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1.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500622

RESUMO

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 µg/mL, which can be considered as highly active.


Assuntos
Proliferação de Células/efeitos dos fármacos , Glucosinolatos/química , Glucosinolatos/farmacologia , Lepidium/química , Linhagem Celular Tumoral , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glioblastoma/tratamento farmacológico , Humanos , Hidrólise , Isotiocianatos/química , Isotiocianatos/farmacologia , Espectrometria de Massas em Tandem/métodos , Tiocianatos/química , Tiocianatos/farmacologia , Tioglucosídeos/química , Tioglucosídeos/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico
2.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443345

RESUMO

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.


Assuntos
Algoritmos , Glicopeptídeos/análise , Glicoproteínas/análise , COVID-19/metabolismo , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Proteômica/métodos , Proteômica/normas , SARS-CoV-2/metabolismo , Software , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/química , Espectrometria de Massas em Tandem/métodos , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/química
3.
Molecules ; 26(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34361591

RESUMO

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid-liquid extraction with only one step-protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r2 ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased.


Assuntos
Adjuvantes Imunológicos/farmacocinética , Antivirais/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Inosina Pranobex/farmacocinética , Espectrometria de Massas em Tandem/métodos , para-Aminobenzoatos/farmacocinética , Adjuvantes Imunológicos/administração & dosagem , Animais , Antivirais/administração & dosagem , Inosina Pranobex/administração & dosagem , Projetos Piloto , Suínos
4.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360885

RESUMO

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo-synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo-synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.


Assuntos
Histonas/biossíntese , Cabeça do Espermatozoide/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Núcleo Celular/metabolismo , Cromatografia Líquida/métodos , Feminino , Fertilização/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Injeções de Esperma Intracitoplásmicas , Espectrometria de Massas em Tandem/métodos
5.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361749

RESUMO

Cefquinome and ceftiofur are ß-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g-1 to 1000 ng g-1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g-1 to 2000 ng g-1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since ß-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL-1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.


Assuntos
Acetamidas/isolamento & purificação , Antibacterianos/isolamento & purificação , Cefalosporinas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/normas , Furanos/isolamento & purificação , Espectrometria de Massas em Tandem/normas , Acetamidas/administração & dosagem , Animais , Antibacterianos/administração & dosagem , Calibragem , Cefalosporinas/administração & dosagem , Cromatografia Líquida de Alta Pressão/métodos , Fezes/química , Feminino , Furanos/administração & dosagem , Injeções Intramusculares , Masculino , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Suínos , Espectrometria de Massas em Tandem/métodos , Tazobactam/química
6.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360905

RESUMO

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana Múltipla , Listeria/efeitos dos fármacos , Listeria/patogenicidade , Proteoma/química , Fatores de Virulência/química , Transportadores de Cassetes de Ligação de ATP/química , Cromatografia Líquida/métodos , Genes Bacterianos , Listeria/classificação , Listeria/genética , Peptídeos/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360914

RESUMO

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.


Assuntos
Caseínas/metabolismo , Glicopeptídeos/química , Lactação/metabolismo , Leite Humano/química , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/química , Aleitamento Materno , Cromatografia Líquida/métodos , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Lactente , Estudos Longitudinais , Fosforilação , Espectrometria de Massas em Tandem/métodos
8.
Nutrients ; 13(8)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34444931

RESUMO

Over the centuries, humans have traditionally used garlic (Allium sativum L.) as a food ingredient (spice) and remedy for many diseases. To confirm this, many extensive studies recognized the therapeutic effects of garlic bulbs. More recently, black garlic (BG), made by heat-ageing white garlic bulbs, has increased its popularity in cuisine and traditional medicine around the world, but there is still limited information on its composition and potential beneficial effects. In this study, the metabolite profile of methanol extract of BG (BGE) was determined by high-performance liquid chromatography coupled to tandem mass spectrometry in high-resolution mode. Results allowed to establish that BGE major components were sulfur derivatives, saccharides, peptides, organic acids, a phenylpropanoid derivative, saponins, and compounds typical of glycerophospholipid metabolism. Characterization of the BGE action in cancer cells revealed that antioxidant, metabolic, and hepatoprotective effects occur upon treatment as well as induction of maturation of acute myeloid leukemia cells. These results are interesting from the impact point of view of BG consumption as a functional food for potential prevention of metabolic and tumor diseases.


