A real-life pediatric experience of Crohn's disease exclusion diet at disease onset and in refractory patients.

OBJECTIVES: We aimed to appraise the real-life efficacy of Crohn's disease exclusion diet (CDED) coupled with partial enteral nutrition (PEN) in inducing clinical and biochemical remission at disease onset and in patients with loss of response to biologics and immunomodulators. METHODS: We retrospectively gathered data of patients aged less than 18 years of age with a diagnosis of Crohn's disease (CD), who received CDED coupled with PEN at a tertiary level pediatric inflammatory bowel disease center. RESULTS: Sixty-six patients were identified. Forty (60.6%) started CDED plus PEN at disease onset and 26 (39.4%) received CDED with PEN as add-on therapy. Forty-six (69.7%) patients achieved clinical remission (weighted Pediatric Crohn's Disease Activity Index < 12.5) at the end of Phase 1, 44 (66.7%) normalized c-reactive protein levels (<0.5 mg/dL) and 18 (27.2%) patients normalized calprotectin levels (<150 microg/g). Nine of 19 (47.3%) of patients with clinically severe disease (defined by Physician Global Assessment) achieved clinical remission at the end of Phase I. Patients with extraintestinal manifestations had statistically lower clinical response rates to the dietary regimen (p = 0.018). Among patients who received CDED + PEN as add-on treatment, a previous successful course of Exclusive Enteral Nutrition was associated with statistically higher clinical remission rates at Week 8 (p = 0.026). Clinical response at Week 4 was an independent predictor of clinical remission and fecal calprotectin normalization at Week 8 (p = 0.002). CONCLUSION: CDED with PEN confirmed its efficacy in a real-life setting, proving to be effective also in refractory patients and those with severe disease. Early clinical response predicts clinical remission at the end of Phase 1.

Endometriosis and the diagnosis of different forms of migraine: an association with dysmenorrhoea.

RESEARCH QUESTION: Women with endometriosis are frequently affected by headache. How many of these have a clear diagnosis of migraine? Are the different forms of migraine related to the phenotypes and/or characteristics of endometriosis? DESIGN: This was a prospective nested case-control study. A consecutive series of 131 women with endometriosis who attended the endometriosis clinic were enrolled and examined for the presence of headache. A headache questionnaire was used to determine the characteristics of the headaches, and the diagnosis of migraine was confirmed by a specialist. The case group included women with endometriosis and a diagnosis of migraine, while the control group included women with only endometriosis. History, symptoms and other comorbidities were collected. A pelvic pain score and associated symptoms were assessed using a visual analogue scale. RESULTS: A diagnosis of migraine was made in 53.4% (70/131) of participants. Pure menstrual migraine was reported by 18.6% (13/70), menstrually related migraine by 45.7% (32/70) and non-menstrual migraine by 35.7% (25/70). Dysmenorrhoea and dysuria were significantly more frequent in patients with endometriosis and migraine than in those without migraine (P = 0.03 and P = 0.01). No difference was found for other variables, including age at diagnosis and duration of endometriosis, endometriosis phenotype, the presence of other autoimmune comorbidities or heavy menstrual bleeding. In most patients with migraine (85.7%) the headache symptoms had started years before the diagnosis of endometriosis. CONCLUSION: The occurrence of headache in many patients with endometriosis is associated with the presence of different forms of migraine, is related to pain symptoms and often precedes the diagnosis of endometriosis.

Fully automated on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry for the simultaneous analysis of alkylphenol polyethoxylates and their carboxylic and phenolic metabolites in wastewater samples.

