Immunoproteomic profile of Malus domestica in Mexican pediatric patients. Evidence of new allergen prospects.

Background: Apple (Malus domestica) is a fruit commonly associated with allergic oral symptoms in the Mexican pediatric population; however, knowledge of its allergenic proteins is limited. This information is crucial as sensitization frequencies to specific allergens can vary among different populations. The main allergic symptomatology before apple ingestion derives from primary sensitizations induced by pollen, promoting cross-reactivity with the main allergenic protein of apple. Therefore, this study aims to identify new potential sensitizing proteins to apple using immunoproteomic techniques. Methods: We collected serum samples from 14 pediatric patients with confirmed immunoglobulin E (IgE)-mediated apple allergy and used these samples to assess immunoreactivity to apple protein extracts through 2D-western blot assays. The spots corresponding to the 2D-SDS-PAGE were analyzed using nanoLC-MS/MS. Results: We identified 11 non-redundant proteins, including Mal d 2 and Mal d 1, the latter showing a high frequency of sensitization (79%) in our patients, and being considered the main apple allergenic protein. The remaining identified proteins have not been previously described as apple allergens in the International Union of Immunological Societies databases. However, three of these may be categorized as pan-allergens. Conclusions: This study shows evidence that the repertoire of apple allergens in the Mexican population could differ from those reported internationally, highlighting the importance of studies in different countries to improve the certainty of allergy diagnosis and allow the implementation of precision medicine.

Pathogenesis of Experimental Infection of Nile Tilapia (Oreochromis niloticus) with Nucleospora Braziliensis Pathology and Proteomic of Microsporidia.

The recent discovery of disease caused by Nucleospora braziliensis in Nile tilapia (Oreochromis niloticus) is important as it has highlighted the high prevalence of infection and associated mortality in cultured fish. Thus, this study conducted an experimental infection of this microsporidium to evaluate pathological alterations and conduct proteomic analysis. For pathological observation, samples of brain, eyes, gall bladder, gut, heart, kidney, liver, muscle, skin, spleen, and stomach tissue, were collected, and liquid chromatography-mass spectrometry (LC-MS/MS) was performed for proteomic analysis. The most prevalent lesions were brownish color of the liver, gill filament fusion, gut ischemia, hemorrhage of the lips and fins, hepatomegaly, spleen atrophy, splenomegaly, and stomach congestion. The most common microscopic lesions were degeneration, hemorrhage, and inflammation in the brain, gills, gut, kidney, liver, muscle, spleen, and stomach. The digested peptides were identified by LC-MS/MS and the intersection of each group showed that in the spleen there were 121 exclusive proteins in the infected sample and 252 in the control, while in the kidney, 129 proteins were identified in the infected specimen compared to 83 in the control. In conclusion, this study demonstrates the proteome profile of O. niloticus kidney and spleen tissue in response to infection with N. braziliensis.

Antioxidant activity of Zuccagnia-type propolis: A combined approach based on LC-HRMS analysis of bioanalytical-guided fractions and computational investigation.

This study reports a combined approach to assess the antioxidant activity of Zuccagnia-type propolis. Fractions exhibiting the highest antioxidant activities evidenced by DPPH, a ß-carotene bleaching and superoxide radical scavenging activity-non-enzymatic assays, were processed by LC-HRMS/MS to characterize the relevant chemical compounds. A computational protocol based on the DFT calculations was used to rationalize the main outcomes. Among the 28 identified flavonoids, caffeic acids derivatives were in the fraction exhibiting the highest antioxidant activity, with 1-methyl-3-(4'-hydroxyphenyl)-propyl caffeic acid ester and 1-methyl-3-(3',4'-dihydroxyphenyl)-propyl caffeic acid ester as major components. Results clearly showed roles of specific chemical motifs, which can be supported by the computational analysis. This is the first report ascribing the antioxidant ability of Zuccagnia-type propolis to its content in specific caffeic acid derivatives, a potential source of radical scavenging phytochemicals. The proposed protocol can be extended to the study of other plant-products to address the most interesting bioactive compounds.

