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1.
PLoS One ; 15(9): e0238954, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941505

RESUMO

Desvenlafaxine (DES) and Alprazolam (ALP) are the drugs commonly prescribed together for the treatment of Major Depressive Disorders (MDD). A literature survey revealed, there is no method for the simultaneous determination of these two drugs. The purpose of this research was to develop and validate a simple, accurate, precise, robust, and isocratic RP-HPLC method for simultaneous determination of DES and ALP in human spiked plasma using UV-detector in short analysis time. The method utilized Hypersil BDS C18 (250 mm×4.6 mm, 5 µm) through an isocratic mode of elution using HPLC grade acetonitrile and 0.02M KH2PO4 buffer (65:35) and 0.1% Tri Fluoro Acetic acid (TFA) with pH 4.00 adjusted with 1M KOH. The flow rate was 1.00 mLmin-1 and elution of the drugs was monitored at 230nm. The elution time of DES and ALP was 4.011 and 5.182 minutes respectively. The method was linear for the concentration range 10-150 µgmL-1 for DES and 5.0-75.0 µgmL-1 for ALP. According to the validation results, the method is sensitive with Limit of Detection (LOD) 4.740 µgmL-1 and Limit of Quantification (LOQ) of 14.365 µgmL-1 for DES and LOD 1.891 µgmL-1 & LOQ 5.730 µgmL-1 for ALP. The reproducibility of results with minute deliberate variations in method parameters has proven that the method is robust. The data from stability studies show a non-significant change in drugs solutions for 2 months. The optimized method was validated as per International Conference for Harmonisation (ICH) Q2(R1) guidelines. This method can be used for the estimation of DES and ALP in plasma and can evaluate pharmacokinetic parameters of both drugs simultaneously.


Assuntos
Alprazolam/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Succinato de Desvenlafaxina/isolamento & purificação , Alprazolam/análise , Alprazolam/sangue , Succinato de Desvenlafaxina/análise , Succinato de Desvenlafaxina/sangue , Humanos , Limite de Detecção , Soluções Farmacêuticas , Plasma/química , Reprodutibilidade dos Testes
4.
Int J Infect Dis ; 98: 334-346, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32634589

RESUMO

BACKGROUND: Convalescent plasma (CP) has been used successfully to treat many types of infectious disease, and has shown initial effects in the treatment of the emerging 2019 coronavirus disease (COVID-19). However, its curative effects and feasibility have yet to be confirmed by formal evaluation and well-designed clinical trials. To explore the effectiveness of treatment and predict the potential effects of CP with COVID-19, studies of different types of infectious disease treated with CP were included in this systematic review and meta-analysis. METHODS: Related studies were obtained from databases and screened according to the inclusion criteria. The data quality was assessed, and the data were extracted and pooled for analysis. RESULTS: 40 studies on CP treatment for infectious diseases were included. Our study found that CP treatment could reduce the risk of mortality, with a low incidence of adverse events, promote the production of antibodies, lead to a decline in viral load, and shorten the disease course. A meta-analysis of 15 controlled studies showed that there was a significantly lower mortality rate in the group treated with CP (pooled OR=0.32; 95% CI=0.19-0.52; p<0.001, I2=54%) compared with the control groups. Studies were mostly of low or very low quality, with a moderate or high risk of bias. The sources of clinical and methodological heterogeneity were identified. The exclusion of heterogeneity indicated that the results were stable. CONCLUSIONS: CP therapy has some curative effect and is well tolerated in treating infectious diseases. It is a potentially effective treatment for COVID-19.