Three different sorbents (i.e. endcapped octadecylsilane, octasilane and styrene-N-vinylpiperidinone co-polymer) were investigated in order to develop an on-line solid phase extraction-liquid chromatographic tandem mass spectrometric method (on-line SPE-LC-MS/MS) for the simultaneous analysis of alkylphenols polyethoxylate (AP(n)EOs, n = 1-8) and corresponding monocarboxylate (AP1ECs) and phenolic (APs) metabolites. The endcapped octadecylsilane was selected due to its full compatibility with a chromatographic approach, which allowed the elution of positively and negatively ionisable compounds in two distinct retention time windows, using a water-acetonitrile-tetrahydrofuran ternary gradient and a pellicular pentafluorophenyl column. On this SPE sorbent, the composition of the loading/clean-up solution was then optimized in order to achieve the best recoveries of target analytes. Under the best experimental conditions, the total analysis time per sample was 25 min and method detection limits (MDLs) were in the sub-nanograms per litre to nanograms per litre range (0.0081-1.0 ng L(-1)) for AP(n)EOs with n = 2-8, AP1ECs and APs, whereas for AP1EOs, an MDL of about 50 ng L(-1) was found. Using the mass-labelled compound spiking technique, the method performance was tested on inlet and outlet wastewater samples from three activated sludge treatment plants managing domestic and industrial sewages of the urban areas and the textile district of Prato and Bisenzio valley (Tuscany, Italy); in most cases, apparent recovery percentages approximately in the ranges of 50-110% and 80-120% were found for inlet and outlet samples, respectively. The on-line SPE-LC-MS/MS analysis of wastewater samples highlighted the presence of target analytes at concentrations ranging from few nanograms per litre to thousands nanograms per litre, depending on the compound and matrix analysed. AP2ECs were also tentatively identified in outlet samples.

The successful binomium of multivariate strategies and electrophoresis for the Quality by Design separation of a class of drugs: the case of triptans.

Quality by Design (QbD) approach was followed having as analytical target profile the determination of different antimigraine drugs (seven triptans, TRP) available on the market. Multivariate strategies were used for defining the design space and solvent-modified MEKC was the selected analytical method. The versatility of electrophoretic technique, allowing a fine modulation of experimental parameters, made it possible to define the design space (DS). The DS limited a suitable range of experimental parameters in which all possible combinations of variables led to an electrokinetic method able to determine all the considered analytes with a predefined quality. Design of experiments and risk analysis fully assisted the method development from the initial investigation of MEKC knowledge space to the DS definition and finally to the control strategy. Applying the working operative conditions, the baseline separation of TRP was obtained in less than 9 min. The method was finally used for the quantification of three TRP in different pharmaceutical products.

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.

Analytical quality by design in the development of a cyclodextrin-modified capillary electrophoresis method for the assay of metformin and its related substances.

Quality by Design (QbD) is a new paradigm of quality to be applied to pharmaceutical products and processes, recently encouraged by International Conference on Harmonisation guidelines. In this paper QbD approach was applied to the development of a CE method for the simultaneous assay of metformin hydrochloride (MET) and its main impurities. QbD strategy was focused on electrophoretic process understanding, and the analytical method was thoroughly evaluated by applying risk assessment and chemometric tools. Method scouting allowed CD-CZE based on the addition of carboxymethyl-ß-CD to Britton-Robinson acidic buffer to be chosen as operative mode. Seven critical process parameters (CPPs) were selected, related to capillary, injection, BGE and instrumental settings. The effect of the different levels of the CPPs on critical quality attributes (CQAs), e.g. critical resolution values and analysis time, was evaluated in a screening study. Response surface methodology led to draw contour plots and sweet spot plots. The definition of design space was accomplished by applying Monte-Carlo simulations, thus identifying by risk of failure maps a multivariate zone where the CQAs fulfilled the requirements with a selected probability. Finally, a control strategy was designed and the method was applied to a real sample of MET tablets.

Optimization of hydrolysis conditions of amino acid analysis for UHPLC-UV antigens content determination: Bexsero vaccine a case study.

In the present study the compositional analysis of the amino acids released by the acidic hydrolysis of the vaccine antigens was approached as an alternative to the dye-binding methods, for improvement of the quality control. In particular, the Analytical Quality by Design principles were undertaken in optimizing the hydrolysis conditions of the antigens to be applied prior to the quantitation by UHPLC-UV. Bexsero was used as a case study; it is a recombinant meningococcal B vaccine and one of its critical quality attributes is the content of the three core protein antigens, namely Neisseria Heparin Binding Antigen, factor H binding protein and Neisseria adhesin A, in the final formulation. Conventionally, the proteins quantitation is carried out by dye-binding assays. Analytical Target Profile was defined as the accurate determination of amounts of the Bexsero antigens. The Critical Method Parameters were chosen by means of the cause-effect matrix. A Face Centered Design was used to select the experiments to investigate the process and finally a Method Operable Design Region with a risk of failure of 5% was defined. The selected working point for routine use was: hydrolysis time, 17 hrs; temperature, 112 °C; 6 M HCl volume, 300 µl; antioxidant 90% phenol volume, 5 µl.

Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling.

Electrokinetic chromatography (EKC) allows the separation of closely related substances by the detection of fine effects in analyte-separation system interactions. With the goal of understanding the fine effects involved in separation using a dual cyclodextrin-microemulsion EKC system, an integrated study of NMR and molecular modeling was carried out. The above dual cyclodextrin-microemulsion system was previously used in the separation of clemastine and its related substances and was prepared by the addition of methyl-ß-cyclodextrin (MßCD) and heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DMßCD) to an oil-in-water microemulsion. The use of DMßCD was shown to be essential in the separation of clemastine from one of its related substance (I(B) ). A molecular modeling study allowed the different affinities of clemastine and I(B) for the two cyclodextrins to be explained. Furthermore, rotating-frame Overhauser effect spectroscopy NMR experiments clearly indicated that besides the primary pseudostationary phase, namely the ionic microemulsion, cyclodextrins acted as a secondary pseudostationary phase. In addition, it was shown that inclusion complexation of sodium dodecyl sulfate (SDS) monomers into the cyclodextrins cavity occurs; differently, the oil (n-heptane) used in the preparation of microemulsion system resulted to be not included into the macrocycle cavity. These experimental results were supported by molecular modeling, which highlighted the preferential inclusion of SDS into DMßCD. On the basis of these results, it was confirmed that, besides its primary role as the ionic carrier in EKC, SDS is involved in inclusion equilibria toward CDs, which can be effective in increasing the system selectivity.

Analytical quality by design in the development of a solvent-modified micellar electrokinetic chromatography method for the determination of sitagliptin and its related compounds.

A solvent-modified micellar electrokinetic chromatography method was developed following the Quality by Design approach for the simultaneous determination of sitagliptin (SIT), an oral antihyperglycemic drug, and its main impurities derived from the synthesis process. The separation system was identified in the scouting phase and was made by sodium dodecyl sulphate (SDS) micelles with the addition of n-butanol and methanol. The knowledge space was investigated through an asymmetric screening matrix, taking into consideration eight critical method parameters (CMPs) involving the composition of the background electrolyte in terms of buffer concentration and pH, the concentration of surfactants and organic modifiers, and voltage. The critical method attributes (CMAs) were identified as analysis time and the distance between the tail of the electroosmotic flow system peak and the front edge of impurity I1 (sitagliptin triazole hydrochloride). A Box-Behnken Design was used in response surface methodology for calculating the quadratic models relating the CMPs to the CMAs. From the models it was possible to compute the method operable design region (MODR) through Monte-Carlo simulations. The MODR was identified in the probability maps as the multidimensional zone where the risk of failure to achieve the desired values for the CMAs was lower than 10 %. The experimental conditions corresponding to the working point, with the MODR interval, were the following: background electrolyte, 14 (10-18) mM borate buffer pH 9.20, 100 mM SDS, 13.6 (11.1-16.0) %v/v n-butanol, 6.7 (4.5-8.8) %v/v methanol; voltage and temperature were set to 28 kV and 22 °C, respectively. The developed CE method was validated in accordance with International Council for Harmonisation guidelines and was applied to the analysis of SIT tablets. The routine analysis for the quality control of the pharmaceutical product could be conducted in about 11 min.

Quality by Design as a risk-based strategy in pharmaceutical analysis: Development of a liquid chromatography-tandem mass spectrometry method for the determination of nintedanib and its impurities.

