Analytical Quality by Design in pharmaceutical quality assurance: Development of a capillary electrophoresis method for the analysis of zolmitriptan and its impurities.

A fast and selective CE method for the determination of zolmitriptan (ZOL) and its five potential impurities has been developed applying the analytical Quality by Design principles. Voltage, temperature, buffer concentration, and pH were investigated as critical process parameters that can influence the critical quality attributes, represented by critical resolution values between peak pairs, analysis time, and peak efficiency of ZOL-dimer. A symmetric screening matrix was employed for investigating the knowledge space, and a Box-Behnken design was used to evaluate the main, interaction, and quadratic effects of the critical process parameters on the critical quality attributes. Contour plots were drawn highlighting important interactions between buffer concentration and pH, and the gained information was merged into the sweet spot plots. Design space (DS) was established by the combined use of response surface methodology and Monte Carlo simulations, introducing a probability concept and thus allowing the quality of the analytical performances to be assured in a defined domain. The working conditions (with the interval defining the DS) were as follows: BGE, 138 mM (115-150 mM) phosphate buffer pH 2.74 (2.54-2.94); temperature, 25°C (24-25°C); voltage, 30 kV. A control strategy was planned based on method robustness and system suitability criteria. The main advantages of applying the Quality by Design concept consisted of a great increase of knowledge of the analytical system, obtained throughout multivariate techniques, and of the achievement of analytical assurance of quality, derived by probability-based definition of DS. The developed method was finally validated and applied to the analysis of ZOL tablets.

Fast analysis of glibenclamide and its impurities: quality by design framework in capillary electrophoresis method development.

A fast capillary zone electrophoresis method for the simultaneous analysis of glibenclamide and its impurities (I(A) and I(B)) in pharmaceutical dosage forms was fully developed within a quality by design framework. Critical quality attributes were represented by I(A) peak efficiency, critical resolution between glibenclamide and I(B), and analysis time. Experimental design was efficiently used for rapid and systematic method optimization. A 3(5)//16 symmetric screening matrix was chosen for investigation of the five selected critical process parameters throughout the knowledge space, and the results obtained were the basis for the planning of the subsequent response surface study. A Box-Behnken design for three factors allowed the contour plots to be drawn and the design space to be identified by introduction of the concept of probability. The design space corresponded to the multidimensional region where all the critical quality attributes reached the desired values with a degree of probability π ≥ 90%. Under the selected working conditions, the full separation of the analytes was obtained in less than 2 min. A full factorial design simultaneously allowed the design space to be validated and method robustness to be tested. A control strategy was finally implemented by means of a system suitability test. The method was fully validated and was applied to real samples of glibenclamide tablets.

Physicochemical properties and sorption capacities of sawdust-based biochars and commercial activated carbons towards ethoxylated alkylphenols and their phenolic metabolites in effluent wastewater from a textile district.

Three biochars were produced using sawdust from waste biomass, via a simple pyrolysis thermal conversion at 450, 650, and 850 °C (BC450, BC650, and BC850), without any activation process. These materials, together with vegetal and mineral commercial activated carbons (VAC and MAC), were characterized for their elemental composition, Brunauer-Emmett-Teller surface area, t-plot microporosity and Barrett-Joyner-Halenda mesoporosity. Moreover, iodine, phenol and methylene blue porosity indexes were measured. The materials were also evaluated for their pH of the point of zero charge, as well as near-surface chemical composition and surface functionality by means of X-ray photoelectron and Fourier-transform infrared spectroscopy. Ash content, water-extractable metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. BC650 showed a much higher surface area (319 m2 g-1) compared to BC450 (102 m2 g-1), as well as an increase in aromatization and the residual presence of functional polar groups. BC850 exhibited a loss of polar and aromatic groups, with the dominance of graphitic carbon and the highest value of surface area (419 m2 g-1). Biochars comply with the EN 12915-1/2009 limits for metal and PAH release in water treatment. Biochars and MAC were tested using Langmuir and Freundlich isotherms for the sorption in real effluent wastewater of a mixture of 14 branched ethoxylated 4-t-octyl and 4-nonylphenols, as well as 4-t-octyl and 4-nonylphenol, the latter representing persistent, endocrine disrupting contaminants, widespread in the effluents from wastewater treatment plants and listed as priority/priority hazardous substances in the Directive 2013/39/EU. Biochars showed a lower sorption efficiency compared to MAC. The best performance was found for BC650 towards the alkylphenols (9-13 times less efficient than the MAC). Considering the lower market price of biochar compared to MAC (estimated as at least 16 times less expensive by a small market survey), the former can be considered more competitive than the latter.