Assuntos
Alho/química , Leucemia Mieloide Aguda/tratamento farmacológico , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Peptídeos/análise , Raízes de Plantas/química , Polissacarídeos/análise , Saponinas/análise , Especiarias/análise , Enxofre/análise , Espectrometria de Massas em Tandem/métodos , Células U937
9.
Nutrients ; 13(8)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34444934

RESUMO

Vitamin D deficiency is a global problem with many health consequences, and it is currently recommended to supplement vitamin D. Change of diet should also be considered to ensure adequate vitamin D in the human body. The aim of this study was to assess the concentration of vitamin D metabolites in two different groups: one group on the low-carbohydrate-high-fat (LCHF) diet and the other group on the Eastern European (EE) diet. In the first stage, 817 participants declaring traditional EE diet or LCHF diet were investigated. Nutrition (self-reported 3-day estimated food record) and basic anthropometric parameters were assessed. After extra screening, 67 participants on the EE diet and 41 on the LCHF diet were qualified for the second stage. Plasma 25-hydroxycholecalciferol (25(OH)D3) and (25(OH)D2) concentration was measured by the validated HPLC-MS/MS method. Plasma 25(OH)D3 concentration was significantly higher in the group on the LCHF diet (34.9 ± 15.9 ng/mL) than in the group on the EE diet (22.6 ± 12.1 ng/mL). No statistical differences were observed in plasma 25(OH)D2 concentration between the study groups (p > 0.05). Women had a higher plasma 25(OH)D2 concentration than men regardless of diet type. The LCHF diet had a positive influence on plasma vitamin D concentration. However, long-term use of the LCHF diet remains contentious due to the high risk of cardiovascular disease. This study confirmed that the type of diet influences the concentration of vitamin D metabolites in the plasma.


Assuntos
Dieta com Restrição de Carboidratos/métodos , Dieta Hiperlipídica/métodos , Deficiência de Vitamina D/sangue , Vitamina D/sangue , Idoso , Calcifediol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Dieta/métodos , Suplementos Nutricionais , Ergocalciferóis/sangue , Grupo com Ancestrais do Continente Europeu , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estado Nutricional , Projetos Piloto , Polônia , Espectrometria de Massas em Tandem/métodos , Vitaminas/sangue
10.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445532

RESUMO

The specificity of a diagnostic assay depends upon the purity of the biomolecules used as a probe. To get specific and accurate information of a disease, the use of synthetic peptides in diagnostics have increased in the last few decades, because of their high purity profile and ability to get modified chemically. The discovered peptide probes are used either in imaging diagnostics or in non-imaging diagnostics. In non-imaging diagnostics, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), lateral flow devices (i.e., point-of-care testing), or microarray or LC-MS/MS are used for direct analysis of biofluids. Among all, peptide-based ELISA is considered to be the most preferred technology platform. Similarly, peptides can also be used as probes for imaging techniques, such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The role of radiolabeled peptides, such as somatostatin receptors, interleukin 2 receptor, prostate specific membrane antigen, αß3 integrin receptor, gastrin-releasing peptide, chemokine receptor 4, and urokinase-type plasminogen receptor, are well established tools for targeted molecular imaging ortumor receptor imaging. Low molecular weight peptides allow a rapid clearance from the blood and result in favorable target-to-non-target ratios. It also displays a good tissue penetration and non-immunogenicity. The only drawback of using peptides is their potential low metabolic stability. In this review article, we have discussed and evaluated the role of peptides in imaging and non-imaging diagnostics. The most popular non-imaging and imaging diagnostic platforms are discussed, categorized, and ranked, as per their scientific contribution on PUBMED. Moreover, the applicability of peptide-based diagnostics in deadly diseases, mainly COVID-19 and cancer, is also discussed in detail.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Peptídeos/análise , COVID-19/virologia , Bases de Dados Factuais , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Tomografia por Emissão de Pósitrons/métodos , Receptores de Somatostatina , SARS-CoV-2/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos
11.
Nat Commun ; 12(1): 4798, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376656

RESUMO

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [13C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [13C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.


