorate on the chromatographic behavior of ropinirole and its impurities is examined. By the extended thermodynamic approach, it was shown that the separation in the given system was predominantly governed by electrostatic interactions between the protonated analytes and the charged surface of the stationary phase, but the ion-pair complex formation in the eluent also proved to be significant. Further, the employment of face-centered central composite design enabled the understanding of the effect of chaotropic agent concentration and its interactions with other factors (acetonitrile content and pH of the water phase) that influence the given chromatographic system. Finally, the same data was used for multi-objective optimization based on the grid point search method. After the method validation, the adequacy of the suggested approach in development of methods for routine pharmaceutical analysis was proven.

Chemometrical tools in the study of the retention behavior of azole antifungals.

Certain chemometrical tools allow an efficient way to provide valuable data to evaluate the retention behavior of analytes in liquid chromatography. In this study of the retention behavior of azole antifungals, the experimental design was applied in combination with artificial neural networks (ANNs). Three potentially significant factors (methanol content, pH of the mobile phase and column temperature) were incorporated in the plan of experiments, defined by central composite design. As the system outputs, the retention factors of all six investigated substances (fluconazole, ketoconazole, bifonazole, clotrimazole, econazole and miconazole) were determined. The pattern for the analyzed behavior of the system was created by employing ANNs. The final, optimized topology of the highly predictive network was 3-8-6. Twelve experiments were used in a training set, whereas a back-propagation algorithm was optimal for network training. The ability of the defined network to predict the retention of the investigated azoles was confirmed by correlations higher than 0.9912 for all analytes. The presented approach allowed the adequate prediction of the retention behavior of azoles, in addition to the extraction of important information for a better understanding of the analyzed system.

Chaotropic agents in liquid chromatographic method development for the simultaneous analysis of levodopa, carbidopa, entacapone and their impurities.

The simultaneous pharmaceutical analysis of multi-component drugs represents a challenge due to a large total number of analytes present in the sample. These analytes are not only the active pharmaceutical ingredients, but also the impurities that might follow the active substances. The aim of this study was to develop an efficient reversed-phase LC method for the simultaneous analysis of antiparkinsonian drugs levodopa, carbidopa and entacapone along with their six related impurities. For the achievement of desirable separation, different acids with anions possessing different properties according to Hofmeister classification (ortho-phosphoric, trifluoroacetic and perchloric acid) were tested. Finally, in order to draw the unbiased conclusions when optimizing the analytical method, for the final tuning of the gradient program, Box-Behnken experimental design and Derringer's desirability function were used. The experiments were performed on Zorbax Extend C18, 150 mm × 4.6 mm, 5 µm particle size column with the UV detection at 280 nm and mobile phase flow rate of 1 mL/min. The optimal mobile phase consisted of methanol and 20mM trifluoroacetic acid (pH 2.0 adjusted with NaOH), while their ratio is changed according to previously defined gradient program. The method was tested for selectivity, sensitivity, linearity, accuracy and precision, and proved to be suitable for routine qualitative and quantitative analysis of levodopa, carbidopa, entacapone and their impurities in their mixture.

Validation of a column liquid chromatographic method for the analysis of pramipexole and its five impurities.

In this paper, a previously optimized method for HPLC analysis of pramipexole and its impurities was subjected to method validation in accordance with official regulations. The optimized chromatographic conditions were as follows: mobile phase acetonitrile-water phase [15 + 85, v/v, water phase contained 1% triethylamine (TEA), pH adjusted to 7.0 with orthophosphoric acid]; detection at 262 nm for pramipexole, BI-II 751 xx, BI-I 786 BS, BI-II 820 BS, and 2-aminobenzothiazole and at 326 nm for BI-II 546 CL; column temperature, 25 degrees C; and flow rate, 1 ml/min. Acetonitrile and TEA content, pH of the water phase, flow rate, column temperature, and column type were factors studied in robustness testing. According to the experimental plan defined by a Plackett-Burman design, five dummy variables were added in order to have 12 factors. As output, resolution factor was chosen. Robustness was assessed by graphical (half-normal probability plots and Pareto charts) and statistical (t-test) methods. Also, nonsignificance intervals for significant factors were estimated, and limits for the system suitability test were determined. Finally, linearity, accuracy, and precision of the proposed HPLC method were defined. LOD and LOQ values for analyzed impurities were determined. The method was completely defined by these experiments.