Nintedanib (NIN) is a tyrosine kinase inhibitor recently approved for the treatment of idiopathic pulmonary fibrosis. As a new drug, no monograph is available so far in official compendia. A liquid chromatography-tandem mass spectrometry method is presented for the simultaneous determination of NIN and its seven potential impurities. The risk-based approach of Analytical Quality by Design was applied in method development. The critical method parameters (CMPs) were the type of organic solvent in the mobile phase, formic acid percentage, column flow rate, oven temperature, gradient slope of organic eluent. The critical method attributes (CMAs) were selected as analysis time and selectivity between the main compound NIN and the adjacent peaks. Design of Experiments methodology was effectively employed for establishing the relationship between the CMPs and the CMAs. In the scouting step, a Restek Ultra AQ C18 (100 × 2.1 mm, 2.7 µm) core-shell column was selected, and then the effects of different levels of the five CMPs on the CMAs were evaluated by means of a 35//16 symmetric screening matrix. A Box-Behnken Design made it possible to obtain detailed maps of predicted CMAs throughout the investigated experimental domain, pointing out the presence of interaction and quadratic effects. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations, performing a risk analysis and drawing risk of failure maps, which were used to visualize and define the method operable design region (MODR) with a probability π ≥ 90%. The final working conditions (enclosing the MODR interval) were as follows: methanol as organic solvent; formic acid percentage, 0.15% v/v; flow rate, 0.40 mL min-1 (0.37-0.43 mL min-1); oven temperature, 40 °C (38-40 °C); gradient slope of organic eluent, 14.00% eluent B min-1 (12.85-15.15% eluent B min-1). The resulting analysis time was about 10 min. Validation was carried out according to International Council for Harmonisation guidelines and the optimized method was applied to the analysis of NIN soft capsules for quality control purposes.

Analytical quality by design: Development and control strategy for a LC method to evaluate the cannabinoids content in cannabis olive oil extracts.

Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) are considered as the most interesting cannabinoids in Cannabis sativa L. for the clinical practice. Since 2013, the Italian law allows pharmacists to prepare and dispense cannabis extracts to patients under medical prescription, and requires the evaluation of CBD and Δ9-THC content in cannabis extracts before sale. Cannabis olive oil extracts are prepared from dried female cannabis inflorescences, but a standard protocol is still missing. In this study, a fast RP-HPLC/UV method has been developed to quantify CBD and Δ9-THC in cannabis olive oil extracts. The analytical quality by design strategy has been applied to the method development, setting critical resolution and total analysis time as critical method attributes (CMAs), and selecting column temperature, buffer pH and flow rate as critical method parameters. Information from Doehlert Design in response surface methodology combined to Monte-Carlo simulations led to draw the risk of failure maps and to identify the method operable design region. The method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and then implemented in routine analysis. A control strategy based on system control charts was planned to monitor the developed method performances. Evaluation data were recorded over a period of one year of routine use, and both the CMAs showed values within the specifications in every analysis performed. Hence, a new risk evaluation for the future performances of the method was achieved by using a Bayesian approach based on the routine use data, computing the future distribution of the two CMAs. Finally, a study focusing on the monitoring of CBD and Δ9-THC concentrations in cannabis olive oil extracts was carried out. The developed method was applied to 459 extracts. The statistical analysis of the obtained results highlighted a wide variability in terms of concentrations among different samples from the same starting typology of cannabis, underlining the compelling need of a standardised procedure to harmonise the preparation of the extracts.

Development and Optimization by Quality by Design Strategies of Frovatriptan Orally Disintegrating Tablets for Migraine Management.

BACKGROUND: Frovatriptan is a potent anti-migraine agent with unfavourable slow onset of action, available on the market as film-coated tablets. OBJECTIVE: Optimization, by Quality by Designs strategies, of an orally disintegrating tablet (ODT) formulation of frovatriptan aimed to make its oral administration easier and its dissolution faster than the commercial tablets, thus improving its effectiveness in migraine management. METHOD: A screening D-optimal design was applied to investigate the effects of different levels of kind and amount of ODT special excipient and disintegrant agents (identified as the critical variables) on disintegration time (DT) and % drug dissolved at 30 s (%Diss), selected as the responses to optimize. The best excipients combination, emerged by the screening step, was in-depth investigated by a Response Surface Methodology. RESULTS: A design space was defined where every combination of the selected variables fulfilled the required values for the responses with P ≥ 95%. In particular, the optimized formulation (Pharmaburst® 60% and Na alginate 15%), showed DT = 1.62±0.08 s and %Diss= 9.02±0.47%, with good agreement between measured and calculated values. Moreover, the developed ODT complied with the USP uniformity weight and drug content requirements, exhibited proper hardness and low friability, and provided 100 % dissolved drug within 5 min. CONCLUSION: A frovatriptan ODT formulation was successfully developed by Quality by Design. It represents an effective alternative to conventional tablets, allowing easier oral administration (also to paediatric and geriatric people) and very faster drug dissolution, enhancing patient compliance and facilitating an earlier treatment of migraine attacks.