Assuntos
Anticorpos Antivirais/administração & dosagem , Betacoronavirus/fisiologia , Infecções por Coronavirus/terapia , Plasma/química , Pneumonia Viral/terapia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Imunização Passiva , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Resultado do Tratamento , Carga Viral
5.
PLoS One ; 15(6): e0234502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525915

RESUMO

Preservation of blood plasma in the dried state would facilitate long-term storage and transport at ambient temperatures, without the need of to use liquid nitrogen tanks or freezers. The aim of this study was to investigate the feasibility of dry preservation of human plasma, using sugars as lyoprotectants, and evaluate macromolecular stability of plasma components during storage. Blood plasma from healthy donors was freeze dried using 0-10% glucose, sucrose, or trehalose, and stored at various temperatures. Differential scanning calorimetry was used to measure the glass transition temperatures of freeze-dried samples. Protein aggregation, the overall protein secondary structure, and oxidative damage were studied under different storage conditions. Differential scanning calorimetry measurements showed that plasma freeze-dried with glucose, sucrose and trehalose have glass transition temperatures of respectively 72±3.4°C, 46±11°C, 15±2.4°C. It was found that sugars diminish freeze-drying induced protein aggregation in a dose-dependent manner, and that a 10% (w/v) sugar concentration almost entirely prevents protein aggregation. Protein aggregation after rehydration coincided with relatively high contents of ß-sheet structures in the dried state. Trehalose reduced the rate of protein aggregation during storage at elevated temperatures, and plasma that is freeze- dried plasma with trehalose showed a reduced accumulation of reactive oxygen species and protein oxidation products during storage. In conclusion, freeze-drying plasma with trehalose provides an attractive alternative to traditional cryogenic preservation.


Assuntos
Proteínas Sanguíneas/metabolismo , Plasma/química , Preservação Biológica/métodos , Conservantes Farmacêuticos/química , Trealose/química , Proteínas Sanguíneas/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Humanos , Agregados Proteicos , Conformação Proteica em Folha beta , Estabilidade Proteica , Temperatura de Transição , Vitrificação
7.
Int J Infect Dis ; 97: 365-370, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32553717

RESUMO

OBJECTIVES: The aim was to fully characterize the plasma and urine washout pharmacokinetics of tenofovir (TFV) in adults following 6 weeks of controlled levels of tenofovir disoproxil fumarate (TDF) adherence, in order to inform the utility of clinic-based adherence testing. DESIGN: This was a three-arm, randomized, open-label study in adult volunteers. Participants were randomized to receive TDF 300 mg/emtricitabine (FTC) 200 mg as (1) 7 doses/week (perfect adherence), (2) 4 doses/week (moderate adherence), or (3) 2 doses/week (low adherence). Plasma and urine samples were collected regularly during the 6-week dosing phase and for 4 weeks following drug cessation. RESULTS: Twenty-eight adults were included in this analysis. Median (range) age was 33 (20-49) years. No differences in TFV pharmacokinetic parameters during the washout were observed across the study arms. Small differences in TFV plasma concentrations occurred across arms between 4 and 10 h post-dose. The cumulative amount of TFV excreted in urine was not different at 24 h post-dose, but at 148 h it was 24.8 mg, 21.0 mg, and 17.2 mg for the perfect, moderate, and low adherence arms, respectively (p = 0.043). CONCLUSIONS: Among adults with different TDF adherence patterns, relative differences in plasma concentrations and cumulative urine extraction of TFV were minor following cessation. TFV measurement in plasma or urine is more indicative of last drug ingestion, rather than prior dose patterns.


Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/psicologia , Tenofovir/farmacocinética , Adulto , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/urina , Emtricitabina/administração & dosagem , Emtricitabina/sangue , Emtricitabina/farmacocinética , Feminino , Infecções por HIV/sangue , Infecções por HIV/urina , Comportamentos Relacionados com a Saúde , Humanos , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Plasma/química , Tenofovir/sangue , Tenofovir/uso terapêutico , Tenofovir/urina , Adulto Jovem
8.
PLoS One ; 15(5): e0233947, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-432093

