Assessment of bioaccumulation of glyphosate and aminomethylphosphonic acid in marine mussels using capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Glyphosate or N-(phosphonomethyl)glycine, widely used as herbicide in agriculture to control weeds and to facilitate harvesting, has been included in Group 2A pollutants (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC). In intensive agricultural areas, runoff and soil leaching are likely to drive glyphosate to surface waters, where the compound is often detected together with its main microbial metabolite, aminomethylphosphonic acid (AMPA). In the present study a method based on capillary electrophoresis coupled with light-emitting diode-induced fluorescence detection has been developed and validated for the determination of the two compounds in whole soft mass of marine mussels (Mytilus galloprovincialis). The method is based on the acidic hydrolysis of lyophilized tissue using 6 M HCl (oven at 110 °C for 22 h) to release the target analytes; their subsequent derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole, was found to be suitable for the sensitive fluorescence detection. To achieve optimum separation of the analytes from the matrix and degradation reagent interferences, the background electrolyte constituted by borate buffer (pH 9.2, 30 mM) was supplemented with 10 mM heptakis(2,6-di-O-methyl)-ß-cyclodextrin. The method was validated for linearity, precision, accuracy, robustness and sensitivity showing LOQ of 0.2 and 1.0 µg/g in fresh tissues, for AMPA and glyphosate, respectively; the recovery values ranged within 88.5 - 94.6% for glyphosate and 70.4 - 76.6% for AMPA. Experimental samples of Mediterranean mussels M. galloprovincialis treated with 100 µg/L or 500 µg/L of both glyphosate and AMPA, showed a dose dependent bioaccumulation of the compounds reaching maximum level of 77.0 µg/g and 11.3 µg/g of AMPA and glyphosate, respectively. The study demonstrates for the first time M. galloprovincialis as potential sentinel organisms for the environmental occurrence of these small amphoteric pollutants.

Quality by Design in optimizing the extraction of (poly)phenolic compounds from Vaccinium myrtillus berries.

Quality by Design was adopted for developing an effective extraction procedure of (poly)phenolic compounds from bilberry (Vaccinium myrtillus L.) fruits, using a pooled sample of berries from different regions of Ukraine. Mechanical solvent extraction, microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) were investigated by screening matrices. Extraction time (Time, from 5 to 15 min), organic solvent type (OS type, methanol, ethanol and acetone), organic solvent percentage (OS%, from 50% to 90%), sample/extractant ratio (S/E ratio, from 0.025 to 0.1 g mL-1), and, only for MAE, extraction temperature (T, from 30 to 60°C), were selected as critical method parameters (CMPs). The spectrophotometric assays total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), and radical scavenging activity (evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), the 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), and the ferric reducing antioxidant power methods) were chosen as critical method attributes (CMAs). The screening procedure allowed for selecting UAE and methanol, while the other CMPs underwent further optimization through Response Surface Methodology. Target values for TSP, TMA and DPPH were selected and the method operable design region (MODR) was defined by means of Monte-Carlo simulations. The optimized conditions, with the corresponding MODR intervals in bracket, were the following: (i) Time, 17 min (15-23 min); OS%, 56% (44-59%); S/E ratio, 0.030 (0.022-0.034) g mL-1. Under these experimental conditions, CMAs values of the pooled sample were the following (n = 3): TSP=4433±176 mg (+)-catechin eq/100 g dry weight (d.w.); TMA=3575±194 mg cyanidin-3-glucoside eq/100 g d.w.; DPPH=273±5 µg DPPH inhib./mg d.w. The optimized extraction method was tested for matrix effect (ME%) in the UHPLC-MS/MS analysis of 15 anthocyanins and 20 non-anthocyanins individual (poly)phenols commonly found in bilberries, as well as for luteolin, sinapic acid, and pelargonidin-3-glucoside, absent in this fruit and therefore added to the extracts as surrogate standards for evaluating apparent recovery (AR%). |ME%| was in any case ≤ 23% and AR% of the surrogate standards in the range 91-95%, confirming the very good performances of the optimized extraction method.

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.

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 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.

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.

Amidated pectin-based wafers for econazole buccal delivery: formulation optimization and antimicrobial efficacy estimation.

Combinations of low-methoxy amidated pectin (LMAP) and carboxymethylcellulose (CMC) were used to develop a lyophilized wafer formulation, aimed to obtain prolonged residence and controlled release of econazole nitrate (ECN) in the oral cavity. Ternary ECN/sulphobutylether-ß-cyclodextrin/citric acid complex, resulted as the most efficient system against selected Candida strains, was loaded into this formulation. The final product with the desired and predicted quality was developed by an experimental design strategy. The experimental values of mucoadhesion strength (28.37 ± 0.04 mg/cm(2)) and residence time (88.1 ± 0.1 min) obtained for the optimized wafer formulation were very close to the predicted ones, thus demonstrating the actual reliability and usefulness of the assumed model in the preparation of buccal wafers. The optimized formulation provided a constant ECN in situ release of 5mg/h and was efficacious against selected Candida strains in vitro. This clearly proved its potential as a novel effective delivery system for the therapy of oral candidiasis.

Application of quality by design to the development of analytical separation methods.

