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1.
J Environ Sci (China) ; 97: 19-24, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32933735

RESUMO

Bisphenol A (BPA) is one of the environmental endocrine disruptors (EDCs), and BPA contamination in environment can cause high risks to human health. Rapid determination of BPA on sites is in high demand in environmental analysis. Taking advantage of aptamers as affinity ligands and fluorescence anisotropy (FA) analysis, we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine (TMR) labeled short 35-mer DNA aptamer against BPA. The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases, resulting in change of FA signals. We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition. The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA. By using this TMR-labeled aptamer, we achieved FA detection of BPA with a detection limit of 0.5 µmol/L under the optimized conditions. This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample, showing the potential applications on water samples.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Compostos Benzidrílicos , Polarização de Fluorescência , Humanos , Limite de Detecção , Fenóis
2.
Proc Natl Acad Sci U S A ; 117(37): 22727-22735, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868442

RESUMO

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Mucosa Nasal/virologia , Pandemias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Smartphone
3.
Environ Monit Assess ; 192(10): 662, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32979107

RESUMO

The centrifuge-less dispersive liquid-liquid microextraction (DLLME) technique was used to separate selenium species in aqueous samples. According to the salting-out effect, a simple approach was used to eliminate the centrifugation step. The optimization of the independent variables was performed using chemometric methods. Under optimal conditions, this methodology was statistically validated. The linearity was between 20 and 300 µg L-1. The limit of detection and quantification were calculated 3.4 µg L-1 and 10.4 µg L-1, respectively. The values of reproducibility and repeatability were determined ≤ 9.5% and ≤ 6.4, respectively. The possibility of the method was successfully assessed by analyzing the analytes in real samples clarified satisfactory recoveries (98.1-101.4% for Se (IV) and 98.4-101.5% for Se (VI)).


Assuntos
Microextração em Fase Líquida , Selênio/análise , Poluentes Químicos da Água/análise , Centrifugação , Monitoramento Ambiental , Limite de Detecção , Reprodutibilidade dos Testes
4.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Biomarcadores/análise , Proteína C-Reativa/análise , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotecnologia/métodos
5.
Sci Adv ; 6(39)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32978154

RESUMO

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , DNA de Cadeia Simples/genética , Eletroforese em Gel de Ágar/métodos , Pneumonia Viral/diagnóstico , RNA Viral/genética , Análise de Sequência de RNA/métodos , Sequência de Bases , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/genética , Eletroforese em Gel de Ágar/economia , Humanos , Limite de Detecção , Pandemias , Pneumonia Viral/virologia , Saliva/virologia , Análise de Sequência de RNA/economia , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia
6.
PLoS One ; 15(9): e0236311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32898153

RESUMO

Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Índice de Gravidade de Doença , Carga Viral
7.
J Nanobiotechnology ; 18(1): 130, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32912236

RESUMO

Fast point-of-care (POC) diagnostics represent an unmet medical need and include applications such as lateral flow assays (LFAs) for the diagnosis of sepsis and consequences of cytokine storms and for the treatment of COVID-19 and other systemic, inflammatory events not caused by infection. Because of the complex pathophysiology of sepsis, multiple biomarkers must be analyzed to compensate for the low sensitivity and specificity of single biomarker targets. Conventional LFAs, such as gold nanoparticle dyed assays, are limited to approximately five targets-the maximum number of test lines on an assay. To increase the information obtainable from each test line, we combined green and red emitting quantum dots (QDs) as labels for C-reactive protein (CRP) and interleukin-6 (IL-6) antibodies in an optical duplex immunoassay. CdSe-QDs with sharp and tunable emission bands were used to simultaneously quantify CRP and IL-6 in a single test line, by using a single UV-light source and two suitable emission filters for readout through a widely available BioImager device. For image and data processing, a customized software tool, the MultiFlow-Shiny app was used to accelerate and simplify the readout process. The app software provides advanced tools for image processing, including assisted extraction of line intensities, advanced background correction and an easy workflow for creation and handling of experimental data in quantitative LFAs. The results generated with our MultiFlow-Shiny app were superior to those generated with the popular software ImageJ and resulted in lower detection limits. Our assay is applicable for detecting clinically relevant ranges of both target proteins and therefore may serve as a powerful tool for POC diagnosis of inflammation and infectious events.