RESUMO

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products. STUDY DESIGN AND METHODS: SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3). RESULTS: Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units. CONCLUSION: Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/efeitos da radiação , Riboflavina/farmacologia , Raios Ultravioleta , Betacoronavirus/crescimento & desenvolvimento , Análise Química do Sangue , Transfusão de Sangue , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Humanos , Imunização Passiva , Pandemias , Plasma/química , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , RNA Viral/análise , Carga Viral
9.
PLoS One ; 15(5): e0233947, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470046

RESUMO

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products. STUDY DESIGN AND METHODS: SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3). RESULTS: Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units. CONCLUSION: Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/efeitos da radiação , Riboflavina/farmacologia , Raios Ultravioleta , Betacoronavirus/crescimento & desenvolvimento , Análise Química do Sangue , Transfusão de Sangue , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Humanos , Imunização Passiva , Pandemias , Plasma/química , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , RNA Viral/análise , Carga Viral
10.
Poult Sci ; 99(5): 2508-2518, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359587

RESUMO

The present study was conducted to investigate the effects of genetic selection and threonine levels on meat quality in Pekin ducks. At 15 D of age, 192 lean ducks and 192 fatty ducks were selected and allotted to one of three treatments with 8 replicates with similar BW (8 ducks/cage), respectively. All ducks were fed the experimental diets (0.00, 0.15, and 0.30% added threonine) for 21 D from 15 to 35 D of age. The results showed that fatty ducks had higher (P < 0.001) feed intake, feed/gain ratio, abdominal fat percentage, and sebum percentage and lower (P = 0.001) breast muscle percentage compared with that of lean ducks. The fatty-type and lean-type ducks had similar weight gain and BW. Dietary threonine supplementation improved (P < 0.05) growth performance and increased breast muscle percentage in lean-type ducks, but it did not affect (P > 0.05) those indices in fatty-type ducks. Lean ducks had higher (P < 0.001) hepatic contents of total lipids, triglyceride, cholesterol, and plasma low-density lipoprotein cholesterol concentration, and dietary threonine supplementation decreased (P < 0.05) hepatic total lipid, cholesterol, and triglyceride contents in lean ducks, but it had no influence on hepatic lipids in fatty ducks (P > 0.05). Lean ducks had higher (P < 0.05) concentrations of monounsaturated fatty acid (MUFA), and C18-polyunsaturated fatty acid (PUFA) in the liver, PUFA in the breast muscle, and C18:3n6 and C18:3n3 in plasma and lower C20-PUFA and C22-PUFA in the liver and MUFA in plasma, compared with fatty ducks. Threonine supplementation increased PUFA, N3-PUFA, and n6-PUFA in plasma and hepatic fatty acids profiles in lean ducks (P > 0.05) but had on influence on total MUFA and total PUFA in the liver, breast muscle, and plasma in fatty ducks (P > 0.05). In conclusion, genetic selection toward meat production and threonine supplementation increases meat production and PUFA contents, which would influence eating quality, but it is benefit for human health.


Assuntos
Patos/fisiologia , Carne/análise , Seleção Genética , Treonina/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Patos/sangue , Patos/genética , Patos/crescimento & desenvolvimento , Lipídeos/análise , Fígado/química , Músculos Peitorais/química , Plasma/química , Distribuição Aleatória , Treonina/administração & dosagem
11.
Artigo em Inglês | MEDLINE | ID: mdl-32371328

RESUMO

INTRODUCTION: Ultrafiltration (UF) is used to separate unbound drugs; however, non-specific binding (NSB) may be a limiting factor of this technique. Pretreatment of UF devices has been suggested to reduce NSB. Therefore, the pretreatment methodologies for UF devices were evaluated in order to test their effectiveness in reducing NSB of protease inhibitors (PIs). METHODOLOGY: Two PIs (lopinavir-LPV and ritonavir-RTV) were tested. UF devices were pretreated with ultrapure water, Tween-20 or Tween-80. To evaluate the NSB, after UF devices being pretreated, ultrafiltrate solutions containing the analytes at two concentrations (low and high) were used. Samples were quantified by LC-MS/MS. RESULTS: UF devices pretreated with Tween-5% had the lowest NSB for both analytes. NSB values varied between 7 and 11% at low concentration 16-34% at high LPV concentration, respectively. For RTV, NSB was approximately 6% for low concentration and 18% for high concentration. Failure to completely remove Tween in UF devices could results in an overestimation of NSB. CONCLUSION: Pretreatment of UF device with Tween and subsequent removal proved to be effective in reducing NSB of PI.


