Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

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.

Metabolic Bile Acid Profile Impairments in Dogs Affected by Chronic Inflammatory Enteropathy.

Bile acids (BAs), endogenous acidic steroids synthetized from cholesterol in the liver, play a key role in the gut-liver axis physiopathology, including in hepatotoxicity, intestinal inflammatory processes, and cholesterol homeostasis. Faecal Oxo-BAs, relatively stable intermediates of oxidation/epimerization reactions of the BA hydroxyls, could be relevant to investigating the crosstalk in the liver-gut axis and the relationship between diseases and alterations in microbiota composition. A paucity of information currently exists on faecal BA profiles in dogs with and without chronic inflammatory enteropathy (CIE). Comprehensive assessment of 31 molecules among faecal BAs and related microbiota metabolites was conducted with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Odds ratios (ORs) for associations of BAs with CIE were estimated using logistic regression. Principal component analysis was performed to find differences between the control and pathological dogs. Higher levels of primary BAs and muricholic acids, and lower levels of secondary BAs were found in pathological dogs. Higher concentrations in faecal oxo-metabolites were associated with the absence of CIE (OR < 1). This study shows a marked difference in faecal BA profiles between dogs with and without CIE. Further research will be needed to better understand the role of oxo-BAs and muricholic acids in CIE dogs.

Extraction method for the multiresidue analysis of legacy and emerging pollutants in marine mussels from the Adriatic Sea.

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

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.

Magnolia officinalis L. bark extract and respiratory diseases: From traditional Chinese medicine to western medicine via network target.

The understanding of the use of Magnolia officinalis L. (Magnoliaceae) as a possible dietary supplement for supporting the treatment of airway pathologies might be of clinical interest. Two commercially available bark extracts (M. officinalis extract [MOE]) were characterized by quantitation in honokiol and magnolol content by means of high-performance liquid chromatography with UV detection. MOE effects, as well as those of the reference compounds per se, on some targets connected to airway pathologies (antibacterial- and lung and trachea relaxing- activities) were investigated. Results showed that MOE possessed interesting antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae. This was accompanied by a spasmolytic and antispasmodic activity, possibly owing to its ability to concurrently modulate different targets such as H1 -, ß2 - and muscarinic receptors and l-type calcium channels involved in bronchodilation. All these effects were directly related to the MOE content in honokiol and magnolol. In conclusion, the properties of MOE highlighted here strongly encourage its application as dietary supplement in the treatment of airway diseases.

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.

Glyphosate and its breakdown product AMPA elicit cytoprotective responses in haemocytes of the Mediterranean mussel (Mytilus galloprovincialis).

This study investigates the effects of glyphosate (GLY) and its metabolite AMPA on cytoprotective and detoxification mechanisms in haemocytes of Mytilus galloprovincialis. Cells were treated in vitro with 0.1 and 1.0 µg/L GLY, 0.1 µg/L, 0.1 and 1.0 µg/L AMPA, or two mixtures GLY+AMPA (0.1 µg/L GLY + 0.1 µg/L AMPA, 1.0 µg/L GLY + 1.0 µg/L AMPA). GLY and AMPA increased MXR efflux activity and modulated expression of the ABCB transcript encoding a MXR related ABC transporter P-glycoprotein. The mixtures GLY+AMPA reduced efflux activity with ABCB down-regulation (at 1 µg/L GLY/AMPA). Modulation of lysosomal and immune related transcripts generally agree with known effects of the chemicals on these physiological functions. Given their cumulative action as chemosensitizers of the MXR system, and their interactive effects on haemocyte parameters, glyphosate and AMPA at environmental concentrations should be addressed as a concern factor for the biological vulnerability of marine habitats.

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.

Determination of Free Amino Acids in Milk, Colostrum and Plasma of Swine via Liquid Chromatography with Fluorescence and UV Detection.

Amino acids are ubiquitous components of mammalian milk and greatly contribute to its nutritional value. The compositional analysis of free amino acids is poorly reported in the literature even though their determination in the biological fluids of livestock animals is necessary to establish possible nutritional interventions. In the present study, the free amino acid profiles in mature swine milk, colostrum and plasma were assessed using a targeted metabolomics approach. In particular, 20 amino acids were identified and quantified via two alternative and complementary reversed-phase HPLC methods, involving two stationary phases based on core-shell technology, i.e., Kinetex C18 and Kinetex F5, and two detection systems, i.e., a diode array detector (DAD) and a fluorescence detector (FLD). The sample preparation involved a de-proteinization step, followed by pre-chromatographic derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). The two optimized methods were validated for specificity, linearity, sensitivity, matrix effect, accuracy and precision and the analytical performances were compared. The analytical methods proved to be suitable for free amino acid profiling in different matrices with high sensitivity and specificity. The correlations among amino acid levels in different biological fluids can be useful for the evaluation of physio-pathological status and to monitor the effects of therapeutic or nutritional interventions in humans and animals.

Sustainable production of pharmaceutical, nutraceutical and bioactive compounds from biomass and waste.