Assuntos
Biomarcadores/análise , Proteína C-Reativa/análise , Imunoensaio/métodos , Interleucina-6/análise , Pontos Quânticos/química , Sepse/diagnóstico , Anticorpos/imunologia , Betacoronavirus/isolamento & purificação , Proteína C-Reativa/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Humanos , Interleucina-6/imunologia , Limite de Detecção , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Sepse/metabolismo , Software , Raios Ultravioleta
8.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751106

RESUMO

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.


Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , Carga Viral
9.
Biosensors (Basel) ; 10(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847008

RESUMO

Cytokines are a family of proteins which play a major role in the regulation of the immune system and the development of several diseases, from rheumatoid arthritis to cancer and, more recently, COVID-19. Therefore, many efforts are currently being developed to improve therapy and diagnosis, as well as to produce inhibitory drugs and biosensors for a rapid, minimally invasive, and effective detection. In this regard, even more efficient cytokine receptors are under investigation. In this paper we analyze a set of IL-6 cytokine receptors, investigating their topological features by means of a theoretical approach. Our results suggest a topological indicator that may help in the identification of those receptors having the highest complementarity with the protein, a feature expected to ensure a stable binding. Furthermore, we propose and discuss the use of these receptors in an idealized experimental setup.


Assuntos
Técnicas Biossensoriais/métodos , Interleucina-6/análise , Receptores de Interleucina-6/análise , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Aptâmeros de Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/imunologia , Limite de Detecção , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Receptores de Interleucina-6/imunologia
10.
J Environ Radioact ; 222: 106340, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763727

RESUMO

As the number of nuclear power plants (NPPs) being decommissioned increases internationally, many issues are being raised. One such issue is related to site soil analyses for the determination of residual risk for license termination. In a typical site-cleanup analysis, the majority of soil samples at the site are at or below the detection limit (BDL). Conventional approaches to managing BDL data are to simply ignore or substitute the data with a value of zero or the detection limit itself. However, these approaches are statistically biased, limiting their usefulness. Within the environmental science community, the issue of how to treat BDL data has been examined by a number of investigators. This study reviewed the issue of BDL data in nuclear decommissioning using the analytical methods suggested by studies in the environmental science, including the Kaplan-Meier method, robust regression on order statistics, and maximum likelihood estimation. The use of these methods to handle BDL data was examined using a case study with respect to its potential impact on dose/risk assessment, the soil volume removal estimate, and the associated costs. The case study was based on the Colorado School of Mines Research Institute's site soil data. Our analysis included the consideration of the uncertainties associated with residual dose/risk, waste soil volume estimation, and costs. An uncertainty analysis was conducted using a Latin hypercube sampling approach. Results showed that using BDL data can have a large impact on the estimation of dose/risk, waste volume, and waste disposal cost of a NPP decommissioning project.


Assuntos
Centrais Nucleares , Monitoramento de Radiação , Poluentes Radioativos do Solo , Colorado , Limite de Detecção , Medição de Risco , Poluentes Radioativos do Solo/análise
11.
J Environ Sci (China) ; 96: 93-98, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32819703

RESUMO

A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg2+ as the reporter module. The developed biosensor showed response for Hg2+ in a relatively wide range of 1-10,000 nmol/L, and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs. In addition, the biosensor could distinguish Hg2+ easily from multiple metal ions and displayed strong adaptability to extensive pH conditions (pH 4.0-10.0). More importantly, the developed biosensor was able to provide an initial assessment of Hg2+ spiked in the environmental water with the recoveries between 85.70% and 112.50%. The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other whole-cell biosensors, meeting the practical requirements with sufficient sensing performance.