Optimal Brassicaceae family microgreens from a phytochemical and sensory perspective.

Microgreens, also called superfoods, emerge because of their high levels of nutrients, diverse flavour profiles, and sustainable cultivation methods, which make them culinary delights and valuable to a healthy and flavorful diet. The present study investigated Brassicaceae family microgreens, proposing a novel system (quality indices) that allows scoring among them. Fourteen Brassica microgreen species were morphological, phytochemical, and sensorial investigated. The morphological assessment revealed that radish microgreens exhibited the highest leaf area (p < 0.05), while red mizuna demonstrated superior yield. Cauliflower microgreens contained the highest concentrations of ascorbic acid (HPLC-DAD) and total phenolic content (p < 0.05). Phytochemical analysis using HPLC-MS/MS identified over 18 glucosinolates and phenolic compounds. Red mustard and red cabbage showed the highest glucosinolate content (p < 0.05). Watercress exhibited the highest phenolic compound content (p < 0.05), primarily flavonoids, while broccoli and radish contained the highest isothiocyanate levels. Cauliflower microgreens resulted in the most consumer-accepted variety. Appling quality indices scoring system identified radish, cauliflower, and broccoli microgreens as the most promising species. This study underscores the potential of Brassica microgreens as an excellent source of health-promoting phytochemicals with favorable market acceptance, providing valuable insights for both nutritional research and commercial applications.

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Characterization of the Polyphenolic Profile in Tomato (Lycopersicon esculentum P. Mill) Peel and Seeds by LC-HRMS/MS.

Peel and seeds are the main byproducts from tomato (Lycopersicon esculentum P. Mill) processing with high concentrations of polyphenols that have been underexploited. Herein, polyphenolic profiles in tomato peel and seeds were elucidated by untargeted liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) with an LTQ Orbitrap analyzer. Samples from two Spanish regions─"Murcia" and "Almería"─were analyzed to obtain complementary results. 57 compounds were found, mainly phenolic acids and flavonoids, of which eight were identified for the first time in tomato. Polyphenols were more abundant in byproducts from "Murcia" samples than in those from"Almería" samples, where the abundance of compounds like coutaric, caffeic, neochlorogenic, dicaffeoylquinic and ferulic acids, vanillic acid hexoside, catechin, naringenin, prunin, apigenin-O-hexoside, rutin, and rutin-O-pentoside was even much higher in byproducts than that in whole fruits. These results reveal the wide range of polyphenols found in tomato byproducts, with potential applications in pharmaceutical research, food preservation, and cosmetic development, among others.

A chemical analysis of the Pelargonium species: P. odoratissimum, P. graveolens, and P. zonale identifies secondary metabolites with activity against gram-positive bacteria with multidrug-resistance.

The Pelargonium genus encompasses around 280 species, most of which are used for medicinal purposes. While P. graveolens, P. odoratissimum, and P. zonale are known to exhibit antimicrobial activity, there is an evident absence of studies evaluating all three species to understand their chemical differences and biological effects. Through the analysis of the hydroalcoholic extracts of P. graveolens, P. odoratissimum, and P. zonale, using HPLC-DAD-MS/MS, quercetin and kaempferol derivatives were identified in these three species. Conversely, gallotannins and anthocyanins were uniquely detected in P. zonale. P. graveolens stood out due to the various types of myricetin derivatives that were not detected in P. odoratissimum and P. zonale extracts. Evaluation of their biological activities revealed that P. zonale displayed superior antibacterial and antibiofilm activities in comparison to the other two species. The antibacterial efficacy of P. zonale was observed towards the clinically relevant strains of Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus (MRSA) 333, Enterococcus faecalis ATCC 29212, and the Vancomycin-resistant E. faecalis INSPI 032. Fractionation analysis of P. zonale suggested that the antibacterial activity attributed to this plant is due to the presence of quercetin derivatives and kaempferol and its derivatives, alongside their synergistic interaction with gallotannins and anthocyanins. Lastly, the three Pelargonium species exhibited notable antioxidant activity, which may be attributed to their high content of total phenolic compounds.