The aim of this tutorial review is to provide a general overview of processes, technologies and challenges in the production of pharmaceutical and bioactive compounds from food waste and lignocellulosic residues. Particular attention is given to benign-by-design processes instinctively devoted to environmental sustainability for the recovery of bioactive compounds from food waste as well as for the production of alcohols, acids, polyols, furans and aromatic compounds from lignocellulosic residues. At the same time, novel green synthetic routes for the production of active pharmaceutical ingredients and the development of novel bioactive compounds are discussed. Recent success industrial stories on the use of food waste and lignocellulosic residues for pharmaceutical and nutraceutical applications are also discussed.

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.

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.

Functional, nutritional, antioxidant, sensory properties and comparative peptidomic profile of faba bean (Vicia faba, L.) seed protein hydrolysates and fortified apple juice.

Enzymatic hydrolysis of plant-derived proteins can improve their quality by offering opportunities for food applications. In this study, three proteolytic enzymes (pepsin, trypsin, Alcalase®) were used, alone or combined, to produce faba bean protein hydrolysates (PHs). Their functional, nutritional and antioxidant properties were evaluated, and the peptidomic profile was assessed by LC-MS/MS. Hydrolysis improved solubility of faba proteins at acidic and neutral pH, and their antioxidant properties. Peptidomic analysis identified 2031 peptides in the different PHs. Among them, 9 showed 100% homology with previously known antioxidant peptides and several others had antioxidant motifs in their sequences. Sensory data analysis showed that after addition of PHs to apple juice, no significant differences were perceived between control and some of the PHs. This study demonstrates that enzymatic hydrolysis enhances the functional and antioxidant properties of faba bean proteins. Specifically, hydrolysates can be used as functional food ingredients to produce fortified beverages.

Apoptotic-Induced Effects of Acacia Catechu Willd. Extract in Human Colon Cancer Cells.

The research for innovative treatments against colon adenocarcinomas is still a great challenge. Acacia catechu Willd. heartwood extract (AC) has health-promoting qualities, especially at the gastrointestinal level. This study characterized AC for its catechins content and investigated the apoptosis-enhancing effect in human colorectal adenocarcinoma HT-29 cells, along with its ability to spare healthy tissue. MTT assay was used to describe the time course, concentration dependence and reversibility of AC-mediated cytotoxicity. Cell cycle analysis and AV-PI and DAPI-staining were performed to evaluate apoptosis, together with ROS formation, mitochondrial membrane potential (MMP) changes and caspase activities. Rat ileum and colon rings were tested for their viability and functionality to explore AC effects on healthy tissue. Quantitative analysis highlighted that AC was rich in (±)-catechin (31.5 ± 0.82 mg/g) and (-)-epicatechin (12.5 ± 0.42 mg/g). AC irreversibly decreased cell viability in a concentration-dependent, but not time-dependent fashion. Cytotoxicity was accompanied by increases in apoptotic cells and ROS, a reduction in MMP and increases in caspase-9 and 3 activities. AC did not affect rat ileum and colon rings' viability and functionality, suggesting a safe profile toward healthy tissue. The present findings outline the potential of AC for colon cancer treatment.

Assessment of gut microbiota fecal metabolites by chromatographic targeted approaches.

Gut microbiota, the specific microbial community of the gastrointestinal tract, by means of the production of microbial metabolites provides the host with several functions affecting metabolic and immunological homeostasis. Insights into the intricate relationships between gut microbiota and the host require not only the understanding of its structure and function but also the measurement of effector molecules acting along the gut microbiota axis. This article reviews the literature on targeted chromatographic approaches in analysis of gut microbiota specific metabolites in feces as the most accessible biological matrix which can directly probe the connection between intestinal bacteria and the (patho)physiology of the holobiont. Together with a discussion on sample collection and preparation, the chromatographic methods targeted to determination of some classes of microbiota-derived metabolites (e.g., short-chain fatty acids, bile acids, low molecular masses amines and polyamines, vitamins, neurotransmitters and related compounds) are discussed and their main characteristics, summarized in Tables.

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.

Enteral Nutrition in Pediatric Patients Undergoing Hematopoietic SCT Promotes the Recovery of Gut Microbiome Homeostasis.

Hematopoietic stem cell transplantation (HSCT) is the first-line immunotherapy to treat several hematologic disorders, although it can be associated with many complications reducing the survival rate, such as acute graft-versus-host disease (aGvHD) and infections. Given the fundamental role of the gut microbiome (GM) for host health, it is not surprising that a suboptimal path of GM recovery following HSCT may compromise immune homeostasis and/or increase the risk of opportunistic infections, with an ultimate impact in terms of aGvHD onset. Traditionally, the first nutritional approach in post-HSCT patients is parenteral nutrition (PN), which is associated with several clinical adverse effects, supporting enteral nutrition (EN) as a preferential alternative. The aim of the study was to evaluate the impact of EN vs. PN on the trajectory of compositional and functional GM recovery in pediatric patients undergoing HSCT. The GM structure and short-chain fatty acid (SCFA) production profiles were analyzed longitudinally in twenty pediatric patients receiving HSCT-of which, ten were fed post-transplant with EN and ten with total PN. According to our findings, we observed the prompt recovery of a structural and functional eubiotic GM layout post-HSCT only in EN subjects, thus possibly reducing the risk of systemic infections and GvHD onset.

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.