Assuntos
Técnicas Biossensoriais , Mercúrio , Íons , Limite de Detecção
12.
PLoS One ; 15(8): e0237583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804936

RESUMO

Identification and quantification of plant flavonoids are critical to pharmacokinetic study and pharmaceutical quality control due to their distinct pharmacological functions. Here we report on a novel plant flavonoid electrochemical sensor for sensitive and selective detection of dihydromyricetin (DMY) based on double- layered membranes consisting of gold nanoparticles (Au) anchored on reduced graphene oxide (rGO) and molecularly imprinted polymers (MIPs) modified glassy carbon electrode (GCE). Both rGO-Au and MIPs membranes were directly formed on GCE via in-situ electrochemical reduction and polymerization processes step by step. The compositions, morphologies, and electrochemical properties of membranes were investigated with X-ray powder diffractometry (XRD), Fourier transform infrared spectrum (FTIR), Field emission scanning electron microscopy (FESEM) combined with various electrochemical methods. The fabricated electrochemical sensor labeled as GCE│rGO-Au/MIPs exhibited excellent performance in determining of DMY under optimal experimental conditions. A wide linear detection range (LDR) ranges from 2.0×10-8 to 1.0×10-4 M together with a low limit of detection (LOD) of 1.2×10-8 M (S/N = 3) were achieved. Moreover, the electrochemical sensor was employed to determine DMY in real samples with satisfactory results.


Assuntos
Carbono/química , Técnicas Eletroquímicas/instrumentação , Flavonoides/análise , Flavonóis/análise , Grafite/química , Técnicas Biossensoriais/métodos , Eletrodos , Ouro , Limite de Detecção , Nanopartículas Metálicas , Impressão Molecular , Extratos Vegetais/análise , Polímeros/síntese química , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
13.
PLoS One ; 15(8): e0237345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813724

RESUMO

BACKGROUND: Mixed/polyclonal infections due to different genotypes are reported in Tuberculosis. The current study was designed to understand the fate of mixed infections during the course of treatment and follow-up and its role in disease pathogenesis. METHODS: Sputum samples were collected on 0,1,2,3,6,12 and 24 months from 157 treatment-naïve patients, cultures subjected to Drug-Susceptibility-testing (MGIT 960), spoligotyping, MIRU-VNTR and SNP genotyping. All isolated colonies on thin layer agar (7H11) were subjected to spoligotyping. FINDINGS: One thirty three baseline cultures were positive (133/157, 84.7%), 43(32.3%) had mixture of genotypes. Twenty-four of these patients (55.8%) showed change in genotype while six showed different drug-susceptibility patterns while on treatment. Twenty-three (53.5%) patients with polyclonal infections showed resistance to at least one drug compared to 10/90 (11.1%) monoclonal infections (P<0.0001). Eight patients had recurrent TB, two with a new genotype and two with altered phenotypic DST. CONCLUSIONS: The coexistence of different genotypes and change of genotypes during the same disease episode, while on treatment, confirms constancy of polyclonal infections. The composition of the mixture of genotypes and the relative predominance may be missed by culture due to its limit of detection. Polyclonal infections in TB could be a rule rather than exception and challenges the age-old dogma of reactivation/reinfection.


Assuntos
Antituberculosos/farmacologia , Coinfecção/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Antituberculosos/uso terapêutico , Técnicas de Tipagem Bacteriana , Evolução Clonal , Coinfecção/epidemiologia , Coinfecção/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Feminino , Seguimentos , Técnicas de Genotipagem , Humanos , Limite de Detecção , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/patogenicidade , Filogenia , Polimorfismo de Nucleotídeo Único , Prevalência , Recidiva , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/epidemiologia , Adulto Jovem
14.
J Chromatogr A ; 1626: 461374, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797852