Assuntos
Inibidores da Protease de HIV/química , Lopinavir/química , Ritonavir/química , Ultrafiltração/métodos , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Humanos , Plasma/química , Ligação Proteica , Padrões de Referência , Espectrometria de Massas em Tandem
12.
Mol Biol (Mosk) ; 54(2): 244-251, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392193

RESUMO

The study of extracellular miRNA is one of the most dynamic areas of modern biomedical research. Oftentimes, there is a need to isolate miRNAs associated with a particular carrier, for example, a ribonucleoprotein complex. The most thoroughly investigated protein component of these complexes is Ago2. Complexes are commonly isolated by immunoprecipitation with specific antibodies. Here we compare three methods for immunoprecipitating Ago2/microRNA complexes from blood plasma. In the first protocol, anti-Ago2-antibodies are added to the plasma following protein A-sepharose. In second protocol, anti-Ago2-antibodies are bound to sepharose from the very beginning, and then mixed with plasma. The third protocol differs from the second in that sepharose is blocked by non-specific antibodies at the final stage. To compare the efficiency of these protocols, the levels of miR-16-5p, miR-21-5p, and miR-144-3p were analyzed after precipitation with anti-Ago2 antibodies and control antibodies. For miR-16-5p all protocols were efficient, for miR-21-5p only the second technique yielded results, while for miR-144-3p none of the protocols resulted in extraction. Thus, we conclude that different protocols for immunoprecipitation of microRNA/Ago2 complexes favor different miRNAs.


Assuntos
Proteínas Argonauta/sangue , Imunoprecipitação/métodos , MicroRNAs/sangue , Plasma/química , Proteínas Argonauta/isolamento & purificação , Humanos , MicroRNAs/isolamento & purificação
13.
PLoS Negl Trop Dis ; 14(3): e0008138, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226013

RESUMO

The changes in host lipid metabolism during leprosy have been correlated to fatty acid alterations in serum and with high-density lipoprotein (HDL) dysfunctionality. This is most evident in multibacillary leprosy patients (Mb), who present an accumulation of host lipids in Schwann cells and macrophages. This accumulation in host peripheral tissues should be withdrawn by HDL, but it is unclear why this lipoprotein from Mb patients loses this function. To investigate HDL metabolism changes during the course of leprosy, HDL composition and functionality of Mb, Pb patients (paucibacillary) pre- or post-multidrug therapy (MDT) and HC (healthy controls) were analyzed. Mb pre-MDT patients presented lower levels of HDL-cholesterol compared to HC. Moreover, Ultra Performance Liquid Chromatography-Mass Spectrometry lipidomics of HDL showed an altered lipid profile of Mb pre-MDT compared to HC and Pb patients. In functional tests, HDL from Mb pre-MDT patients showed impaired anti-inflammatory and anti-oxidative stress activities and a lower cholesterol acceptor capacity compared to other groups. Mb pre-MDT showed lower concentrations of ApoA-I (apolipoprotein A-I), the major HDL protein, when compared to HC, with a post-MDT recovery. Changes in ApoA-I expression could also be observed in M. leprae-infected hepatic cells. The presence of bacilli in the liver of a Mb patient, along with cell damage, indicated hepatic involvement during leprosy, which may reflect on ApoA-I expression. Together, altered compositional and functional profiles observed on HDL of Mb patients can explain metabolic and physiological changes observed in Mb leprosy, contributing to a better understanding of its pathogenesis.