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.

Quality by design approach in the development of an ultra-high-performance liquid chromatography method for Bexsero meningococcal group B vaccine.

Bexsero is the first approved vaccine for active immunization of individuals from 2 months of age and older to prevent invasive disease caused by Neisseria meningitidis serogroup B. The active components of the vaccine are Neisseria Heparin Binding Antigen, factor H binding protein, Neisseria adhesin A, produced in Escherichia coli cells by recombinant DNA technology, and Outer Membrane Vesicles (expressing Porin A and Porin B), produced by fermentation of Neisseria meningitidis strain NZ98/254. All the Bexsero active components are adsorbed on aluminum hydroxide and the unadsorbed antigens content is a product critical quality attribute. In this paper the development of a fast, selective and sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of the Bexsero antigens in the vaccine supernatant is presented. For the first time in the literature, the Quality by Design (QbD) principles were applied to the development of an analytical method aimed to the quality control of a vaccine product. The UHPLC method was fully developed within the QbD framework, the new paradigm of quality outlined in International Conference on Harmonisation guidelines. Critical method attributes (CMAs) were identified with the capacity factor of Neisseria Heparin Binding Antigen, antigens resolution and peak areas. After a scouting phase, aimed at selecting a suitable and fast UHPLC operative mode for the vaccine antigens separation, risk assessment tools were employed to define the critical method parameters to be considered in the screening phase. Screening designs were applied for investigating at first the effects of vial type and sample concentration, and then the effects of injection volume, column type, organic phase starting concentration, ramp time and temperature. Response Surface Methodology pointed out the presence of several significant interaction effects, and with the support of Monte-Carlo simulations led to map out the design space, at a selected probability level, for the desired CMAs. The selected working conditions gave a complete separation of the antigens in about 5min. Robustness testing was carried out by a multivariate approach and a control strategy was implemented by defining system suitability tests. The method was qualified for the analysis of the Bexsero vaccine.

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.

Combining excitation-emission matrix fluorescence spectroscopy, parallel factor analysis, cyclodextrin-modified micellar electrokinetic chromatography and partial least squares class-modelling for green tea characterization.

In this study, an alternative analytical approach for analyzing and characterizing green tea (GT) samples is proposed, based on the combination of excitation-emission matrix (EEM) fluorescence spectroscopy and multivariate chemometric techniques. The three-dimensional spectra of 63 GT samples were recorded using a Perkin-Elmer LS55 luminescence spectrometer; emission spectra were recorded between 295 and 800 nm at excitation wavelength ranging from 200 to 290 nm, with excitation and emission slits both set at 10 nm. The excitation and emission profiles of two factors were obtained using Parallel Factor Analysis (PARAFAC) as a 3-way decomposition method. In this way, for the first time, the spectra of two main fluorophores in green teas have been found. Moreover, a cyclodextrin-modified micellar electrokinetic chromatography method was employed to quantify the most represented catechins and methylxanthines in a subset of 24 GT samples in order to obtain complementary information on the geographical origin of tea. The discrimination ability between the two types of tea has been shown by a Partial Least Squares Class-Modelling performed on the electrokinetic chromatography data, being the sensitivity and specificity of the class model built for the Japanese GT samples 98.70% and 98.68%, respectively. This comprehensive work demonstrates the capability of the combination of EEM fluorescence spectroscopy and PARAFAC model for characterizing, differentiating and analyzing GT samples.

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.