RESUMO

This work demonstrates the development of a compact, modular, cost-effective separation system configured to address a specific separation problem. The principles of the separation are based on gradient capillary liquid chromatography where the system consists of precision stepper motor-driven portable syringe pumps with interchangeable glass syringes (100 µL to 1000 µL). Excellent flow-rate precision of < 1% RSD was achieved with typical flow-rates ranging from 1 µL/min to 100 µL/min, which was ideal for capillary columns. A variable external loop volume and electrically actuated miniature injection valve was used for sample introduction. Detection was based upon a commercial Z-type UV absorbance flow-cell housed within a custom-built cooling enclosure (40 mm x 40 mm) which also contained a UV-LED light-source and a photodiode. System and chromatographic performance was evaluated using linear gradient elution, with day to day repeatability of <1.5% RSD (n = 6) for peak area, and < 0.4% RSD (n = 6) for retention time, for the separation of a 5 component mixture using a 50 mm X 530 µm ID C18 3 µm particle capillary column. The system can run any commercial or in-house packed columns from 50 mm to 100 mm length with IDs ranging from 200 to 700 µm. The developed portable system was operated using custom-built windows-based chromatography software, complete with data acquisition and system control.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cafeína/análise , Carbamazepina/análise , Análise Custo-Benefício , Limite de Detecção , Miniaturização , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta , Sulfametazina/análise
15.
J Chromatogr A ; 1626: 461381, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797855

RESUMO

A reliable and sensitive analytical approach has been optimized for the extraction of seven polychlorinated biphenyls (PCBs) from human breast milk. Hollow fiber liquid phase microextraction (HF-LPME) was applied for the first time for the extraction and pre-concentration of the analytes. Analytes were separated by gas chromatography with electron capture detector (GC-µECD) for the sensitive detection and mass spectrometry for the unequivocal identification. A rotable central composite design (RCCD) was performed for the multivariate optimization of the method. The best results were obtained at 40 °C during 30 min and 600 rpm of stirring speed using a hollow fiber length of 5 cm and toluene as an extractant phase and salt addition was not required. The detection limits were in the range 7-14 ng L-1 for PCBs. The coefficients of determination of the calibration curves indicated good linearity (R2> 0.96) and the enrichment factors ranged from 74 to 143. This type of study is of great importance due to the deleterious effect that the presence of contaminants can produce in infants health related to the immature character of the defense system. Moreover, exclusive breastfeeding is recommended by neonatologists up to six months of life and as complementary food during the first two years.


Assuntos
Microextração em Fase Líquida/métodos , Leite Humano/química , Bifenilos Policlorados/análise , Cromatografia Gasosa , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Bifenilos Policlorados/isolamento & purificação , Porosidade , Reprodutibilidade dos Testes , Sais/química
16.
J Chromatogr A ; 1626: 461386, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797858

RESUMO

In this study, the microcrystalline cellulose/metal-organic framework 199 hybrid (MCC/MOF-199) was applied as sorbent for the dispersive micro-solid phase-extraction (D-µSPE) of chlorophenols. The D-µSPE method combined with high-performance liquid chromatography- ultraviolet detection (HPLC-UV) was employed to determine of four chlorophenols including 2-chlorophenol (2-CP), 4-chlorophenol (4-CP), 2,3-dichlorophenol (2,3-DCP), and 2,5-dichlorophenol (2,5-DCP) in aqueous. The main parameters of the D-µSPE process that influence the extraction (i.e. the amount of sorbent, elution condition, extraction time, and pH) were investigated and optimized. Based on the outputs, the presence of MCC on the surface of MOF-199 leads to improve the properties of MOF-199 and the MCC/MOF-199 has the highest sorption capacity, durability, and porosity in comparison with MCC and MOF-199. According to the validation study at the optimized conditions, the linearity for the analytes was achieved in the range from 0.1 to 200 ng mL-1 for 2-CP and 4-CP and 0.15 to 200 ng mL-1 for 2,3-DCP and 2,5-DCP with correlation coefficients between 0.9928 and 0.9965. The limits of detection calculated at S/N=3 were in the range of 0.03-0.05 ng mL-1. Besides, the relative standard deviations (RSDs) for three spiking levels (0.2, 10,100 ng mL-1) do not exceed 6.8% and extraction recoveries are between 81.0% and 88.3%. Finally, the D-µSPE-HPLC-UV method was successfully applied to the analysis of CPs in real water samples (mineral, river and wastewater samples) with good recoveries (95.8 to 99.5%) and satisfactory precisions (RSD < 6.8%).