Recent pharmaceutical regulatory documents have stressed the critical importance of applying quality by design (QbD) principles for in-depth process understanding to ensure that product quality is built in by design. This article outlines the application of QbD concepts to the development of analytical separation methods, for example chromatography and capillary electrophoresis. QbD tools, for example risk assessment and design of experiments, enable enhanced quality to be integrated into the analytical method, enabling earlier understanding and identification of variables affecting method performance. A QbD guide is described, from identification of quality target product profile to definition of control strategy, emphasizing the main differences from the traditional quality by testing (QbT) approach. The different ways several authors have treated single QbD steps of method development are reviewed and compared. In a final section on outlook, attention is focused on general issues which have arisen from the surveyed literature, and on the need to change the researcher's mindset from the QbT to QbD approach as an important analytical trend for the near future.

Differentiation of green tea samples by chiral CD-MEKC analysis of catechins content.

A chiral CD-MEKC method, enantioselective for catechin and gallocatechin, was developed, validated and applied to the analysis of tea samples. The method was addressed to the fast and simultaneous quantitation of the most represented and biologically important green tea catechins and methylxanthines. The CD-MEKC was based on SDS as surfactant (90 mM) and hydroxypropyl-beta-CD (25 mM) as chiral selector, under acidic conditions (25 mM borate-phosphate buffer, pH 2.5). The method was first applied to study the thermal epimerisation of epi-structured catechins, (-)-epicatechin and (-)-epigallocatechin, to non-epi-structured (-)-catechin and (-)-gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea samples subjected to thermal treatment. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes. Finally, factor analysis was used to visualise the useful information contained in the data set, showing that it was possible to distinguish tea samples on the basis of their different contents of native and non-native catechins.

Quenching of alpha,beta-unsaturated aldehydes by green tea polyphenols: HPLC-ESI-MS/MS studies.

The aim of this work was to investigate in vitro the quenching activity of green tea polyphenols against alpha,beta-unsaturated aldehyde, using 4-hydroxy-nonenal (HNE) as prototype and HPLC-ESI-MS/MS techniques. HNE is the most abundant and genotoxic product of oxidation of dietary polyunsaturated fatty acids, and is believed to be involved in the early stage of colorectal carcinogenesis on account of its genotoxic potential. Both epigallocatechin gallate (EGCG, 1.0-3.5mM), the main constituent of green tea polyphenols, and a green tea aqueous extract are able to quench HNE (50 microM) in colorectal physiomimetic conditions (10mM phosphate buffer, pH 8.0, 37 degrees C), giving rise to the formation of six diastereomeric covalent adducts at the ring A of EGCG, as indicated by their ESI-MS/MS fragmentation pathways. The specificity of the adduction positions was explained by (1)H NMR experiments. HNE quenching is pH-dependent and maximum at pH 8.0. ESI-MS analysis showed no formation of 4-hydroxy-2,3-epoxy-nonanal, or adduction of the epoxide to EGCG. This implies that too little hydrogen peroxide (1mM, 24h incubation, FOX-2 method) develops from auto-oxidation of EGCG in our aerobic experimental conditions to oxidize HNE to its corresponding epoxide, so this mechanism is not responsible for the compound's disappearance. EGCG and green tea extract also quenched acrolein, another genotoxic alpha,beta-unsaturated aldehyde, giving one predominant adduct and minor isobaric species, probably due the adduction of acrolein at different positions of the EGCG ring A. These results suggest that EGCG and green tea extract, beside the proposed mechanisms of chemoprevention that target multiple cell-signaling pathways that control cell proliferation and apoptosis in cancer cells, can also prevent protein carbonylation in the tumor tissue environment, depending on the pH of the medium surrounding the tissue, the type of tumor, the stage of dysregulation of lipid peroxidation and, finally, the stage of carcinoma development.

Development and evaluation of an in vitro method for prediction of human drug absorption I. Assessment of artificial membrane composition.

A study was undertaken aimed at developing a dynamic in vitro method based on the use of artificial membranes for screening and prediction of the absorption properties of drugs. The development of a suitable artificial membrane simulating the behaviour of the natural gastrointestinal one is the critical step for the fulfillment of this research and it has been the aim of the present work. A series of filters of different chemical nature and different characteristics (pore size, percent of porosity, thickness) were evaluated and compared in order to select the most suitable one to use as support for the artificial lipid membrane preparation. Permeation experiments were performed by using naproxen as reference model drug. The target was to achieve a naproxen apparent permeability (P(app)) value close to that previously obtained using a Caco-2 cell dynamic methodology (4.88 x 10(-5)cms(-1)), which related well to the fraction of dose absorbed in vivo in human. Filters of cellulose nitrate-acetate mixture with a porosity of 70% and 0.025 microm pore size were selected on the basis of the highest reproducibility of results and suitable drug P(app) value obtained. Mixture design experiments were then carried out in order to optimize the composition of the lipid mixture to use for porous support impregnation to obtain the desired P(app) value. Sixteen different lipid mixtures were prepared according to mixture experimental plan and evaluated by diffusion experiments. The contour plot obtained was used to select an optimum lipidic mixture (cholesterol 2.10%; Lipoid((R)) E 80 1.70%; n-octanol 96.2%) for the cellulose nitrate-acetate membrane impregnation. The experimental response obtained from diffusion experiments performed by utilising the optimised lipidic impregnation mixture, was 4.88 x 10(-5)cms(-1), matching exactly the prefixed target.