Removal efficiency and mass balance of polycyclic aromatic hydrocarbons, phthalates, ethoxylated alkylphenols and alkylphenols in a mixed textile-domestic wastewater treatment plant.

In this work the occurrence and fate of polycyclic aromatic hydrocarbons (PAHs), phthalic acid esters (PAEs), mono and diethoxylate alkylphenols (AP1-2EOs) and alkylphenols (APs) have been investigated during a two-weeks period in a facility treating mixed textile-domestic wastewater (Prato, Italy). The wastewater treatment plant (WWTP) consists of primary sedimentation, activated sludge biological oxidation, secondary sedimentation, clariflocculation and ozonation. The sludge is treated within the facility by thickening, dewatering and final incineration, thus providing the almost quantitative removal of the adsorbed micropollutants. Naphthalene (50%), di(2-ethylhexyl) phthalate (74%) and branched 4-nonylphenols (59%) were the individual main representative compounds of each class in the influent wastewater, which showed concentration ranges of 5.6-66, 85-290 and 21-133µg/L for PAHs, PAEs and APs+AP1-2EOs, respectively. The WWTP efficiently removed PAHs, PAEs and APs+AP1-2EOs, providing effluent concentrations of 0.075-0.16ng/L 0.38-9.9µg/L and 0.53-1.4µg/L. All targeted priority and priority-hazardous micropollutants showed effluent concentrations in line with the European environmental quality standards (EQS), even though for di(2-ethylhexyl) phthalate and benzo(a)pyrene after correction for the dilution factor of the recipient. The WWTP performance was evaluated by mass balance, verifying its accuracy by monitoring Pb and Cd as conservative species. The biological treatment sections provided mass losses of 85.5%, 74.5% and 56.8% for APs+AP1-2EOs, PAEs and PAHs, highlighting efficient biotransformation performances of the activated sludge process. However, for the more volatile PAHs (e.g. naphthalene), a significant contribution of stripping cannot be excluded. A remarkable mass loss was also determined in the ozonation stage for PAEs (72.9%) and especially PAHs (97.0%), whereas a lower efficiency was observed for APs+AP1-2EOs (41.3%). The whole plant allowed for obtaining an almost quantitative removal (96.7-98.4%) for all targeted compounds.

Chiral analysis of theanine and catechin in characterization of green tea by cyclodextrin-modified micellar electrokinetic chromatography and high performance liquid chromatography.

Monomeric catechins are important compounds in green tea accounting for potential bioactivity against a wide range of diseases. Besides catechins, l-Theanine (γ-glutamylethylamide), a characteristic amino acid in tea leaves, has become a further focus of the phytochemical research for the reported beneficial effects mainly on cognitive performance, emotional state and sleep quality. In the present study has been developed a CD-MEKC method based on sodium dodecyl sulfate (SDS) and Heptakis (2,6-di-O-methyl)-ß-cyclodextrin for the separation of six major green tea catechins and enantiomers of theanine. The latter, because of the poor detectability was derivatized prior analysis by o-phthaldialdehyde in the presence of N-acetyl-l-cysteine which, under mild conditions (neutral pH, in two minutes) allowed two diastereomers isoindole derivatives to be obtained. The derivatization reaction was directly carried out on tea infusion and derivatized samples were analysed by CD-MEKC involving 65 mM SDS and 28 mM cyclodextrin in acidic buffer (pH 2.5). The separation of six major green tea catechins including enantioresolution of (±)-Catechin and d/l-Theanine was obtained in about 5 min allowing d-Theanine to be quantified at least at 0.5% m/m level with respect to l-Theanine. Since (-)-Catechin and d-Theanine can be considered as non-native enantiomers (distomers), their presence in real samples provides an indication of tea leaves treatments (thermal treatment, fermentation, etc.) and could represent an opportunity for grading tea. The obtained results were confirmed by a RP-HPLC approach; even though the chromatography was developed in achiral conditions, the derivatization approach applied to theanine (diastereomers formation), allowed for d/l-Theanine chiral analysis.

Exploring the intermolecular interactions acting in solvent-modified MEKC by Molecular Dynamics and NMR: The effect of n-butanol on the separation of diclofenac and its impurities.