Assuntos
Acetatos/metabolismo , Clostridiales/metabolismo , Inositol/metabolismo , Intestinos/química , Propionatos/metabolismo , Animais , Clostridiales/classificação , Clostridiales/fisiologia , Dieta , Fezes/microbiologia , Interações entre Hospedeiro e Microrganismos , Humanos , Intestinos/microbiologia , Espectroscopia de Ressonância Magnética/métodos , Masculino , Camundongos Endogâmicos C57BL , Ácido Fítico/metabolismo , Espectrometria de Massas em Tandem/métodos
12.
J Am Chem Soc ; 143(31): 12014-12024, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34328324

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry, and the S protein glycosylation plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants as well as other O-glycoproteins in general.


Assuntos
Polissacarídeos/análise , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Sequência de Carboidratos , Polissacarídeos/química , Domínios Proteicos , Espectrometria de Massas em Tandem/métodos
13.
Anal Bioanal Chem ; 413(23): 5811-5820, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34302183

RESUMO

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C18 column (50 × 2.1 mm, 3.5 µm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg-1). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Adenosina/sangue , Adenosina/farmacocinética , Adenosina/normas , Monofosfato de Adenosina/sangue , Monofosfato de Adenosina/farmacocinética , Monofosfato de Adenosina/normas , Alanina/sangue , Alanina/farmacocinética , Alanina/normas , Animais , Antivirais/farmacocinética , Antivirais/normas , Limite de Detecção , Masculino , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes
14.
J Chromatogr A ; 1652: 462355, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34233246

RESUMO

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.


Assuntos
Neoplasias Colorretais/diagnóstico , Eletroforese Capilar/métodos , Poliaminas/análise , Saliva/química , Espectrometria de Massas em Tandem/métodos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/isolamento & purificação , Neoplasias Colorretais/química , Humanos , Poliaminas/isolamento & purificação
15.
J Chromatogr A ; 1652: 462360, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34246057

RESUMO

The misuse of propofol for recreational purposes has become a serious social issue. Accordingly, practical and sensitive analytical methods to investigate the chronic abuse and toxicity of propofol are required. However, current propofol determination methods using liquid chromatography-mass spectrometry (LC-MS/MS) suffer from problems associated with loss in sample preparation due to its volatility and its poor ionization efficiency and collision-induced dissociation in mass spectrometry. Herein, we have developed a sensitive and accurate fluoride-assisted LC-MS/MS method combined with direct-injection for propofol determination. Ionization via fluoride-ion attachment/induced deprotonation, effected by ammonium fluoride in the mobile phase, was found to dramatically improve the sensitivity of propofol without derivatization. Furthermore, direct injection without derivatization enables the simultaneous analysis of propofol and its phase II metabolites without analyte loss. The optimal concentration of ammonium fluoride in the mobile phase was found to be 1 mM under methanol conditions. The linearity is good (R2 ≥ 0.999) and the intra- and inter-day precisions for propofol determination are between 1.9 and 8.7%. The accuracies range from 87.5% to 105.4% and the limits of detection and quantitation for propofol in urine are 0.15 and 0.44 ng mL-1, respectively. The present method was successfully applied to human urine and showed a sufficient sensitivity to determine propofol and five phase II metabolites over 48 h in human urine after administration. Consequently, the fluoride-assisted LC-MS/MS method was demonstrated to be sensitive, accurate, and practical for the determination of propofol and its metabolites.


Assuntos
Cromatografia Líquida/métodos , Propofol/urina , Espectrometria de Massas em Tandem/métodos , Compostos de Amônio/química , Fluoretos/química , Humanos , Propofol/análise , Propofol/metabolismo
16.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299017

RESUMO

Oxygen deficiency in cells, tissues, and organs can not only prevent the proper development of biological functions but it can also lead to several diseases and disorders. In this sense, the kidney deserves special attention since hypoxia can be considered an important factor in the pathophysiology of both acute kidney injury and chronic kidney disease. To provide better knowledge to unveil the molecular mechanisms involved, new studies are necessary. In this sense, this work aims to study, for the first time, an in vitro model of hypoxia-induced metabolic alterations in human proximal tubular HK-2 cells because renal proximal tubules are particularly susceptible to hypoxia. Different groups of cells, cultivated under control and hypoxia conditions at 0.5, 5, 24, and 48 h, were investigated using untargeted metabolomic approaches based on reversed-phase liquid chromatography-mass spectrometry. Both intracellular and extracellular fluids were studied to obtain a large metabolite coverage. On the other hand, multivariate and univariate analyses were carried out to find the differences among the cell groups and to select the most relevant variables. The molecular features identified as affected metabolites were mainly amino acids and Amadori compounds. Insights about their biological relevance are also provided.


