Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.

Olive Oil Waste as a Source of Functional Food Ingredients: Assessing Polyphenolic Content and Antioxidant Activity in Olive Leaves.

Around two million tons of olive oil are produced in Europe annually, with Portugal being among the top five European olive oil-producing countries. Olive oil production results in a substantial amount of waste in the form of olive leaves. These discarded olive leaves contain valuable phenolic compounds with antioxidant, anti-inflammatory, hypoglycaemic, neuroprotective, and antiproliferative properties. Due to their richness in polyphenols with health-promoting properties, olive leaves can be considered a potential functional food ingredient. Thus, sustainable practices for reusing olive leaf waste are in demand. In this study, the polyphenolic content in olive leaves from different Portuguese locations was determined using HPLC-UV-Vis after defining the best fit-for-purpose liquid extraction strategy. The differences in the in vitro antioxidant activity in these samples were determined by several methodologies based on radical scavenging (against 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-2-picrylhydrazyl (DPPH), and peroxyl radical (ORAC)) and on reducing properties (cupric-reducing antioxidant capacity (CUPRAC), and Folin-Ciocalteu assay (FC)), to unveil the relationship between the profile and quantity of polyphenols with antioxidant mechanisms and their capacity. At last, the stability of extracted compounds upon lyophilization and exposition to surrogate biological fluids was assessed, envisioning the future incorporation of olive leaves extracted compounds in food products.

Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds.

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme ß-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-ß-d-galactopyranoside, is proposed, allowing for the assessment of EDCs' presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17ß-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Assessing the differences of two vineyards soils' by NIR spectroscopy and chemometrics.

Soil properties influence greatly the status of vine plants which consequently influences the quality of wine. Therefore, in the context of viticulture management, it is extremely important to assess the physical and chemical parameters of vineyards soils. In this study, the soils of two vineyards were analysed by near-infrared (NIR) spectroscopy and established analytical reference procedures. The main objective of this study was to verify if NIR spectroscopy is a potential tool to discriminate the soils of both vineyards as well as to quantify differences of soil's parameters. For that, a total of eight sampling spots were selected at each vineyard taking into consideration the soil type and sampled at different depths. The data analysis was performed using analysis of variance (ANOVA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) and partial least squares (PLS) regression. The ANOVA results revealed that 12 out of the 18 parameters analysed through the reference procedures can be considered statistically different (p < 0.05). Regarding PCA, the obtained results revealed a clear separation between the scores of both vineyards either considering NIR spectra or the chemical parameters. The PLS-DA model was able to obtain 100 % of correct predictions for the discrimination of both vineyards. PLS regression analysis using NIR spectra revealed R2P and RER values higher than 0.85 and 10, respectively, for 8 (pH (H2O), N, Ca2+, Mg2+, SB, CEC, ECEC and GSB) of the 18 chemical parameters evaluated. Concluding, these results demonstrate that it is possible to discriminate the soils of the different vineyards through NIR spectroscopy as well as to quantify several chemical parameters through soils NIR spectra in a rapid, accurate, cost-effective, simple and environmentally friendly way when compared to the reference procedures.

Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior in vivo, for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier. The observed differences between cryo-XPS and standard XPS measurements indicate the potential of cryo-XPS for providing quantitative measurements of such nanoparticle systems in hydrated conditions.

Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry.

Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (µSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the µSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the µSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed µSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 µg mL-1 in plasma. LOQ values were 2 µg mL-1 for INDS and 7 µg mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

Combining orthogonal measurements to unveil diclofenac encapsulation into polymeric and lipid nanocarriers.