An integrated approach involving CE experiments, Molecular Dynamics (MD) simulations and two-dimensional NOE spectroscopy (2D-NOESY) experiments was employed to elucidate the intermolecular interactions and the separation mechanisms involved in a solvent-modified MEKC method for the simultaneous determination of diclofenac sodium and its impurities. The CE findings indicated that the addition of n-butanol (nBuOH) to the SDS micellar solution played a primary role for controlling the partitioning into the mixed micelles and the migration of the analytes and that the presence of nBuOH as cosurfactant was compulsory for achieving the complete separation of the compounds. The different capacity factors of the analytes were calculated and a change in solute association with the mixed micelle when changing the SDS/nBuOH molar ratio was highlighted. The optimal SDS/nBuOH molar ratio for the electrophoretic separation was 1:8. On the other hand, both MD simulations and NMR experiments indicated that the most favorable molar ratio for the formation of mixed SDS/nBuOH micelles was 1:2. These results suggested that probably there is an excess of nBuOH in the background electrolyte, both as free molecules and in form of aggregates, which is able to interact with the analytes, and thus may compete with mixed micelles for the considered compounds. The calculated values of gain in potential energy of the analytes when included in mixed micelles were in agreement with the observed migration order of the compounds. The role of methyl-ß-cyclodextrin (MßCyD) in the background electrolyte was also investigated, since the addition of this CyD to the solvent-modified MEKC system was found to be useful to reduce the analysis time. MD simulations and 2D-NOESY spectra highlighted the formation of inclusion complexes with MßCyD not only with the analytes, but also with SDS. MßCyD may lower the availability of both SDS and nBuOH for forming micelles and mostly may compete with the mixed micelle as a second pseudostationary phase.

Chiral capillary zone electrophoresis in enantioseparation and analysis of cinacalcet impurities: Use of Quality by Design principles in method development.

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity and of impurities of the chiral calcimimetic drug cinacalcet hydrochloride has been developed following Quality by Design principles. The scouting phase was aimed to select the separation operative mode and to identify a suitable chiral selector. Among the tested cyclodextrins, (2-carboxyethyl)-ß-cyclodextrin and (2-hydroxypropyl)-γ-cyclodextrin (HPγCyD) showed good chiral resolving capabilities. The selected separation system was solvent-modified capillary zone electrophoresis with the addition of HPγCyD and methanol. Voltage, buffer pH, methanol concentration and HPγCyD concentration were investigated as critical method parameters by a multivariate strategy. Critical method attributes were represented by enantioresolution and analysis time. A Box-Behnken Design allowed the contour plots to be drawn and quadratic and interaction effects to be highlighted. The Method Operable Design Region (MODR) was identified by applying Monte-Carlo simulations and corresponded to the multidimensional zone where both the critical method attributes fulfilled the requirements with a desired probability π≥90%. The working conditions, with the MODR limits, corresponded to the following: capillary length, 48.5cm; temperature, 18°C; voltage, 26kV (26-27kV); background electrolyte, 150mM phosphate buffer pH 2.70 (2.60-2.80), 3.1mM (3.0-3.5mM) HPγCyD; 2.00% (0.00-8.40%) v/v methanol. Robustness testing was carried out by a Plackett-Burman matrix and finally a method control strategy was defined. The complete separation of the analytes was obtained in about 10min. The method was validated following the International Council for Harmonisation guidelines and was applied for the analysis of a real sample of cinacalcet hydrochloride tablets.

Combined approach using capillary electrophoresis, NMR and molecular modeling for ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation and separation mechanism.

A comprehensive investigation on the CE separation mechanisms and on the inclusion complexation with CyDs of the chiral drug S-ambrisentan (S-AMB), its R-enantiomer and other impurities was performed by different techniques. A CE method was previously set up allowing the simultaneous determination of the enantiomeric purity and of impurities of S-AMB, based on the addition of SDS micelles and γ-cyclodextrin (γCyD) to borate buffer. In this study, the electrophoretic behavior of the analytes in terms of selectivity and mobility with respect to the addition of different CyDs was first investigated, evidencing the presence of interactions for all the CyDs, but the unique ability of γCyD for obtaining the separation of all the compounds. By molecular modeling, aggregates between SDS micelles and analytes, and inclusion complexes between CyDs, SDS and/or analytes of different stoichiometries were simulated. The potential and the gain energy of complexes were calculated on the minimized conformations, showing the great tendency of γCyD of forming mixed complexes with one or two SDS molecules and with the analyte, even if with different affinities among the analytes. For 1:1:1 mixed complexes with different CyDs, the highest difference of potential energy between the enantiomers' complexes was observed for γCyD. Two-dimensional NOE spectroscopy experiments were performed for S-AMB and I1 and pointed out the interactions of the aromatic moiety of the analytes and of SDS aliphatic chain with γCyD protons, confirming the existence of γCyD mixed complexes. The high affinity of SDS for the γCyD cavity was suggested to justify the fundamental role of SDS in modulating and achieving the CE separation, due to its influence both on the stability and on the type of complexes between γCyD and the analytes.

