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ObjectiveTo establish a rapid screening method for influenza virus neuraminidase（NA） inhibitors sourced from Chinese medicines based on fluorescence detection. MethodThe method was constructed based on the principle that after the reaction of the test sample and a certain amount of NA， the activity of some NA will be inhibited by the test sample， and the NA that is still active after the addition of the substrate can generate fluorescence at a specific wavelength when combined with the fluorescent substrate， and the inhibition rate of the test sample on NA was calculated according to the measured fluorescence intensity， so as to evaluate the in vitro inhibitory activity of the test sample on NA. A total of 49 high-purity chemical components from 12 Chinese medicines were used to evaluate the in vitro anti-NA activity by the established method. The theoretical calculated values of binding energy and inhibition constant after docking between the NA protein receptor and the test sample were used to prove the accuracy of the experimental results. The established method was applied to detect the in vitro NA inhibitory activity of different batches of Banlangen granules and Kangbingdu granules， so as to evaluate the quality consistency among different batches of samples. ResultThe methodological examination results showed that the method had good accuracy and repeatability. The screening results of 49 components showed that 22 of them had strong in vitro inhibitory activity against NA than peramivir ［half inhibitory concentration（IC50） was 131.2 μmol·L-1］， such as schaftoside， isoorientin， chebulinic acid， menthone and isoschaftoside. The inhibitory activity of the remaining 27 components was weaker than that of peramivir. The molecular docking results showed that the theoretical calculation results of binding energies and inhibition constants of most compounds were basically consistent with the experimental results. The test results of the inhibitory activity of 12 batches of Banlangen granules on NA showed that the quality consistency among samples A1， A2， B2， C1， C2， E2 and F2 was good. The analysis results of the inhibitory activity of 9 batches of Kangbingdu granules produced by the same manufacturer on NA showed that the inhibitory rates of samples K1 to K9 were 37.68%， 36.18%， 31.37%， 33.98%， 40.36%， 33.76%， 40.69%， 41.08%， 40.06% when the concentration of 0.02 g·mL-1， and the average inhibitory rate was 37.24%. ConclusionIn this paper， we successfully established an analytical method that can be used to rapidly evaluate whether Chinese medicines （derived from chemical components of traditional Chinese medicine or proprietary Chinese medicines） have in vitro anti-NA activity， which can be a powerful supplement to the existing screening methods for influenza virus NA inhibitors. And this method was used to screen 22 compounds from 12 Chinese medicines with good in vitro inhibitory activity against NA， which can provide candidate compounds for the development of anti-influenza small molecule drugs.
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A incerteza de medição representa o nível de confiança no resultado. Para a estimativa da incerteza de medição foi empregado o cálculo do desvio padrão da reprodutibilidade intralaboratorial de 48 ensaios de contagem de bactérias heterotróficas pela técnica da membrana filtrante com detecção por fluorescência pelo uso de substrato fluorogênico em amostras de água purificada contaminadas artificialmente entre 10 e 100 UFC/mL. O valor obtido, 1,3 x 10-3 (log10), indica que a técnica utilizada pode ser uma alternativa para a estimativa da incerteza de medição em ensaios microbiológicos quantitativos de contagem de bactérias heterotróficas em amostras de água purificada. (AU)
Measurement uncertainty represents the confidence level in the result. To estimate the expanded measurement uncertainty, the standard deviation of intra-laboratory reproducibility of 48 heterotrophic bacterial count assays by fluorescence detection by the use of fluorogenic substrate on artificially contaminated purified water samples between 10 and 100 CFU/mL was used. The value obtained, 1.3 x 10-3 (log10), indicates that the technique used can be an alternative to estimate measurement uncertainty in quantitative microbiological heterotrophic bacterial count assays in purified water samples using fluorogenic substrate. (AU)
Subject(s)
Colony Count, Microbial , Water Purification , Uncertainty , Heterotrophic Bacteria , FluorescenceABSTRACT
A incerteza de medição representa o nível de confiança no resultado. Para a estimativa da incerteza de medição foi empregado o cálculo do desvio padrão da reprodutibilidade intralaboratorial de 48 ensaios de contagem de bactérias heterotróficas pela técnica da membrana filtrante com detecção por fluorescência pelo uso de substrato fluorogênico em amostras de água purificada contaminadas artificialmente entre 10 e 100 UFC/mL. O valor obtido, 1,3 x 10-3 (log10), indica que a técnica utilizada pode ser uma alternativa para a estimativa da incerteza de medição em ensaios microbiológicos quantitativos de contagem de bactérias heterotróficas em amostras de água purificada.