The quantification of the drug associated to nanoparticle carriers, often expressed in terms of encapsulation efficiency, is a regulatory requirement. The establishment of independent methods to evaluate this parameter provides a means for measurement validation, which is critical in providing confidence in the methods and enabling the robust characterization of nanomedicines. Chromatography is traditionally used to measure drug encapsulation into nanoparticles. Here, we describe an additional independent strategy based on analytical centrifugation. The encapsulation of diclofenac into nanocarriers was quantified based on the mass difference between placebo (i.e. unloaded) and loaded nanoparticles. This difference was estimated using particle densities measured by differential centrifugal sedimentation (DCS) and size and concentration values measured by particle tracking analysis (PTA). The proposed strategy was applied to two types of formulations, namely poly(lactic-co-glycolic acid) (PLGA) nanoparticles and nanostructured lipid carriers, which were analysed by DCS operated in sedimentation and flotation modes, respectively. The results were compared to those from high performance liquid chromatography (HPLC) measurements. Additionally, X-ray photoelectron spectroscopy analysis was used to elucidate the surface chemical composition of the placebo and loaded nanoparticles. The proposed approach enables the monitoring of batch-to-batch consistency and the quantification of diclofenac association to PLGA nanoparticles from 0.7 ng to 5 ng of drug per 1 µg of PLGA, with good linear correlation between DCS and HPLC results (R2 = 0.975). Using the same approach, similar quantification in lipid nanocarriers was possible for a loading of diclofenac ≥1.1 ng per 1 µg of lipids, with results in agreement with the HPLC method (R2 = 0.971). Hence, the strategy proposed here expands the analytical tools available for evaluating nanoparticles encapsulation efficiency, being thus significant for increasing the robustness of drug-delivery nanocarriers characterization.

Exploiting Kinetic Features of ORAC Assay for Evaluation of Radical Scavenging Capacity.

The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,2'-azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.

Lab-on-Valve Automated and Miniaturized Assessment of Nanoparticle Concentration Based on Light-Scattering.

Nanoparticles (NPs) concentration directly impacts the dose delivered to target tissues by nanocarriers. The evaluation of this parameter is required during NPs developmental and quality control stages, for setting dose-response correlations and for evaluating the reproducibility of the manufacturing process. Still, faster and simpler procedures, dismissing skilled operators and post-analysis conversions are needed to quantify NPs for research and quality control operations, and to support result validation. Herein, a miniaturized automated ensemble method to measure NPs concentration was established under the lab-on-valve (LOV) mesofluidic platform. Automatic NPs sampling and delivery to the LOV detection unit were set by flow programming. NPs concentration measurements were based on the decrease in the light transmitted to the detector due to the light scattered by NPs when passing through the optical path. Each analysis was accomplished in 2 min, rendering a determination throughput of 30 h-1 (6 samples h-1 for n = 5) and only requiring 30 µL (≈0.03 g) of NPs suspension. Measurements were performed on polymeric NPs, as these represent one of the major classes of NPs under development for drug-delivery aims. Determinations for polystyrene NPs (of 100, 200, and 500 nm) and for NPs made of PEGylated poly-d,l-lactide-co-glycolide (PEG-PLGA, a biocompatible FDA-approved polymer) were accomplished within 108-1012 particles mL-1 range, depending on the NPs size and composition. NPs size and concentration were maintained during analysis, as verified for NPs eluted from the LOV by particle tracking analysis (PTA). Moreover, concentration measurements for PEG-PLGA NPs loaded with an anti-inflammatory drug, methotrexate (MTX), after their incubation in simulated gastric and intestinal fluids were successfully achieved (recovery values of 102-115%, as confirmed by PTA), showing the suitability of the proposed method to support the development of polymeric NPs targeting intestinal delivery.

Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.

Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.

Rapid and sustainable HPLC method for the determination of uremic toxins in human plasma samples.

Protein-bound uremic toxins, mainly indoxyl sulfate (3-INDS), p-cresol sulfate (pCS), and indole-3-acetic acid (3-IAA) but also phenol (Pol) and p-cresol (pC), are progressively accumulated during chronic kidney disease (CKD). Their accurate measurement in biomatrices is demanded for timely diagnosis and adoption of appropriate therapeutic measures. Multianalyte methods allowing the establishment of a uremic metabolite profile are still missing. Hence, the aim of this work was to develop a rapid and sensitive method based on high-performance liquid chromatography with fluorescence detection for the simultaneous quantification of Pol, 3-IAA, pC, 3-INDS, and pCS in human plasma. Separation was attained in 12 min, using a monolithic C18 column and isocratic elution with acetonitrile and phosphate buffer containing an ion-pairing reagent, at a flow rate of 2 mL min-1. Standards were prepared in plasma and quantification was performed using the background subtraction approach. LOQ values were ≤ 0.2 µg mL-1 for all analytes except for pCS (LOQ of 2 µg mL-1). The method proved to be accurate (93.5-112%) and precise (CV ≤ 14.3%). The multianalyte application of the method, associated to a reduced sample volume (50 µL), a less toxic internal standard (eugenol) in comparison to the previously applied 2,6-dimethylphenol and 4-ethylphenol, and a green extraction solvent (ethanol), resulted in the AGREE score of 0.62 which is in line with the recent trend of green and sustainable analytical chemistry. The validated method was successfully applied to the analysis of plasma samples from control subjects exhibiting normal levels of uremic toxins and CKD patients presenting significantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.