Assuntos
Celulose/química , Clorofenóis/análise , Estruturas Metalorgânicas/química , Microextração em Fase Sólida/métodos , Clorofenóis/química , Clorofenóis/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Água Doce/análise , Concentração de Íons de Hidrogênio , Limite de Detecção , Rios/química , Espectrofotometria Ultravioleta , Águas Residuárias/análise
17.
J Chromatogr A ; 1626: 461388, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797859

RESUMO

A reversed-phase high performance liquid chromatographic method was developed and validated for the simultaneous determination of the related substances of S-dapoxetine, including R-dapoxetine, (3S)-3-(dimethylamino-3-phenyl-1-propanol), S-3-amino-3-phenyl-1-propanol, 1-naphtol, 4-phenyl-2H,3H,4H-naphtho[1,2-b]pyran and 1-(2E)-Cinnamyloxynaphthalene. During the screening experiments seven different polysaccharide-type chiral stationary phases (amylose-based Lux-Amylose-1, Lux-i-Amylose-1 and Lux-Amylose-2, as well as cellulose-based Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3 and Lux-Cellulose-4) were tested in polar organic mode using a mobile phase consisting of 0.1% diethylamine in methanol, ethanol, 2-propanol and acetonitrile with 0.5 mL min-1 flow rate at 20 °C. Best results were obtained on Lux Cellulose-3 column with the ethanol-based mobile phase. To increase the retention factor of two, early-eluting impurities, water was added to the mobile phase. In order to counterbalance the increased total analysis time, higher column temperature (40 °C) and gradient elution, combined with flow-programming` was applied. Using the optimized conditions baseline separations were achieved for all compounds within 30 min. The method was validated according to the International Council on Harmonization guideline Q2(R1) and applied to the analysis of an approved, tablet formulation and dapoxetine-containing products sold on the internet. As expected, in the case of the pharmacy-acquired product, all of the monitored impurities were below 0.1%. However, interesting results were obtained when internet-acquired samples were analyzed. These tablets contained racemic dapoxetine and/or high concentration of R-dapoxetine impurity. Based on this work polysaccharide-based chiral stationary phases can be successfully applied for the simultaneous determination of achiral and chiral impurities in reversed-phase mode applying gradient elution and flow-rate programs. The study further underlines the importance of not only achiral, but also enantiomeric quality control, whenever counterfeiting of a single enantiomeric agent is suspected.


Assuntos
Benzilaminas/análise , Cromatografia Líquida de Alta Pressão/métodos , Naftalenos/análise , Cromatografia de Fase Reversa , Limite de Detecção , Espectrometria de Massas , Preparações Farmacêuticas/química , Estereoisomerismo , Comprimidos/química , Temperatura
18.
J Chromatogr A ; 1627: 461413, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823111

RESUMO

Innovations in extraction phases, extraction modes and hyphenated instrument configurations, are the most important issues to address for progress in the solid phase microextraction (SPME) methodology. In this regard, we have embarked on the development of a novel biocompatible 96-monolithic inorganic hollow fiber (96-MIHF) array as a new configuration for high-throughput SPME on a 96-well plate system. An arrangement of highly ordered 96 titania/Hydroxyapatite (TiO2/HAP) nanocomposite hollow fibers and corresponding stainless-steel needles on a Teflon plate holder were used as the extraction module. The inorganic hollow fibers were prepared via a rapid and reproducible template approach (Polypropylene hollow fiber) in combination with a sol-gel method in the presence of polyvinyl alcohol (PVA), as a network maker. The hollow fiber-shape sorbents were obtained with excellent precision by weight (RSD% = 4.98, n = 10) and length (RSD% = 1.08, n = 10) criteria. The proposed design can overcome a number of geometrically dependent drawbacks of conventional high-throughput SPME methods, mainly the ones related to sorbent amount and surface area due to possessing inner/outer surfaces without additional internal supports. The SPME platform, for the first time, was successfully applied for the extraction and preconcentration of doxorubicin from urine and water media without requiring sample preparation and free from significant matrix effect. The extracted analyte was analyzed by liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS). Highly satisfactory analytical figures of merit were obtained under optimized conditions. The limit of detection (LOD), limit of quantification (LOQ) and linearity of determination were 0.1 ng mL-1, 0.25 ng mL-1 and 0.25 to 4000 ng mL-1, respectively. The interday, intraday and inter sorbent precisions for three concentration levels ranged from 2.01 to 8.09 % (n = 3), 1.02 to 8.65 % (n = 5) and 0.99 to 1.02% (n = 15), respectively. The mean intra-well RSD value for 96 individual wells in 96-MIHF-SPME-LC-MS/MS (n = 3) at the medium concentration level was 7.81%.