Comprehensive assessment of clean-up strategies for optimizing an analytical multi-method to determine pesticides and mycotoxins in Brazilian medicinal herbs using QuEChERS-LC-TQ-MS/MS.

The use of medicinal herbs has increased significantly. However, the presence of pesticide residues and mycotoxins in medicinal herbs has generated constant discussion and concern among regulatory agencies. Developing and validating an analytical method for determining pesticides and mycotoxins in medicinal plants is challenging due to the naturally occurring substances in these plants. The purpose of this work was to develop and to optimize a sensitive, accurate, precise, effective QuEChERS method for simultaneous determination of over 160 pesticide and mycotoxin residues in complex medicinal plant matrices using LC-TQ-MS/MS. A comprehensive comparison of clean-up procedures and other parameters was conducted to achieve this goal. The validation procedure was performed according to SANTE 11312/2021. More polar analytes, such as acephate, methamidophos and omethoate, presented a higher negative matrix effect in both Melissa officinalis L. and Malva sylvestris L. However, other molecules, such as spirodiclofen, showed a 24% signal enhancement in M. officinalis and a 46% signal suppression in M. sylvestris, indicating that a representative matrix-matched calibration would lead to inaccurate quantification of the analyte. Accuracy and precision were satisfactory according to SANTE 11312/2021 for 157 pesticide residues and mycotoxins in M. officinalis and for 152 molecules in M. sylvestris. LOQs at 10 µg kg-1 were achieved for 117 pesticides in M. officinalis and 99 pesticides in M. sylvestris. Among the mycotoxins, all four aflatoxins (B1, B2, G1 and G2) presented LOQs of 5 µg kg-1, and ochratoxin A had an LOQ of 10 µg kg-1 in M. officinalis. The same LOQ values were shown for these mycotoxins in M. sylvestris, except for aflatoxin B2 and ochratoxin A, which had LOQs of 20 µg kg-1. Moreover, in Southern Brazil, there has been no previous study on mycotoxin and pesticide contamination in medicinal herbs. Therefore, the application of this method was assessed through the analysis of forty-two real samples. Imidacloprid was found in M. officinalis, and methyl pirimiphos was found in M. sylvestris. The proposed method not only serves as a helpful tool for routine monitoring but also offers a basis for further research on risk assessment and control in food safety.

"Cocaine Shark": First report on cocaine and benzoylecgonine detection in sharks.

Cocaine (COC) and benzoylecgonine (BE), the main COC metabolite, have been detected in aquatic ecosystems. Studies focusing on wild fish are, however, very limited, and no reports concerning elasmobranchs are available. This study investigated COC and BE levels in Brazilian Sharpnose sharks (Rhizoprionodon lalandii) (n = 13) using LC-MS/MS. All samples (13/13) tested positive for COC, with 92 % (12/13) testing positive for BE. COC concentrations (23.0 µg kg-1) were over 3-fold higher than BE (7.0 µg kg-1). COC levels were about three-fold significantly higher in muscle (33.8 ± 33.4 g kg-1) compared to liver (12.2 ± 14.2 µg kg-1). Females presented higher COC concentrations in muscle (40.2 ± 35.8 µg kg-1) compared to males (12.4 ± 5.9 µg kg-1). Several positive statistical correlations were noted between COC and BE (rho = 0.84) in females, indicating systemic COC transport and metabolization, as well as between BE and weight (rho = 0.62), and between COC and the Condition Factor (rho = 0.73). A strong correlation was noted between BE and COC in the muscle of non-pregnant females (rho = 1.00). This study represents the first COC and BE report in free-ranging sharks, and the findings point to the potential impacts of the presence of illicit drugs in environments.