Vascular Calcification and the Gut and Blood Microbiome in Chronic Kidney Disease Patients on Peritoneal Dialysis: A Pilot Study.

Vascular calcification (VC) is a frequent condition in chronic kidney disease (CKD) and a well-established risk factor for the development of cardiovascular disease (CVD). Gut dysbiosis may contribute to CVD and inflammation in CKD patients. Nonetheless, the role of gut and blood microbiomes in CKD-associated VC remains unknown. Therefore, this pilot study aimed to explore the link between gut and blood microbiomes and VC in CKD patients on peritoneal dialysis (CKD-PD). Our results showed relative changes in specific taxa between CKD-PD patients with and without VC, namely Coprobacter, Coprococcus 3, Lactobacillus, and Eubacterium eligens group in the gut, and Cutibacterium, Pajaroellobacter, Devosia, Hyphomicrobium, and Pelomonas in the blood. An association between VC and all-cause mortality risk in CKD-PD patients was also observed, and patients with higher mortality risk corroborate the changes of Eubacterium eligens in the gut and Devosia genus in the blood. Although we did not find differences in uremic toxins, intestinal translocation markers, and inflammatory parameters among CKD-PD patients with and without VC, soluble CD14 (sCD14), a nonspecific marker of monocyte activation, positively correlated with VC severity. Therefore, gut Eubacterium eligens group, blood Devosia, and circulating sCD14 should be further explored as biomarkers for VC, CVD, and mortality risk in CKD.

Automatic and renewable micro-solid-phase extraction based on bead injection lab-on-valve system for determination of tranexamic acid in urine by UHPLC coupled with tandem mass spectrometry.

An automatic micro-solid-phase extraction (µSPE) method using on-line renewable sorbent beads followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established for the determination of tranexamic acid (TXA) in urine. The µSPE method was based on the bead injection (BI) concept combined with the mesofluidic lab-on-valve (LOV) platform. All steps of the µSPE-BI-LOV were implemented by computer programming, rendering enhanced precision on time and flow events. Several parameters, including the type of sorbent, volume and composition of the conditioning solution, washing solution, and eluent composition, were evaluated to improve the extraction efficiency. The best results were obtained with a hydrophilic-lipophilic balanced mixed-mode sorbent, decorated with sulfonic acid groups (Oasis MCX), and 99% acetonitrile-water (50:50, v/v)-1% ammonium hydroxide as eluent. Chromatographic separation was performed using a BEH amide column coupled to MS/MS detection in positive ionization mode. Good linearity was achieved (R2 > 0.998) for TXA concentrations in urine ranging from 300 to 3000 ng mL-1, with LOD and LOQ of 30 and 65 ng mL-1, respectively. Dilution integrity was observed for dilution factors up to 20,000 times, providing the extension of the upper limit of quantification to 12 mg mL-1. The method was validated according to international guidelines and successfully applied to urine samples collected during scoliosis surgery of pediatric patients treated with TXA.

Acetonitrile Adducts of Tranexamic Acid as Sensitive Ions for Quantification at Residue Levels in Human Plasma by UHPLC-MS/MS.

The quantitative analysis of pharmaceuticals in biomatrices by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is often hampered by adduct formation. The use of the molecular ion resulting from solvent adducts for quantification is uncommon, even if formed in high abundance. In this work, we propose the use of a protonated acetonitrile adduct for the quantitative analysis of tranexamic acid (TXA) by LC-MS/MS. The high abundance of the protonated acetonitrile adduct [M + ACN + H]+ was found to be independent of source-dependent parameters and mobile phase composition. The results obtained for TXA analysis in clinical samples were comparable for both [M + ACN + H]+ and [M + H]+, and no statistically significant differences were observed. The relative stability and structure of the [M + ACN + H]+ ions were also studied by analyzing probable structures from an energetic point of view and by quantum chemical calculations. These findings, and the studied fragmentation pathways, allowed the definition of an acetimidium structure as the best ion to describe the observed acetonitrile protonated adduct of TXA.

Effects of Zinc Source and Enzyme Addition on the Fecal Microbiota of Dogs.

