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.

Quantitative amino acids profile of monofloral bee pollens by microwave hydrolysis and fluorimetric high performance liquid chromatography.

Bee pollen is an attractive resource in the field of alternative remedies and thanks to the content of carbohydrates, crude fibers, proteins and lipids must be considered as a supplementary food of high potential rate. In characterization of bee pollen with the aim to define its value in human nutrition, the amino acids profile is one of the most important attributes. In the present study, the determination of amino acids composition of different monofloral bee pollen samples was obtained by an approach combining microwave acidic hydrolysis (60 min at 150 °C instead of 22 h at 120 °C in conventional oven) followed by derivatization using 9-fluorenylmethylchloroformate (FMOC-Cl) and separation of amino acids derivatives using a Phenomenex Kinetex core-shell 5 µm C18 (150 x 4.6 mm i.d.) column under a ternary gradient elution. Separation of 19 amino acids was achieved in about 40 min and fluorimetric detection (λexc = 265 nm λem = 315 nm) allowed selective and sensitive quantitation with LOQ values ranging within 0.14-3.00 µg/mL. Interestingly, the present approach allowed determination of some amino acids e.g., tryptophan and trans-4-hydroxyproline that are often lost by other methods of analysis. Significant differences in the composition of the considered samples were found confirming the impact of botanical origin of the product on its nutritional value. Principal Component Analysis was applied to treat the obtained data, highlighting the importance for discrimination, of detecting low abundance amino acids. The proposed method can be used as an advantageous alternative to the existing ones for characterization of bee pollen as an important source of dietary proteins.

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.

Polyphenolic profiles and antioxidant and antiradical activity of Italian berries from Vaccinium myrtillus L. and Vaccinium uliginosum L. subsp. gaultherioides (Bigelow) S.B. Young.

Total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), radical scavenging activity (RSA), ferric reducing antioxidant power (FRAP), and a number of anthocyanins, phenolic acids, coumarins, flavanols, dihydrochalcones and flavonols were investigated in Tuscan bilberry (i.e. Vaccinium myrtillus) and "false bilberry" (i.e. Vaccinium uliginosum subsp. gaultherioides Bigelow). V. myrtillus berries showed much higher TSP, TMA, RSA and FRAP values than V. uliginosum subsp. gaultherioides fruits. Moreover, very different profiles of individual phenolics were observed in the two species, being V. myrtillus mainly characterised by delphinidin and cyanidin glycosides, together with chlorogenic acid, and V. uliginosum subsp. gaultherioides dominated by malvidin derivatives and flavonols. Strong differences between the two species regarded also metabolites investigated herein for the first time, such as scopoletin, which was approximately two magnitude orders higher in V. uliginosum subsp. gaultherioides than in V. myrtillus berries. Very different abundances were also highlighted for cryptochlorogenic acid and quercetin-3-rhamnoside that were about ten-fold higher in bilberry than in "false bilberry". When the anthocyanin composition pattern of Tuscan "false bilberry" was compared to those elsewhere reported for V. uliginosum fruits harvested in different world areas, some important differences were observed.

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.

QuEChERS sample preparation for the determination of pesticides and other organic residues in environmental matrices: a critical review.

Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) is an extraction and clean-up technique originally developed for recovering pesticide residues from fruits and vegetables. Since its introduction, and until December 2013, about 700 papers have been published using the QuEChERS technique, according to a literature overview carried out using SciFinder, Elsevier SciVerse, and Google search engines. Most of these papers were dedicated to pesticide multiresidue analysis in food matrices, and this topic has been thoroughly reviewed over recent years. The QuEChERS approach is now rapidly developing beyond its original field of application to analytes other than pesticides, and matrices other than food, such as biological fluids and non-edible plants, including Chinese medicinal plants. Recently, the QuEChERS concept has spread to environmental applications by analyzing not only pesticides but also other compounds of environmental concern in soil, sediments, and water. To the best of our knowledge, QuEChERS environmental applications have not been reviewed so far; therefore, in this contribution, after a general discussion on the evolution and changes of the original QuEChERS method, a critical survey of the literature regarding environmental applications of conventional and modified QuEChERS methodology is provided. The overall recoveries obtained with QuEChERS and other extraction approaches (e.g., accelerated solvent extraction, ultrasonic solvent extraction, liquid/solid extraction, and soxhlet extraction) were compared, providing evidence for QuEChERS higher recoveries for various classes of compounds, such as biopesticides, chloroalkanes, phenols, and perfluoroalkyl substances. The role of physicochemical properties of soil (i.e., clay and organic carbon content, as well as cation exchange capacity) and target analytes (i.e., log KOW, water solubility, and vapor pressure) were also evaluated in order to interpret recovery and matrix effect data.

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.