Measurement uncertainty represents the confidence level in the result. To estimate the expanded measurement uncertainty, the standard deviation of intra-laboratory reproducibility of 48 heterotrophic bacterial count assays by fluorescence detection by the use of fluorogenic substrate on artificially contaminated purified water samples between 10 and 100 CFU/mL was used. The value obtained, 1.3 x 10-3 (log10), indicates that the technique used can be an alternative to estimate measurement uncertainty in quantitative microbiological heterotrophic bacterial count assays in purified water samples using fluorogenic substrate.
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Heterotrophic Bacteria/analysis , Bacterial Load/methods , Uncertainty , Water Purification , FluorescenceABSTRACT
Carbon dots are an emerging nanomaterial with excellent fluorescent properties. Compared with traditional organic dyes and semiconductor quantum dots, carbon dots possess advantages of low toxicity and good biocompatibility. At present, carbon dots are widely used in many fields such as analytical detection, fluorescence imaging and drug delivery. Cervi Cornu Pantotrichum, a rare mammalian organ with ability of full regeneration, has been used as famous traditional Chinese medicine. The biological functions and efficacy of Cervi Cornu Pantotrichum are closely related to their chemical constituents. In this paper, we briefly reviewed the progress of carbon dots research in fluorescence detection, fluorescence imaging and phototherapy, carried out the bioimaging and cell cytotoxicity test by carbon dots, and discussed the feasibility of carbon dots in biological features and chemical composition analysis of Cervi Cornu Pantotrichum.
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Ion-pairing liquid chromatographic method was validated for determination of ketoconazole in shampoo and cream samples as per ICH guidelines. The chromatographic conditions were carried out in the isocratic mode using a mixture of methanol and 8 mM sodium dodecyl sulfate (pH 5.5) in a ratio of 45:55 v/v %, as mobile phase. The flow rate was set at 1.0 mL min-1. Chromolith RP-18e (100×4.6 mm) was used as the analytical column with a fluorescence detection at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The average percentage recovery of shampoo A, shampoo B, shampoo C, cream A and cream B were 99.88, 97.06, 99.58, 96.77 and 97.26, respectively. The limit of detection was 0.12 mg L-1. The drug decomposition under acid degradation, base degradation and oxidative degradation were found to be in the range of 91.63-94.70% indicating that the drug is resistant towards acidic conditions. The drug decomposition under thermal condition and photolysis condition were found to be in the range of 69.05-87.15% and 47.31-66.83% respectively, indicating that the drug decomposition is more sensitive under photolysis conditions. This method is suitable for the quality control of ketoconazole in commercial shampoo and creams.
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Fiber photometry is a sensitive and easy way to detect changes in fluorescent signals. The combination of fiber photometry with various fluorescent biomarkers has substantially advanced neuroscience research over the last decade. Despite the wide use of fiber photometry in biomedical fields, the lack of a detailed and comprehensive protocol has limited progress and sometimes complicated the interpretation of data. Here, we describe detailed procedures of fiber photometry for the long-term monitoring of neuronal activity in freely-behaving animals, including surgery, apparatus setup, data collection, and analysis.
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Animals , Female , Male , Mice , Brain , Metabolism , Calcium Signaling , Neurons , Metabolism , Neurosurgical Procedures , Optical Fibers , Optical Imaging , Methods , Photometry , MethodsABSTRACT
To measure particle size distribution of phenols in mainstream cigarette smoke aerosol,the particles of cigarette smoke aerosol were divided into 12 stages using single channel smoking machine coupled electrical low-pressure impactor (ELPI) and collected by 12 polyester films.The collected particles were weighted and then analyzed by ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLD) to determine the 14 phenols in the different size particles.The results showed that the aerosols collecting method had good stability with relative standard deviation (RSD) of collected particles mass less than 10%.The analyzing results of 14 phenols by UPLC-FLD showed that the linear correlation coefficients(R2) were greater than 0.9959,with detection limits were less than 1.2 ng/cig and recoveries were 80.1%-115.0%.The distributions of 14 phenols with respect to smoke aerosol particle size were investigated.The results indicated that except 4-ethyl-2-methoxy-phenol not detected,the other 13 phenols were detected in mainstream cigarette smoke aerosol.The content of 13 phenols appeared increasing at first and then decreasing with increase of the particle size which distributed in a pattern similar to that of particle mass.All of 13 phenols were present in higher amounts in the medium size particles (0.261-0.722 μm) with peak content in particles 0.431 μm.The distribution of concentrations (ratio of content to particle mass) of 13 phenols in different size particles was different.The concentrations of phenol and mono-substituted phenol appeared to first increase and then decrease with increasing smoke aerosol particle size and were higher in medium size particles (0.261-0.772 μm).The concentrations of benzenediol and mono-substituted benzenediol were uniformly distributed in medium size particles (0.144-1.166 μm),and the concentration of disubstituted phenol was uniform throughout the particles of varying sizes.
