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.

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.

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.

Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry.

Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6 µm, 150 × 2.1 mm) at 30 °C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30 ng mL-1 with LOD and LOQ values as low as 0.1 and 0.3 pg in cell extracts and 16 and 48 pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (µ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL-1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 µg mL-1 (corresponding to 5.0-60 mg mL-1 in undiluted samples), with a detection limit of 33 µg mL-1 (1.7 mg mL-1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulations.

The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.

Human Platelet Antigens in Brazilian Multiethnic Populations: Occurrence of Regional Variation and Frequency in a Large Urban Center (Belo Horizonte).

BACKGROUND: The frequency of human platelet antigens (HPA) varies according to ethnicity, which causes differences in the morbidity of alloimmune and autoimmune thrombocytopenic disorders in different populations. Studies on HPA frequencies in Brazil have reported differences among Brazilian populations produced by the diverse degrees of admixture throughout the country. METHODS: In the present study, we investigated the variation of HPA distribution in Brazil, compared with worldwide populations, and describe the frequencies of HPA-1, -2, -3, -5, and -15 in a large urban center in Southern Brazil (Belo Horizonte) based on a sample of blood donors. RESULTS: The principal component analysis and the dendrogram based on genetic distance revealed a clear relationship between Brazilian populations and the groups formed by European and African populations. The coefficients of variation for HPA allele frequencies suggest that Brazilian populations presented variations for HPA alleles comparable with the populations from continental groups. In Belo Horizonte, the allele a frequencies for HPA-1, -2, -3, -5 and -15 were 0.8575, 0.8400, 0.6225, 0.8525 and 0.5825 respectively. The genotypes with higher frequencies were a/a (72-74%), except for HPA-3 and -15, whose heterozygous a/b genotypes were shown to be more prevalent (43.5 and 44.5%, respectively). CONCLUSION: We confirmed the heterogeneity of HPA antigens in Brazilian populations, reinforcing the importance of HPA panels composed of regional blood donors, or a national panel that contemplates the specificities of the different regions of the country, in order to provide support in platelet transfusions and to minimize the risks associated with HPA alloimmunization. The evaluation of HPA data from Belo Horizonte represents the initial step toward the development of a genotyped platelet donor registry in order to treat HPA alloimmunized patients in this region.

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.

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.

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.

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.

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.

Benefits of Fermented Papaya in Human Health.

Fermented foods have been used for several years all over the world, due to their unique nutritional characteristics and because fermentation promotes conservation and food security. Moreover, fermented foods and beverages have a strong impact on human gut microbiota. Papaya is the fruit of the Carica papaya plant, traditionally used as a medicinal fruit, but there are also references to the use of the fermented form of this fruit. The main purpose of this review is to provide an improved understanding of fermented papaya nutritional and health applications. A literature search was conducted in the PubMed and Google Scholar databases. Both in vitro and in vivo studies were included. According to the retrieved studies, fermented papaya has proven to be an excellent antioxidant and an excellent nutraceutical adjuvant in combined therapies against several diseases, such as Alzheimer's disease, allergic reactions, anticancer activity, and anemias. Therefore, it is concluded that fermented papaya has many benefits for human health and can be used as prevention or aid in the treatment of various diseases.

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.

Bacterial diversity and bioaugmentation in floodwater of a paddy field in the presence of the herbicide molinate.

This work aimed at studying variations on the diversity and composition of the bacterial community of a rice paddy field floodwater, subjected to conventional management, namely by using the herbicide molinate. The promotion of the herbicide biodegradation either by the autochthonous microbiota or by a bioaugmentation process was also assessed. This study comprehended four sampling campaigns at key dates of the farming procedures (seeding, immediately and 6 days after application of the herbicide molinate, and after synthetic fertilization) and the subsequent physic-chemical and microbiological characterization (pH, DOC and molinate contents, total cells, cultivable bacteria and DGGE profiling) of the samples. Multivariate analysis of the DGGE profiles showed temporal variations in the bacterial community structure and the Shannon's index values indicated that the bacterial diversity reached its minimum at the molinate application day. The highest bacterial diversity coincided with the periods with undetectable concentrations of the herbicide, although microcosm assays suggested that other factors than molinate may have been responsible for the decrease of the bacterial diversity. The ability of autochthonous microorganisms to degrade molinate and the influence of the herbicide on the bacterial community composition were assessed in microcosm assays using floodwater collected at the same dates. Given molinate was not degraded by autochthonous microorganisms, and considering it represents an environmental contaminant, bioaugmentation microcosms were assayed aiming the assessment of the feasibility of a bioremediation process to clean contaminated floodwater. A molinate-mineralizing culture, previously isolated, promoted molinate removal, induced alterations in the autochthonous bacterial community structure and diversity, and was undetected after 7 days of incubation, suggesting the feasibility of the process.

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.

Miniaturized Fluorimetric Method for Quantification of Zinc in Dry Dog Food.

Zinc is an essential trace element for animals in several biological processes, particularly in energy production, and it is acquired from food ingestion. In this context, a microplate-based fluorimetric assay was developed for simple, fast, and low-cost determination of zinc in pet food using 2,2'-((4-(2,7-difluoro-3,6-dihydroxy-4aH-xanthen-9-yl)-3-methoxyphenyl)azanediyl)diacetic acid (FluoZin-1) as fluorescent probe. Several aspects were studied, namely, the stability of the fluorescent product over time, the FluoZin-1 concentration, and the pH of reaction media. The developed methodology provided a limit of detection of 1 µg L-1 in sample acid digests, with a working range of 10 to 200 µg L-1, corresponding to 100-2000 mg of Zn per kg of dry dog food samples. Intraday repeatability and interday repeatability were assessed, with relative standard deviation values < 3.4% (100 µg L-1) and <11.7% (10 µg L-1). Sample analysis indicated that the proposed fluorimetric assay provided results consistent with ICP-MS analysis. These results demonstrated that the developed assay can be used for rapid determination of zinc in dry dog food.

Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection.

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.