Assuntos
Hanseníase/patologia , Lipoproteínas HDL/sangue , Adolescente , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Plasma/química , Adulto Jovem
14.
A A Pract ; 14(6): e01182, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32224689

RESUMO

A 35-year-old parturient with antiphospholipid syndrome and a working diagnosis of hemolysis, elevated liver enzyme, and low platelets (HELLP) underwent a cesarean delivery 9 hours after receiving heparin. Her preoperative activated partial thromboplastin time and rotational thromboelastometry (ROTEM) intrinsic pathway (INTEM) clotting time were 120 and 1870 seconds, respectively. Fresh frozen plasma was administered for heparin neutralization. The ROTEM INTEM/heparinase assay (HEPTEM) ratio can help confirm heparin neutralization and guide intraoperative transfusion management.


Assuntos
Síndrome Antifosfolipídica/complicações , Síndrome HELLP/tratamento farmacológico , Heparina/administração & dosagem , Plasma/química , Adulto , Cesárea , Feminino , Heparina/efeitos adversos , Humanos , Tempo de Tromboplastina Parcial , Gravidez , Tromboelastografia
15.
Int J Nanomedicine ; 15: 2303-2314, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280222

RESUMO

Objective: The objective of this study is to evaluate the performance and feasibility of surface-enhanced Raman spectroscopy coupled with a filter membrane and advanced multivariate data analysis on identifying and differentiating benign and malignant thyroid tumors from blood plasma. Patients and Methods: We proposed a membrane filter SERS technology for the differentiation between benign thyroid tumor and thyroid cancer. That is to say, by using filter membranes with optimal pore size, the blood plasma samples from thyroid tumor patients were pretreated with the macromolecular proteins being filtered out prior to SERS measurement. The SERS spectra of blood plasma ultrafiltrate obtained using filter membranes from 102 patients with thyroid tumors (70 thyroid cancers and 32 benign thyroid tumors) were then analyzed and compared. Two multivariate statistical analyses, principal component analysis-linear discriminate analysis (PCA-LDA) and Lasso-partial least squares-discriminant analysis (Lasso-PLS-DA), were performed on the SERS spectral data after background subtraction and normalization, as well as the first derivative processing, to analyze and compare the differential diagnosis of benign thyroid tumors and thyroid cancer. Results: SERS measurements were performed in blood plasma acquired from a total of 102 thyroid tumor patients (benign thyroid tumor N=32; thyroid cancer N=70). By using filter membranes, the macromolecular proteins in blood plasma were effectively filtered out to yield high-quality SERS spectra. 84.3% discrimination accuracy between benign and malignant thyroid tumor was achieved using PCA-LDA method, while Lasso-PLS-DA yields a discrimination accuracy of 90.2%. Conclusion: Our results demonstrate that SERS spectroscopy, coupled with ultrafiltration and multivariate analysis has the potential of providing a non-invasive, rapid, and objective detection and differentiation of benign and malignant thyroid tumors.


Assuntos
Plasma/química , Análise Espectral Raman/métodos , Neoplasias da Glândula Tireoide/sangue , Ultrafiltração/métodos , Adulto , Diagnóstico Diferencial , Análise Discriminante , Humanos , Membranas Artificiais , Nanopartículas Metálicas/química , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente Principal , Estudo de Prova de Conceito , Prata/química , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/patologia , Ultrafiltração/instrumentação
16.
Zhongguo Zhong Yao Za Zhi ; 45(3): 645-654, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32237525