Phytochemical analysis and investigation of analgesic, anti-inflammatory, and antispasmodic activities of hydroethanolic extracts of Alternanthera dentata, Ocimum carnosum, and Plectranthus barbatus, three species with vernacular names derived from analgesic drugs.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant vernacular names can provide clues about the popular use of a species in different regions and are valuable sources of information about the culture or vocabulary of a population. Several medicinal plants in Brazil have received names of medicines and brand-name products. AIM OF THE STUDY: The present work aimed to evaluate the chemical composition and pharmacological activity in the central nervous system of three species known popularly by brand names of analgesic, anti-inflammatory, antispasmodic, and digestive drugs. MATERIALS AND METHODS: Hydroethanolic extracts of Alternanthera dentata (AD), Ocimum carnosum (OC), and Plectranthus barbatus (PB) aerial parts were submitted to phytochemical analysis by HPLC-PAD-ESI-MS/MS and evaluated in animal models at doses of 500 and 1000 mg/kg. Mice were tested on hot plate, acetic acid-induced writing, formalin-induced licking, and intestinal transit tests. Aspirin and morphine were employed as standard drugs. RESULTS: The three extracts did not change the mice's response on the hot plate. Hydroethanolic extracts of AD and PB reduced the number of writhes and licking time, while OC was only effective on the licking test at dose of 1000 mg/kg. In addition, AD and OC reduced intestinal transit, while PB increased gut motility. CONCLUSIONS: Pharmacological tests supported some popular uses, suggesting peripheral antinociceptive and anti-inflammatory effects, while the phytochemical analysis showed the presence of several flavonoids in the three hydroethanolic extracts and steroids in PB, with some barbatusterol derivatives described for the first time in the species.

Discovery of candidate biomarkers from plasma-derived extracellular vesicles of patients with cirrhosis and hepatocellular carcinoma: an exploratory proteomic study.

Extracellular vesicles (EVs) represent an attractive source of biomarkers due to their biomolecular cargo. The aim of this study was to identify candidate protein biomarkers from plasma-derived EVs of patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Plasma-derived EVs from healthy participants (HP), LC, and HCC patients (eight samples each) were subjected to label-free quantitative proteomic analysis using LC-MS/MS. A total of 248 proteins were identified, and differentially expressed proteins (DEPs) were obtained after pairwise comparison. We found that DEPs mainly involve complement cascade activation, coagulation pathways, cholesterol metabolism, and extracellular matrix components. By choosing a panel of up- and down-regulated proteins involved in cirrhotic and carcinogenesis processes, TGFBI, LGALS3BP, C7, SERPIND1, and APOC3 were found to be relevant for LC patients, while LRG1, TUBA1C, TUBB2B, ACTG1, C9, HP, FGA, FGG, FN1, PLG, APOB and ITIH2 were associated with HCC patients, which could discriminate both diseases. In addition, we identified the top shared proteins in both diseases, which included LCAT, SERPINF2, A2M, CRP, and VWF. Thus, our exploratory proteomic study revealed that these proteins might play an important role in the disease progression and represent a panel of candidate biomarkers for the prognosis and diagnosis of LC and HCC.

A miniaturized QuEChERS and UPLC-MS/MS method for the determination of mycotoxins in cashew nuts.

This study aimed to develop and validate a multi-mycotoxin analysis method applied to cashew nuts by employing a miniaturized QuEChERS method followed by determination by ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Satisfactory recoveries for the concentrations 1, 10 and 30 ng g-1, ranging from 66% (fumonisin B1) to 110% (ochratoxin A) and relative standard deviations lower than 9% (fumonisin B2) were obtained for the target compounds. Limits of quantification ranged from 0.004 ng g-1 (sterigmatocystin) to 0.59 ng g-1 (alternariol). The applicability of the analytical method was verified by analyzing 30 cashew nut samples from the city of Rio de Janeiro, RJ, southeastern Brazil. Aflatoxins M1, G2, G1, B2, B1, ochratoxin A and sterigmatocystin were detected, respectively, in 27%, 10%, 17%, 30%, 30%, 30% and 50% of the analyzed samples, at maximum concentrations of 0.56, 0.67, 1.43, 2.02, 4.93, 4.81, and 0.35 ng g-1. The maximum limit established by Brazilian legislation for aflatoxins was not exceeded by any of the analyzed samples.

PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis.

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

Hydroethanolic extracts from Bauhinia guianensis: a study on acute toxicity in Zebrafish embryos and adults.

CONTEXT: The botanical species Bauhinia guianensis Aublet (Leguminosae-Cercidoideae) is traditionally used in the Amazon for medicinal purposes. OBJECTIVE: The acute toxicity of the hydroethanolic extracts from B. guianensis leaves and stems (HELBg and HESBg) was evaluated in zebrafish (Danio rerio), with emphasis on the embryonic developmental stage and adult alterations. MATERIALS AND METHODS: Extracts were analyzed on LC-DAD-MS/MS. Zebrafish eggs were inoculated individually with concentrations of HELBg and HESBg (0.25, 0.5, 0.75, 1.0, and 1.5 µg/mL), observed for 96 h. Adult zebrafish were treated with a single oral dose (100, 200, 500, 1000, and 2000 mg/kg) of HELBg and HESBg, observed for 48 h. RESULTS: HELBg and HESBg analysis detected 55 compounds. Both extracts exhibited toxicity, including embryo coagulation at higher doses of HELBg and absence of heartbeats in embryos at all doses of HESBg. Behavioral variations were observed; tissue alterations in adult zebrafish were found at the highest doses, primarily in the liver, intestine, and kidneys because of HELBg and HESBg effects. The LD50 of HESBg was 1717 mg/kg, while HELBg exceeded the limit dose of 2000 mg/kg. CONCLUSIONS: The study on acute toxicity of B. guianensis extracts exhibits significant toxic potential, emphasizing effects on embryonic and adult zebrafish. The results suggest relative safety of the species preparations, encouraging further clinical trials on potential biological activities.

Pesticides in vegetables and fruits from Brazil and risk assessment.

Levels of 237 pesticides were assessed in 1063 fruit and vegetable samples from 12 São Paulo markets spanning the period May 2015 to December 2022. The QuEChERS method was employed for extraction, followed by GC-MS/MS and LC-MS/MS analysis. Findings indicated that 30% of the samples contained residues below the Maximum Residue Limits (MRLs), while 6% exceeded these. Additionally, 23% exhibited excessive residues for their respective crops and 40% had no detectable residues. Health risk evaluation focused on tomatoes, cabbage and oranges, revealing exposure within 0.002-0.9% of the Acceptable Daily Intake (ADI), indicating no chronic risks. However, pyraclostrobin in orange presented a potential acute risk for adults (112%). These results underscore the necessity for continuous monitoring of pesticide residues in fruits and vegetables to safeguard consumer health, especially considering the significant levels of consumption.

Investigating the etiology of Haff disease: Optimization and validation of a sensitive LC-MS/MS method for palytoxins analysis in directly associated freshwater and marine food samples from Brazil.

Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid-liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 µg/kg), being able to work in a range that includes the provisional ingestion limit (30 µg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS).

Association between Childhood Overweight and Altered Concentrations of Circulating Amino Acids.

(1) Background: Dysregulated serum amino acids (AA) are known to be associated with obesity and risk of Type 2 Diabetes (T2D) in adults, and recent studies support the same notion in the pubertal age. It is, however, unknown whether childhood overweight may already display alterations of circulating AA. (2) Methods: We used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-targeted metabolomics to determine plasma concentrations of AA and AA-related molecules in 36 children aged 7-12 years with normal weight or overweight. Clinical and anthropometric parameters were measured. (3) Results: Overweight in children is associated with an altered AA profile, with increased branched-chain amino acids (BCAA) and decreased glycine levels, with no clinically manifested metabolic conditions. Moreover, z-BMI was positively and negatively correlated with BCAA and glycine levels, respectively, even after adjustment for age and gender. We also found a correlation between the AA profile and clinical parameters such as lipids profile and glycemia. (4) Conclusions: A pattern of low glycine, and increased BCAA is correlated to z-BMI, total cholesterol, and triglycerides in overweight but otherwise healthy children. Our data suggest that, in childhood overweight, AA disturbances may precede other clinical parameters, thus providing an early indicator for the later development of metabolic disease.