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A molecular imprinting polymer technique was successfully applied to precipitation polymerization by using styrene as a functional monomer, curcuminoids as templates, acetonitrile as a porogenic solvent, benzoyl peroxide as the initiator, and ethylene glycol dimethacrylate as the crosslinker. The effects of interaction on the adsorption capacity of the molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) were investigated. A comparison of the adsorption capacity for MIP and NIP indicated that the NIP had the lowest adsorption capacity. The curcuminoid-imprinted polymer (Cur-MIP) was syn-thesized from 0.0237 mmol of styrene, 47.0 g of acetonitrile, 1.0238 mmol of ethylene glycol dimetha-crylate, 0.0325 mmol of curcuminoids, and 0.2480 mmol of benzoyl peroxide. A high-performance liquid chromatography method with fluorescence detection was developed and validated for various chro-matographic conditions for the determination of the curcuminoids in turmeric samples. The sample solution was separated using the Cur-MIP via solid-phase extraction and analyzed on a Brownlee ana-lytical C18 column (150 mm × 6 mm, 5μm) using an isocratic elution consisting of acetonitrile and 0.1%trichloroacetic acid (40:60, v/v). The flow rate was maintained at 1.5 mL/min. The fluorescence detector was set to monitor atλex = 426 nm andλem = 539 nm. The quantification limit values were found to be 16.66, 66.66, and 33.33μg/L for curcumin, demethoxycurcumin, and bisdemethoxycurcumin, respec-tively. Thus, we concluded that the Cur-MIP and high-performance liquid chromatographic-fluorescence method could be applied to selective extraction and could be used as a rapid tool for the determination of curcuminoids in medicinal herbal extracts.
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Objective To understand the cleaning quality of dental handpieces in Suzhou City, analyze the relevant factors that influencing cleaning effect.Methods A cross-sectional study was performed with the proportional system sampling method, questionnaires were adopted to investigate the cleaning location, cleaning method and process of dental handpieces, the ATP fluorescence detection method was conducted to detect cleaning quality.Results In 10 administrative regions of this city, a total of 72 medical institutions were selected, 25 were public medical oral diagnosis and treatment institutions, 47 were private clinics.Cleaning effect of automatic handpiece cleaning machine was better than traditional manual cleaning (unqualified rate :3.95% vs 11.96%, P0.05).The quality of cleaning of handpieces could be improved if waiting time of cleaning ≤30 minutes, enzymes were used during cleaning, and purified water was used at the end rinse(all P<0.05);whether there was drying process and used lubricant, difference were both not significant.Conclusion Using automatic handpiece cleaning machine, cleaning personnel with adequate knowledge, cleaning waiting time ≤30 minutes, enzyme use during the cleaning process, and purified water use at the end rinse can improve the quality of cleaning of dental handpieces.
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Objective: To establish an HPLC-fluorescence detection method for the determination of thioguanosine-monophosphate (TGMP), thioguanosine-diphosphate (TGDP) and thioguanosine-triphosphate (TGTP) in red blood cells (RBC), as well as quantify the individual thioguanine nucleotides metabolites in kidney transplant recipients with azathioprine (AZA) therapy.Methods: The individual thioguanosine phosphates were extracted from RBC by dichloromethane and subsequently oxidized by potassium permanganate.The separation was achieved on a Nucleosil C18 column (150 mm×4.6 mm,5 μm) with an ion pairing reagent and detected by a fluorescence detector (excitation at 315 nm, emission at 390 nm).The mobile phase consisted of 20 mmol·L-1 potassium phosphate buffer (pH was adjusted to 6.8 by 5 mmol·L-1 tetrabutylammonium hydrogensulfate)-acetonitril (80:20) with the flow rate of 1.0 ml·min-1.Results: TGMP, TGDP and TGTP were quantified from RBC within the range of 50-500, 50-1000 and 100-5 000 pmol·ml-1, respectively.The limit of quantification (LOQ) was 50, 50 and 100 pmol·ml-1 RBC for TGMP, TGDP and TGTP, respectively.The intra-and inter-day RSDs were below 7.0% with the method recovery between 95.0% and 103.6%.The mean extraction recovery was above 90%.The assay was applied in the blood samples of 30 kidney transplant recipients with AZA therapy, and the results indicated that TGTP was the predominant phosphate metabolite in RBC.Conclusion: The method is simple, rapid, sensitive and specific, and it can quantitatively determine the individual thioguanosine phosphates in RBC of kidney transplant recipients with AZA therapy.