Supplemental zinc from organic sources has been suggested to be more bioavailable than inorganic ones for dog foods. However, the bioavailability of zinc might be affected by dietary constituents such as phytates. The present study aimed to evaluate the effects of two zinc sources (zinc sulfate and zinc proteinate) and the addition of a multi-enzymatic complex from the solid-state fermentation of Aspergillus niger on end-products of fecal fermentation and fecal microbiota of adult Beagles fed a high-phytate diet. The experimental design consisted of three 4 × 4 Latin Squares with a 2 × 2 factorial arrangement of treatments (n = 12 Beagles), with four periods and four diets: zinc sulfate without (IZ) or with (IZ +) enzyme addition, and zinc proteinate without (OZ) or with (OZ +) enzyme addition. Enzyme addition significantly affected Faith's phylogenetic diversity index, whereas zinc source did not affect either beta or alpha diversity measures. Linear discriminant analysis effect size detected nine taxa as markers for organic zinc, 18 for inorganic source, and none for enzyme addition. However, with the use of a negative binomial generalized linear model, further effects were observed. Organic zinc was associated with a significantly higher abundance of Firmicutes and lower Proteobacteria and Bacteroidetes, although at a genus level, the response varied. The DNA abundance of Clostridium cluster I, Clostridium cluster XIV, Campylobacter spp., Ruminococcaceae, Turicibacter, and Blautia was significantly higher in dogs fed IZ and IZ + diets. Higher abundance of genus Lactobacillus was observed in dogs fed enzyme-supplemented diets. End-products of fecal fermentation were not affected by zinc source or enzymes. An increase in some taxa of the phyla Actinobacteria and Firmicutes was observed in feces of dogs fed organic zinc with enzyme addition but not with inorganic zinc. This study fills a gap in knowledge regarding the effect of zinc source and enzyme addition on the fecal microbiota of dogs. An association of zinc bioavailability and bacteria abundance is suggested, but the implications for the host (dog) are not clear. Further studies are required to unveil the effects of the interaction between zinc sources and enzyme addition on the fecal microbial community.

Miniaturized analytical methods for determination of environmental contaminants of emerging concern - A review.

The determination of contaminants of emerging concern (CECs) in environmental samples has become a challenging and critical issue. The present work focuses on miniaturized analytical strategies reported in the literature for the determination of CECs. The first part of the review provides brief overview of CECs whose monitoring in environmental samples is of particular significance, namely personal care products, pharmaceuticals, endocrine disruptors, UV-filters, newly registered pesticides, illicit drugs, disinfection by-products, surfactants, high technology rare earth elements, and engineered nanomaterials. Besides, an overview of downsized sample preparation approaches reported in the literature for the determination of CECs in environmental samples is provided. Particularly, analytical methodologies involving microextraction approaches used for the enrichment of CECs are discussed. Both solid phase- and liquid phase-based microextraction techniques are highlighted devoting special attention to recently reported approaches. Special emphasis is placed on newly developed materials used for extraction purposes in microextraction techniques. In addition, recent contributions involving miniaturized analytical flow techniques for the determination of CECs are discussed. Besides, the strengths, weaknesses, opportunities and threats of point of need and portable devices have been identified and critically compared with chromatographic methods coupled to mass chromatography. Finally, challenging aspects regarding miniaturized analytical methods for determination of CECs are critically discussed.

Supplemental selenium source on gut health: insights on fecal microbiome and fermentation products of growing puppies.

Selenium is an essential trace element that can modulate the gut microbiome with an impact on host health. The present study aimed to evaluate the effects of organic (selenium-enriched yeast) vs inorganic (sodium selenite) selenium source on fecal end-fermentation products and gut microbiome of puppies from 20 to 52 weeks of age. Alpha and beta diversity of the gut bacterial community were affected by age but not by gender or selenium source. The relative abundance of taxa was differently affected by age, and the DNA concentration of all selected bacterial groups increased with age, although total volatile fatty acids (VFA), acetate, propionate, caproate and lactate concentrations decreased. Organic selenium was associated with a higher concentration of total VFA, propionate and butyrate, a higher number of DNA copies of Lactobacillus, and a trend to lower DNA copies of Escherichia coli. Effects on fecal microbiome during growth differed with selenium source. Females had higher fecal end-fermentation products related to protein degradation, whereas males had higher DNA concentration of Bifidobacterium. Organic selenium might be beneficial over inorganic for dog food supplementation due to the positive modulation of the gut microbiome observed in puppies.