Determination of ethylene-bis-dithiocarbamates and their degradation product ethylenethiourea in dry herbs by UHPLC-MS/MS.

Dithiocarbamates are a class of fungicides widely used in many countries. In this study, methods for determining the ethylene-bis-dithiocarbamate (EBDC) subclass, and their degradation product ethylenethiourea (ETU) were validated by UHPLC-MS/MS in different types of dry herbs, which can be used as food and/or medicinal purposes. Mancozeb was used in the validation of the EBDC method, where it was initially complexed with EDTA, derivatized, extracted with dimethyl sulfate in acetonitrile, magnesium sulfate (MgSO4), and sodium chloride (NaCl), and then purified using primary secondary amine (PSA). In the ETU method, L-cysteine hydrochloride monohydrate was added to the samples before extraction with acetonitrile, MgSO4, and NaCl, followed by purification with PSA. A pesticide-free blend of seven herbs (boldo, artichoke, "espinheira-santa", cat's claw, senna, chamomile, and cascara buckthorn) comprising distinct parts of the plants (leaves, bark, flowers and/or stems) was used as a control for method validation. Recoveries ranged from 79 to 113% for EBDC and 81 to 109% for ETU. Repeatability and intermediate precision were <20% for both methods. The limit of quantification was 0.03 mg kg-1 for EBDC (0.02 mg kg-1 of CS2) and ETU. The limit of detection (LOD) was set at 1/3 of the LOQ (0.01 mg kg-1 for both analytes). In total, 103 samples of 33 different dry herbs were analyzed, of which 19.4% were positive for EBDC (≥LOD), but no ETU residues were found in any of the analyzed samples. Given the absence of registered dithiocarbamates for use in the investigated herbs in Brazil, the positive results suggest potential illegal pesticide use or cross-contamination, especially considering the low concentrations detected in most samples. Although exposure to EBDC through the consumption of medicinal herbs from positive samples did not indicate a health risk to consumers, these plants must be monitored to prevent illicit pesticide usage, particularly when the herbs are intended for therapeutic purposes.

In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

Improving analytical performance for pesticides and mycotoxins determination in Brazilian table olives: one extraction and one analysis.

This paper describes an extensive study in which a multiclass QuEChERS based approach was optimized for determination of 150 pesticides and 7 mycotoxins in table olives. Three versions of QuEChERS were evaluated and compared (unbuffered, citrate and acetate buffering). A combination of EMR-Lipid cartridges and liquid nitrogen or freezer freezing out were tested for clean-up of the oily olive extracts. Analysis of the extracts were performed by LC-MS/MS triple quadrupole. The best results were achieved using acetate QuEChERS with liquid nitrogen for clean-up. For validation, organic olives were ground and spiked at 4 concentrations with pesticides and mycotoxins (n = 5). The linearity of the calibration curves was assessed by analyzing calibration standards of 7 concentrations which were prepared separately in acetonitrile and in blank olive extract (n = 5). The validation study demonstrated that the calculated r2 was ≥0.99 for 144 pesticides and 6 mycotoxins, when the calibration curves were prepared in matrix extract, showing satisfactory linearity. Matrix effects were within the range of ±20% for only 46 pesticides and one mycotoxin. Then, to ensure reliable quantification, calibration standards had to be matrix-matched. In accuracy experiments 138 pesticides and 6 mycotoxins presented recoveries from 70 to 120% and RSD ≤ 20% for at least 2 of the 4 spike concentrations evaluated, being successfully validated. The integrated QuEChERS and LC-MS/MS method meet MRL for 11 of the 21 pesticides regulated for olives in Brazil and for 132 pesticides which are regulated in the EU law. Eleven commercial table olive samples were analyzed and 4 of them tested positive for pesticides. All the positive samples violate the Brazilian law and one sample violates also the European law.