Analytical Quality by Design-Compliant Development of a Cyclodextrin-Modified Micellar ElectroKinetic Chromatography Method for the Determination of Trimecaine and Its Impurities.

In 2022, the International Council for Harmonisation released draft guidelines Q2(R2) and Q14, intending to specify the development and validation activities that should be carried out during the lifespan of an analytical technique addressed to assess the quality of medicinal products. In the present study, these recommendations were implemented in Capillary Electrophoresis method development for the quality control of a drug product containing trimecaine, by applying Analytical Quality by Design. According to the Analytical Target Profile, the procedure should be able to simultaneously quantify trimecaine and its four impurities, with specified analytical performances. The selected operative mode was Micellar ElectroKinetic Chromatography employing sodium dodecyl sulfate micelles supplemented with dimethyl-ß-cyclodextrin, in a phosphate-borate buffer. The Knowledge Space was investigated through a screening matrix encompassing the composition of the background electrolyte and the instrumental settings. The Critical Method Attributes were identified as analysis time, efficiency, and critical resolution values. Response Surface Methodology and Monte Carlo Simulations allowed the definition of the Method Operable Design Region: 21-26 mM phosphate-borate buffer pH 9.50-9.77; 65.0 mM sodium dodecyl sulfate; 0.25-1.29% v/v n-butanol; 21-26 mM dimethyl-ß-cyclodextrin; temperature, 22 °C; voltage, 23-29 kV. The method was validated and applied to ampoules drug products.

New Trends in the Quality Control of Enantiomeric Drugs: Quality by Design-Compliant Development of Chiral Capillary Electrophoresis Methods.

Capillary electrophoresis (CE) is a potent method for analyzing chiral substances and is commonly used in the enantioseparation and chiral purity control of pharmaceuticals from different matrices. The adoption of Quality by Design (QbD) concepts in analytical method development, optimization and validation is a widespread trend observed in various analytical approaches including chiral CE. The application of Analytical QbD (AQbD) leads to the development of analytical methods based on sound science combined with risk management, and to a well understood process clarifying the influence of method parameters on the analytical output. The Design of Experiments (DoE) method employing chemometric tools is an essential part of QbD-based method development, allowing for the simultaneous evaluation of experimental parameters as well as their interaction. In 2022 the International Council for Harmonization (ICH) released two draft guidelines (ICH Q14 and ICH Q2(R2)) that are intended to encourage more robust analytical procedures. The ICH Q14 guideline intends to harmonize the scientific approaches for analytical procedures' development, while the Q2(R2) document covers the validation principles for the use of analytical procedures including the recent applications that require multivariate statistical analyses. The aim of this review is to provide an overview of the new prospects for chiral CE method development applied for the enantiomeric purity control of pharmaceuticals using AQbD principles. The review also provides an overview of recent research (2012-2022) on the applicability of CE methods in chiral drug impurity profiling.

Application of Experimental Design Methodologies in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: A Review.

Chirality is one of the major issues in pharmaceutical research and industry. Capillary electrophoresis (CE) is an interesting alternative to the more frequently used chromatographic techniques in the enantioseparation of pharmaceuticals, and is used for the determination of enantiomeric ratio, enantiomeric purity, and in pharmacokinetic studies. Traditionally, optimization of CE methods is performed using a univariate one factor at a time (OFAT) approach; however, this strategy does not allow for the evaluation of interactions between experimental factors, which may result in ineffective method development and optimization. In the last two decades, Design of Experiments (DoE) has been frequently employed to better understand the multidimensional effects and interactions of the input factors on the output responses of analytical CE methods. DoE can be divided into two types: screening and optimization designs. Furthermore, using Quality by Design (QbD) methodology to develop CE-based enantioselective techniques is becoming increasingly popular. The review presents the current use of DoE methodologies in CE-based enantioresolution method development and provides an overview of DoE applications in the optimization and validation of CE enantioselective procedures in the last 25 years. Moreover, a critical perspective on how different DoE strategies can aid in the optimization of enantioseparation procedures is presented.

Liquid chromatographic/electrospray ionization quadrupole/time of flight tandem mass spectrometric study of polyphenolic composition of different Vaccinium berry species and their comparative evaluation.

Ultra-high-performance liquid chromatography coupled with high-resolution quadrupole-time of flight mass spectrometry with both negative and positive ionization was used for comprehensively investigating the phenolic and polyphenolic compounds in berries from three spontaneous or cultivated Vaccinium species (i.e., Vaccinium myrtillus, Vaccinium uliginosum subsp. gaultherioides, and Vaccinium corymbosum). More than 200 analytes, among phenolic and polyphenolic compounds belonging to the classes of anthocyanins, monomeric and oligomeric flavonols, flavanols, dihydrochalcones, phenolic acids, together with other polyphenolic compounds of mixed structural characteristics, were identified. Some of the polyphenols herein investigated, such as anthocyanidin glucuronides and malvidin-feruloyl-hexosides in V. myrtillus, or anthocyanindin aldopentosides and coumaroyl-hexosides in V. uliginosum subsp. gaultherioides and a large number of proanthocyanidins with high molecular weight in all species, were described for the first time in these berries. Principal component analysis applied on original LC-TOF data, acquired in survey scan mode, successfully discriminated the three Vaccinium berry species investigated, on the basis of their polyphenolic composition, underlying one more time the fundamental role of mass spectrometry for food characterization.