Chiral cyclodextrin-modified micellar electrokinetic chromatography and chemometric techniques for green tea samples origin discrimination.

Catechins and methylxanthines were determined in 92 green tea (GT) samples originating from Japan and China by using micellar electrokinetic chromatography with the addition of (2-hydroxypropyl)-ß-cyclodextrin. GT samples showed high concentrations of (-)-epigallocatechin gallate and caffeine, with (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epicatechin in relevant content and (+)-catechin, (-)-catechin and theobromine in much lower amounts. The amount of all the considered compounds was higher for Chinese GTs, with the exception of (-)-epicatechin gallate. Pattern recognition methods were applied to discriminate GTs according to geographical origin, which is an important factor to determine quality and reputation of a commercial tea product. Data analysis was performed by principal component analysis and hierarchical cluster analysis as exploratory techniques. Linear discriminant analysis and quadratic discriminant analysis were utilized as discrimination techniques, obtaining a very good rate of correct classification and prediction.

Cyclodextrin- and solvent-modified micellar electrokinetic chromatography for the determination of captopril, hydrochlorothiazide and their impurities: A Quality by Design approach.

A fast and selective capillary electrophoresis method has been developed for the simultaneous determination of the antihypertensive drugs captopril and hydrochlorothiazide and their related impurities in a combined dosage form. Method development was carried out implementing each step of Quality by Design workflow, the new paradigm of quality outlined in International Conference on Harmonisation Guidelines. Captopril is characterized by the lack of a strong chromophore and contains a proline-similar moiety, which gives rise to the presence of interconverting cis-trans isomers and leads to the possible interference between electrophoretic migration and reaction of isomerization. The scouting phase was dedicated to the investigation of several operative modes in order to overcome detection and isomerization issues. The best performances were obtained with sodium cholate-based micellar electrokinetic chromatography with the addition of n-butanol and γ-cyclodextrin. Critical quality attributes were represented by the critical resolution values and by analysis time. Critical process parameters were defined as temperature, voltage, concentration and pH of borate buffer, concentration of sodium cholate, n-butanol and γ-cyclodextrin. Screening experimental design was applied for investigating knowledge space. Response surface methodology pointed out several significant interaction effects, and with Monte-Carlo simulations led to map out the design space at a selected probability level. Robustness testing was carried out and a control strategy based on system suitability tests was defined. The selected working conditions gave a complete separation of the analytes in less than three minutes. The method was validated and applied to the analysis of a real sample of coformulation tablets.

Combination of capillary electrophoresis, molecular modeling and NMR to study the enantioselective complexation of sulpiride with double cyclodextrin systems.

The enantioselective complexation of sulpiride by a number of cyclodextrins (CDs) was deeply investigated by different techniques with the aim of evaluating the role of the used chiral selectors involved in the enantioseparation of the eutomer levosulpiride (S-SUL) and its dextro-isomer by capillary electrophoresis (CE). A CE method was previously developed with the aim of determining the optical purity of S-SUL and was based on the use of a dual cyclodextrin system, made by sulfated-ß-cyclodextrin (SßCD) and methyl-ß-cyclodextrin (MßCD). In this paper, a molecular modeling study made it possible to explain the different affinity of sulpiride enantiomers for several CDs, which had been tested during the early phase of CE method development. The potential and the gain energy of the inclusion complexes between the enantiomers and neutral and charged CDs were calculated on the minimized conformations. The calculated docking energies indicated that the most stable complexes were effectively obtained with SßCD and MßCD. A correlation between CE migration time of the last migrating enantiomer S-SUL and the stability of analyte-neutral CDs complexes was postulated. Furthermore, two-dimensional rotating-frame Overhauser effect spectroscopy NMR (2-D ROESY) experiments were carried out, which clearly indicated the formation of complexes and suggested the inclusion of the benzene sulfonamide moiety of S-SUL inside the hydrophobic cavity of the CDs.