Ana Vemić

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.

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 Derringers 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.

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities.

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.

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.

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.

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.

Design of Experiments–Design Space Approach for Development of Chaotropic Chromatography Method for Determination of Trimetazidine Dihydrochloride and Two Impurities

The quality by design approach has been applied to develop a chaotropic chromatography method for determination of trimetazidine dihydrochloride (TMZ) and two impurities (Y-145, Y-234). Baseline separation and accurate determination of the investigated analytes was set as the target analytical profile, with kY-145, kTMZ, and αY-145/TMZ selected as critical quality attributes. The critical process parameters studied in this research were pH of water phase, acetonitrile content in mobile phase, and concentration of perchloric acid in water phase. To compute a design space as a region of the knowledge space having satisfactory values of all critical quality attributes at the desired quality level, Monte Carlo simulations were performed to propagate the error originating from the calculated model coefficients to the predicted responses. At each of 4851 defined grid points, 5000 iterations were performed and the region having probability πxa0≥xa095% of achieving all defined critical quality attributes was computed. A working point situated in the center of the design space was chosen, corresponding to 27% acetonitrile in mobile phase, 63% 170xa0mM perchloric acid in water phase, and pH of water phase adjusted to 3.60. The method was validated to confirm its reliability for use in routine pharmaceutical analysis.Graphical Abstract

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.

Physicochemical factors governing the partition of pramipexole and its five impurities in microemulsion liquid chromatographic systems

Microemulsions when used as mobile phases represent a very powerful chromatographic tool due to their polyphase structure. The aim of this study was to investigate the influence of the mobile phase composition and resulting microemulsion droplets properties, as well as the stationary phase lipophilicity on the chromatographic behavior of pramipexole and its five impurities. Investigation was carried out according to the experimental plan defined by a full factorial design 24 with four central point replications. While the most affected by column characteristics was the last eluting, most lipophilic substance, the composition and characteristics of microemulsion droplets were equally important, so the partition system microemulsion droplets/stationary phase proved to be the most significant. Stationary phase lipophilicity did not affect the most hydrophilic component and only the partition system microemulsion droplets/bulk of the eluent impacted its behavior. For the separation of the critical pair, almost equal effects of the microemulsion droplets/stationary phase and bulk of the eluent/stationary phase partition systems were noted.

Systematical approach in evaluation of LC method for determination of raloxifene hydrochloride and its impurities employing experimental design

Method validation presents a detailed investigation of analytical method and provision of the evidence that the method, when correctly applied, produces results that fit to the purpose. In order to achieve the method validation scope efficiently, experimental design presents a very useful tool. The greatest benefits of such approach could be seen in robustness testing through the provision of very useful data about the control of the chromatographic system during the routine application. In this paper, robustness testing of the LC method proposed for the determination of raloxifene hydrochloride and its four impurities was done employing Plackett–Burman design. Applying this design, the effect of five real factors (acetonitrile content, sodium dodecyl sulfate content, column temperature, pH of the mobile phase and flow rate) on the corresponding resolution factors was investigated through twelve experiments. Furthermore, the insignificance intervals for significant factors were calculated and the parameters for system suitability tests were defined. Eventually, the other validation parameters were tested and the effectiveness of the proposed analytical method with a high degree of accuracy was confirmed.