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.

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.

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.

Development of a chitosan-derivative micellar formulation to improve celecoxib solubility and bioavailability.

CONTEXT: Celecoxib is an anti-inflammatory drug, specific inhibitor of COX-2, classified as a BCS class II compound due to its very low aqueous solubility (3 µg/mL) and good permeability. OBJECTIVE: An innovative micellar formulation of celecoxib has been developed to increase its solubility and, consequently, its oral bioavailability. MATERIALS AND METHODS: Quaternary-ammonium-palmitoyl-glycol-chitosan (GCPQ) was selected as carrier, due to its micelle-forming ability joined to its solubilizing and enhancer properties towards hydrophobic drugs. A Doehlert design was applied to optimize the drug solubilizing efficiency of the micellar formulation. Tested factors were GCPQ concentration and time and power of probe sonication during micelles formation; the response to maximize was the celecoxib solubility. RESULTS: The response-surface study allowed a thorough investigation of the effect of factors variations on the response over the considered experimental domain and identification of the best variable combination in order to maximize the desired improvement in drug solubility. The optimized micellar formulation (GCPQ 4.5 mg/mL; 25 min at 60% power of probe sonication) enabled an about 60-fold increase in celecoxib aqueous solubility. The optimized formulation, tested in vivo in mice by the writhing test, allowed a statistically significant shortening (p < 0.05) of the pain alleviation onset and a more intense effect (p < 0.05) with respect to the celecoxib aqueous suspension obtained by the commercial formulation. CONCLUSIONS: The results proved that the developed GCPQ micellar formulation is a valuable approach for improving the therapeutic effectiveness of celecoxib.

Analytical quality by design-based development of a capillary electrophoresis method for Omeprazole impurity profiling.

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n-butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 37//16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n-butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations.

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.

Analysis of Volatile Hydrocarbons (Pentene Dimers and Terpenes) in Extra Virgin Olive Oil: Optimization by Response Surface Methodology and Validation of HS-SPME-GC-MS Method.

A head space-solid phase microextraction-gas chromatography-mass spectrometery (HS-SPME-GC-MS) method for the simultaneous analysis of pentene dimers from lipoxygenase (LOX) pathway, monoterpenes, and sesquiterpenes in extra virgin olive oil (EVOO) was proposed. A Doehlert design was performed; the conditions of the HS-SPME preconcentration step (extraction temperature, extraction time, sample amount, and desorption time) were optimized by response surface methodology, allowing defining the method operable design region. A quantitative method was set up using the multiple internal standard normalization approach: four internal standards were used, and the most suitable one was selected for area normalization of each external standard. The quantitative method was successfully validated and applied to a series of monocultivar EVOOs. This is the first paper in which a quantitative method using commercial standards has been proposed for the analysis of an important class of molecules of EVOO such as pentene dimers. The optimized method is suitable for routine analysis aimed at characterizing high quality EVOOs.

Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Oral propranolol for retinopathy of prematurity: risks, safety concerns, and perspectives.

OBJECTIVE: To evaluate safety and efficacy of oral propranolol administration in preterm newborns affected by an early phase of retinopathy of prematurity (ROP). STUDY DESIGN: Fifty-two preterm newborns with Stage 2 ROP were randomized to receive oral propranolol (0.25 or 0.5 mg/kg/6 hours) added to standard treatment or standard treatment alone. To evaluate safety of the treatment, hemodynamic and respiratory variables were continuously monitored, and blood samples were collected weekly to check for renal, liver, and metabolic balance. To evaluate efficacy of the treatment, the progression of the disease (number of laser treatments, number of bevacizumab treatments, and incidence of retinal detachment) was evaluated by serial ophthalmologic examinations, and plasma soluble E-selectin levels were measured weekly. RESULTS: Newborns treated with propranolol showed less progression to Stage 3 (risk ratio 0.52; 95% CI 0.47-0.58, relative reduction of risk 48%) or Stage 3 plus (relative risk 0.42 95% CI 0.31-0.58, relative reduction of risk 58%). The infants required fewer laser treatments and less need for rescue treatment with intravitreal bevacizumab (relative risk 0.48; 95% CI 0.29-0.79, relative reduction of risk 52 %), a 100% relative reduction of risk for progression to Stage 4. They also had significantly lower plasma soluble E-selectin levels. However, 5 of the 26 newborns treated with propranolol had serious adverse effects (hypotension, bradycardia), in conjunction with episodes of sepsis, anesthesia induction, or tracheal stimulation. CONCLUSION: This pilot study suggests that the administration of oral propranolol is effective in counteracting the progression of ROP but that safety is a concern.