RESUMO

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS) method was deve-loped for analysis of rutaecarpine(Ru), evodiamine(Ev), rutaevine(Rv), limonin(Li), ginsendside Rb_1(Rb_1), ginsendside Re(Re) in rat plasma and brain tissues of nitroglycerin-induced migraine rats. Male healthy Sprague-Dawley(SD) rats were orally given multiple dose of optimized(OS) and un-optimized Wuzhuyu Decoction(UNOS), and their blood samples and brainstem were collected at different time points after injection of nitroglycerin(10 mg·kg~(-1)) into the frontal region. The drug concentrations of the 6 analytes in plasma and brainstem were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma were calculated by using Phoenix WinNolin 5.2.1 software. The methodological test showed that all of analytes in both plasma and brainstem homogenate exhibited a good linearity within the concentration range(r>0.994 7). The intra-day and inter-day accuracy, precision, matrix effect, stability of the investigated components meet the requirements for biopharmaceutical analysis. The developed method was successfully applied in pharmacokinetic studies on abovementioned ingredients in rat plasma and brain stem. The plasma pharmacokinetic parameters of active ingredients in two different Wuzhuyu Decoction group were compared, it was found that Rb_1 had higher t_(1/2), T_(max), C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. Meanwhile, Ev had higher t_(1/2) and T_(max) but lower C_(max), AUC_(0-24 h) and AUC_(0-∞), Ru has higher t_(1/2 )but lower C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. The brain tissue distribution of each component were compared between the two groups, the component with higher content in OS, such as Ru at 30 min and 2 h after administration, Ev at 30 min, Rb_1 at 30 min and Rb_1 at 2 h after administration have lower brain tissue distribution than those in UNOS group, while the component with higher content in UNOS, such as Rv at 30 min, 2 h and 12 h after administration had higher brain tissue distribution than those in OS group.


Assuntos
Transtornos de Enxaqueca/tratamento farmacológico , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Química Encefálica , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/farmacocinética , Medicamentos de Ervas Chinesas/uso terapêutico , Masculino , Transtornos de Enxaqueca/induzido quimicamente , Nitroglicerina , Plasma/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
17.
Artigo em Inglês | MEDLINE | ID: mdl-32247186

RESUMO

The quantitative determination of intact proteins in biological samples by LC with high-resolution MS detection can be a useful alternative to ligand-binding assays or LC-MS-based quantification of a surrogate peptide after protein digestion. The 22-kDa biopharmaceutical protein somatropin (recombinant human growth hormone) was quantified down to 10 ng/mL (0.45 nM) in 75 µL of rat plasma by the combination of an immunocapture step using an anti-somatropin antibody and LC-MS on a quadrupole-time of flight instrument. Accuracy and precision of the method as well as its selectivity and sensitivity did not depend on the width of the mass extraction window nor on whether only one or a summation of multiple charge states of the protein analyte were used as the detection response. Quantification based on deconvoluted mass spectra showed equally acceptable method performance but with a less favorable lower limit of quantification of 30 ng/mL. Concentrations in plasma after dosing of somatropin to rats correlated well for the deconvolution approach and the quantification based on the summation of the response of the four most intense charge states (14+ to 17+) of somatropin.


Assuntos
Hormônio do Crescimento Humano/sangue , Hormônio do Crescimento Humano/farmacocinética , Proteínas Recombinantes/farmacocinética , Animais , Técnicas Biossensoriais/métodos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Limite de Detecção , Peptídeos/análise , Plasma/química , Ratos , Proteínas Recombinantes/sangue , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem
18.
Artigo em Inglês | MEDLINE | ID: mdl-32278292

RESUMO

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1-1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3-8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.


Assuntos
Compostos Macrocíclicos/sangue , Compostos Macrocíclicos/farmacocinética , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/sangue , Pirazóis/farmacocinética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Animais , Axitinibe/química , Axitinibe/normas , Bioensaio , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Limite de Detecção , Compostos Macrocíclicos/administração & dosagem , Camundongos , Plasma/química , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazóis/administração & dosagem , Receptor trkA/antagonistas & inibidores , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Distribuição Tecidual
19.
Artigo em Inglês | MEDLINE | ID: mdl-32172173