Assuntos
Hipóxia Celular , Cromatografia de Fase Reversa/métodos , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Ativação Metabólica/genética , Ativação Metabólica/fisiologia , Hipóxia Celular/genética , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Rim/citologia , Rim/metabolismo , Rim/patologia , Metaboloma/genética , Análise Multivariada , Análise de Componente Principal
17.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299329

RESUMO

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.


Assuntos
Glutens/análise , Glutens/metabolismo , Triticum/metabolismo , Alelos , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional/métodos , Haplótipos , Peso Molecular , Melhoramento Vegetal/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Triticum/química , Triticum/genética
18.
Molecules ; 26(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34299386

RESUMO

This study was carried out to develop a high-performance liquid chromatography method for short-time analysis of the main cannabinoids in the inflorescence of hemp (Cannabis sativa L.). We also performed decarboxylation of the raw material using our advanced analysis technique. In this study, the UV spectrum was considered to analyze each of the four common cannabinoids, solvents, and samples, where the uniform elution of acidic cannabinoids without peak tailing and acids was tested. Optimal results were obtained when readings were taken at a wavelength of 220 nm using water and methanol containing trifluoroacetic acid as mobile phases in a solvent gradient system. The established conditions were further validated by system suitability, linearity, precision, detection limit, and quantitation limit tests. The decarboxylation index (DT50) confirmed that Δ9-THCA decarboxylated faster than CBDA, and both maintained a linear relationship with time and temperature. In addition, the loss of cannabidiol was better prevented during the decarboxylation process in the natural state than in the extracted state.


Assuntos
Canabinoides/análise , Cannabis/química , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/análise , Solventes/química , Espectrometria de Massas em Tandem/métodos
19.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299551

RESUMO

Forchlorfenuron (CPPU) is a plant growth regulator extensively used in agriculture. However, studies on CPPU pharmacokinetics are lacking. We established and validated a rapid, sensitive, and accurate liquid chromatography-mass spectrometry method for CPPU detection in rat plasma. CPPU pharmacokinetics was evaluated in adult and juvenile rats orally treated with 10, 30, and 90 mg/kg of the compound. The area under the plasma drug concentration-time curve from 0 to 24 h (AUC), at the final time point sampled (AUC0-t), and the maximum drug concentration of CPPU increased in a dose-dependent manner. The pharmacokinetic parameters AUC0-t and absolute bioavailability were higher in the juvenile rats than in adult rats. The mean residence time and AUC0-t of juvenile rats in the gavage groups, except for the 10 mg/kg dose, were significantly higher in comparison to those observed for adult rats (p < 0.001). The plasma clearance of CPPU in juvenile rats was slightly lower than that in the adult rats. Taken together, juvenile rats were more sensitive to CPPU than adult rats, which indicates potential safety risks of CPPU in minors.


Assuntos
Compostos de Fenilureia/farmacocinética , Piridinas/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Masculino , Plasma/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos
20.
Molecules ; 26(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34299437

RESUMO

We examined the application of six different resins with the aim of selecting a macroporous resin suitable for purifying Acanthopanax senticosus total flavonoids (ASTFs) from Acanthopanax senticosus crude extract (EAS) by comparing their adsorption/desorption capacities, which led to the selection of HPD-600. Research on the adsorption mechanism showed that the adsorption process had pseudo-second-order kinetics and fit the Freundlich adsorption model. Moreover, the analysis of thermodynamic parameters indicated that the adsorption process is spontaneous and endothermic. The optimal conditions for purification of ASTFs were determined as sample pH of 3, 60% ethanol concentration, and 3 BV·h-1 flow rate, for both adsorption and desorption, using volumes of 2.5 and 4 BV, respectively. The application of macroporous resin HPD-600 to enrich ASTFs resulted in an increase in the purity of total flavonoids, from 28.79% to 50.57%. Additionally, the antioxidant capacity of ASTFs was higher than that of EAS, but both were lower than that of L-ascorbic acid. The changes in ASTFs compositions were determined using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), with the results illustrating that the levels of seven major flavonoids of ASTFs were increased compared to that in the crude extract.


Assuntos
Eleutherococcus/metabolismo , Flavonoides/química , Adsorção/fisiologia , Antioxidantes/análise , Flavonoides/análise , Extratos Vegetais/química , Folhas de Planta/química , Resinas Vegetais/análise , Espectrometria de Massas em Tandem/métodos
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