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.

Chiral high-performance liquid chromatography analysis of mono-, di-, and triacylglycerols with amylose- and cellulose-phenylcarbamate-based stationary phases.

The ever-increasing technological advancement in the (ultra)high-performance liquid chromatography tandem (high-resolution) mass spectrometry platforms have largely contributed to steeply intensify the interest towards lipidomics research. However, mass spectrometers alone are unable to distinguish between enantiomers. This obstacle is especially evident in the case of glycerolipids analysis due the prochiral nature of glycerol. Until a couple of decades ago, the stereoselective analysis of triacylglycerols (TAGs) was performed on the end products generated either from their enzymatic or chemical hydrolysis, namely on mono- or diacyl-sn-glycerols (MAGs and DAGs, respectively). These were then mostly analyzed with Pirkle-type chiral stationary phases (CSPs) after dedicated multi-step derivatization procedures. One of the most significant drawbacks of these traditional methods for enantioselective TAGs analysis (actually of the produced MAGs and DAGs, often investigated as target species per se) was the difficulty to totally abolish the migration of fatty acyls between glycerol positions. This made difficult to control and keep unaltered the stereochemistry of the original molecules. Over the last two decades, it has been widely demonstrated that the enantioselective analysis of intact TAGs as well as of non-derivatized MAGs and DAGs can be efficiently obtained using polysaccharide-based CSPs incorporating either amylose- or cellulose-phenylcarbamate derivatives chiral selectors. In this paper, the enantioselective methods developed with these CSPs for the enantioselective direct LC analysis of MAGs, DAGs and TAGs embedding different types of fatty acid residues are comprehensively reviewed.

Millet Fermented by Different Combinations of Yeasts and Lactobacilli: Effects on Phenolic Composition, Starch, Mineral Content and Prebiotic Activity.

Millet is the sixth-highest yielding grain in the world and a staple crop for millions of people. Fermentation was applied in this study to improve the nutritional properties of pearl millet. Three microorganism combinations were tested: Saccharomyces boulardii (FPM1), Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius (FPM2) and Hanseniaspora uvarum plus Fructilactobacillus sanfranciscensis (FPM3). All the fermentation processes led to an increase in minerals. An increase was observed for calcium: 254 ppm in FPM1, 282 ppm in FPM2 and 156 ppm in the unfermented sample. Iron increased in FPM2 and FPM3 (approx. 100 ppm) with respect the unfermented sample (71 ppm). FPM2 and FPM3 resulted in richer total phenols (up to 2.74 mg/g) compared to the unfermented sample (2.24 mg/g). Depending on the microorganisms, it was possible to obtain different oligopeptides with a mass cut off ≤10 kDalton that was not detected in the unfermented sample. FPM2 showed the highest resistant starch content (9.83 g/100 g) and a prebiotic activity on Bifidobacterium breve B632, showing a significant growth at 48 h and 72 h compared to glucose (p < 0.05). Millet fermented with Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius can be proposed as a new food with improved nutritional properties to increase the quality of the diet of people who already use millet as a staple food.

Effect of mobile phase pH on liquid chromatography retention of mepartricin related compounds and impurities as support to the structural investigation by liquid chromatography-mass spectrometry.

Mepartricin is a semisynthetic polyene macrolide with antifungal and anti-protozoal activities, and it is widely used for the treatment of benign prostatic hyperplasia. Mepartricin is produced by synthetic methyl esterification of the more toxic partricin, and its activity is due to a complex of related compounds. Among them, the main ones are mepartricin B and mepartricin A which are characterized by the presence of a primary and a secondary amine group, respectively. In this work a previously reported HPLC-UV method was properly modified to make it MS-compatible. The selected conditions entail the use of a C18 reverse phase column, and a mobile phase composed by ammonium formate and acetonitrile, with the addition of heptafluorobutyric acid as modifier. The developed method was applied to the characterization of a mepartricin reference standard and a mepartricin experimental batch. All the UV responding peaks, 30 for the standard and 21 for the experimental batch, were successfully detected by MS, allowing to define their m/z values and acquire their fragmentation spectra. For the structural elucidation of isobaric species and, in particular, the identification of toxic partricin-related impurities, the presence of differently ionisable chemical groups was considered, as partricins contain free caboxy-groups, while mepartricins represent their estherified counterparts. A deep study of the effect of mobile phase pH on the chromatographic retention of partricin and mepartricin related compounds was performed in the pH range 2.5-6.5. This study allowed to successfully cluster all the detected species and asses, in the considered batch, the absence of other partricin-related impurities in addition to partricin B and partricin A.

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.