RESUMO

The current study reports the development of a novel biofluid sampler (BFS) which is capable of sampling and sample preparation of whole blood without converting it into plasma or serum. The sampler can retain a whole blood sample from 10 to 1000 µL. Although the device shares the same working principle of dried blood spot (DBS) cards, it eliminates most of the technological shortcomings of DBS cards such as low maximum sample volume (~50 µL), sample inhomogeneity due to haematocrit, and poor physical adsorption driven analyte retention by incorporating sol-gel derived high efficiency, multi-functional sorbents on cellulose fabric substrate. The performance of BFS was tested via "Mail-in-Analysis" using three non-steroidal anti-inflammatory drugs (NSAIDs, ketoprofen, carprofen and diclofenac) as the test compounds. Human whole blood samples were fortified with the test compounds and sampled on conventional DBS cards and biofluid samplers (BFSs) in the USA. After drying the blood samples at room temperature, the samples were shipped to Italy for chromatographic analysis. The analytes were back-extracted from the DBS cards and BFSs using methanol and subsequently analysed using a short Symmetry C18 column (75 × 4.6 mm, 3.5 µm). Acetonitrile (ACN) and PBS (30 mM; pH = 2.5) were used as the mobile phases and the elution was performed under isocratic conditions. Compared to the classical dried blood spot cards (DBS), BFSs offer better performance in retaining the selected NSAIDs under conventional postal shipment. By substantially expanding the sampling capacity, eliminating most of the shortcomings of classical DBS cards and exploiting the better materials properties of sol-gel based functional sorbents, BFSs offer a new and profoundly simplified approach for whole blood sampling and analysis and is expected to change the current practice of blood analysis, allowing accurate quantitative analyses either in a local laboratory (on site) or using mail-in-analysis (off site) without compromising the quality of bioanalytical data.


Assuntos
Anti-Inflamatórios não Esteroides/análise , Carbazóis/sangue , Diclofenaco/sangue , Cetoprofeno/sangue , Plasma/química , Adsorção , Cromatografia Líquida de Alta Pressão , Teste em Amostras de Sangue Seco , Hematócrito , Humanos , Limite de Detecção , Plasma/metabolismo , Serviços Postais , Reprodutibilidade dos Testes , Manejo de Espécimes , Propriedades de Superfície
20.
PLoS Negl Trop Dis ; 14(3): e0008147, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32155159

RESUMO

BACKGROUND: Echinococcosis is a chronic zoonosis caused by tapeworms of the genus Echinococcus. Treatment of the disease is often expensive and complicated, sometimes requiring extensive surgery. Ultrasonographic imaging is currently the main technique for diagnosis, while immunological analysis provides additional information. Confirmation still needs pathological analysis. However, these diagnostic techniques generally detect infection in late stages of the disease. An accurate, early and non-invasive molecular diagnostic method is still unavailable. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the cell-free DNA (cfDNA) from plasma of echinococcosis patients and confirmed the presence of Echinococcus DNA. To improve detection sensitivity, we developed a method based on targeted next-generation sequencing of repeat regions. Simulation experiments demonstrate that the targeted sequencing is sensitive enough to detect as little as 0.1% of an Echinococcus genome in 1 mL of plasma. Results obtained using patient plasma shows that the Area Under the Curve (AUC) of the method is 0.862, with a detection sensitivity of 62.50% and specificity of 100%, corresponding to a Youden-index of 0.625. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that hydatid cysts release cfDNA fragments into patient plasma. Using the repeat region targeted sequencing method, highly specific detection of Echinococcus infection was achieved. This study paves a new avenue for potential non-invasive screening and diagnosis of echinococcosis.


Assuntos
DNA de Helmintos/sangue , Equinococose/diagnóstico , Echinococcus/genética , Técnicas de Diagnóstico Molecular/métodos , Plasma/química , Sequências Repetitivas de Ácido Nucleico , Adulto , Animais , DNA de Helmintos/química , DNA de Helmintos/genética , DNA de Helmintos/isolamento & purificação , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Sensibilidade e Especificidade
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