Assuntos
Cromatografia Líquida/métodos , Doxorrubicina/urina , Microextração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Água/química , Adolescente , Adulto , Daunorrubicina/urina , Feminino , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Concentração Osmolar , Polipropilenos/química , Porosidade , Reprodutibilidade dos Testes , Fatores de Tempo , Adulto Jovem
19.
J Chromatogr A ; 1627: 461416, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823114

RESUMO

Animal feeds are often reported to be contaminated with chemical residues, and when present above the maximum legal limit, these compounds can cause harmful effects to consumers of animal produce. Thus, animal feed safety is an important regulatory concern. The aim of this study was to optimise a multiresidue method for the simultaneous analysis of multi-class pesticides and a number of frequently used veterinary drugs using LC-MS/MS and GC-MS/MS. The method was validated in a range of feed matrices, including maize feed, poultry feed and mixed feed concentrate. The optimised sample preparation workflow involved extraction of feeds (5 g) with ethyl acetate (10 mL), followed by a freezing step (at -20°C) used for eliminating the matrix co-extractives. The extract was further cleaned by dispersive solid phase extraction with a combination of primary secondary amine, C18 and florisil sorbents. From the cleaned-extract, an aliquot was analysed by GC-MS/MS, while another portion of it was solvent-exchanged to acetonitrile:water (50:50) and then analysed by LC-MS/MS. This method effectively minimised the matrix interferences. A total of 192 pesticides was analysed by GC-MS/MS within a runtime of 22 min. The LC-MS/MS method was validated for 187 compounds including 17 veterinary drugs. For most of the compounds, the limit of quantification (LOQ) was 0.01 mg/kg. The recoveries at LOQ and higher levels ranged between 70% and 120%, with precision-RSDs of < 20%. The method provided a precise analysis in a wide range of market-feed samples. As shown, the method is suitable for regulatory and commercial testing purposes.


Assuntos
Ração Animal/análise , Cromatografia Líquida/métodos , Praguicidas/análise , Espectrometria de Massas em Tandem/métodos , Drogas Veterinárias/análise , Animais , Congelamento , Cromatografia Gasosa-Espectrometria de Massas , Limite de Detecção , Resíduos de Praguicidas/análise , Reprodutibilidade dos Testes , Solventes/química , Água/química
20.
J Chromatogr A ; 1627: 461421, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823116

RESUMO

Herein we present an efficient, column-switching method that relies on a custom-made T-union passive diffusion micromixer to assist water dilution and promote trap solute focusing of a high sample volume dissolved in pure organic solvent using a 0.075 mm i.d. nano-LC column. This method allows injecting 20 µL (or higher) of sample volume, speeding up the analysis time, with a 400-fold increase of the limits of quantitation for selected compounds. Five pesticides in different media were used as model compounds, and the analyses were carried out with a triple quadrupole mass spectrometer equipped with a Liquid Electron Ionization (LEI) LC-MS interface working in multiple reaction monitoring (MRM) mode. The system microfluidics were investigated using COMSOL modeling software. Robustness of the entire system was evaluated using a post-extraction addition soil extracts with limits of detection values spanning from 0.10 to 0.45 µg/L. Reproducible results in terms of peak area, peak shape, and retention times were achieved in soil matrix. Repeatability test on peak area variations were lower than 10%.


Assuntos
Cromatografia de Fase Reversa/métodos , Elétrons , Microfluídica/métodos , Nanopartículas/química , Espectrometria de Massas em Tandem/métodos , Água/química , Acetonitrilos/química , Difusão , Limite de Detecção , Praguicidas/análise , Reprodutibilidade dos Testes , Solventes/química
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