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Objective To explore the effect of ATP fluorescence detection on on-site monitoring and supervision of healthcare-associated infection management .Methods ATP bioluminescence analyzer was used to detect the con-tamination status of hands of health care workers(HCWs),the object surfaces,and the cleaning tools in all quarters of 2015,the detection results were timely given feedback,and improvement measures were put forward.Results A total of 1 294 specimens were detected,the overall qualified rate was 62.75%.The qualified rates of hands of HC-Ws,object surfaces,and cleaning tools increased from 54.35%,50.30%,and 60.26% in the first quarter to 76.42%,64.80%,and 79.52% in the fourth quarter respectively,tendency chi-square test showed that difference was statistically significant (all P <0.05).The median of relative light unit (RLU)of hands of HCWs,object sur-faces,and cleaning tools were 20.00,85.00,and 35.00,respectively.Conclusion ATP fluorescence detection for on-site monitoring and supervision for cleaning and disinfection effect can promote the continuous quality improve-ment of hand hygiene and environmental cleanliness.
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Aliphatic amines in infant food packaging materials were extracted and concentrated by 0 . 5 mL of acidified methanol using gas purge microsyringe extraction ( GP-MSE ) . Pre-column fluorescence labeling of amines was achieved in mild conditions with 10-ethyl-acridine-2-sulfonyl chloride ( EASC ) as labeling reagent. The derivatization was carried out at 60℃ and pH 10. The derivatives were successfully separated on a Hypersil GOLD column with excitation and emission wavelengths of 262 and 430 nm, respectively. The detection limits were in the range of 0. 4-0. 6 μg/kg, and the quantitation limits were in the range of 1. 2-2. 1 μg/kg. All analytes were in good linearity in the concentration range of 2. 0-2000 μg/L with correlation coefficients of higher than 0. 998. The developed method was characterized by celerity, accuracy and high sensitivity. It was successfully applied to the determination of aliphatic amines in infant food packaging materials.
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Objective To study inhibitory effect of serine protease activity by Ulinastatin in vitro .Methods Different chromogenic peptides were designed and synthesized.Highly sensitive fluorescence detection was performed to optimize the concentration of each serine proteases and their chromogenic substrates.Multi-point method was used for the calculation of half maximal inhibitory concentration of Ulinastatin .ResuIts Ulinastain could inhibit Polymorphonuclear leukocyte elastase ( PMNE ) and plasmin with IC50 lower than 100 U/mL.For factor Xa, and Kallikrein, the IC50 of Ulinastatin was higher than 1000U/mL.No thrombin IC50 could be calculated at the present experiments.ConcIusion Similar to Ulinastatin injection from Japan, domestic Ulinastatin shows the strongest inhibitory effects on PMNE among those serine proteases.As important references, this study gives reliable data for dose range of domestic Ulinastatin in anti-inflammation, coagulation/anti-coagulation and anti-shock therapy.
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A simple, rapid, economical and reliable high performance liquid chromatographic method has been developed and successfully applied in simultaneous determination of ethinyl estradiol and drospirenone in coated tablets. The HPLC method was performed on a LiChroCART® 100RP column (125x4 mm i.d., 5 µm) with acetonitrile:water 50:50 (v/v) as mobile phase, pumped at a flow rate of 1.0 mL.min-1. The fluorescence detection for ethinyl estradiol was made at λex= 280 nm and λem= 310 nm and a UV detection for drospirenone was made at 200 nm. The elution time for ethinyl estradiol and drospirenone were 4.0 and 5.7 min, respectively. The method was validated in accordance to USP 34 guidelines. The proposed HPLC method presented advantages over reported methods and is suitable for quality control assays of ethinyl estradiol and drospirenone in coated tablets.
Um método simples, rápido, econômico e confiável foi desenvolvido empregando a cromatografia líquida de alta eficiência para a determinação simultânea de etinilestradiol e drospirenona em comprimidos revestidos. O método foi realizado utilizando coluna LiChroCART® 100RP (125 x 4 mm d.i., 5 µm), a fase móvel constituída de acetonitrila:água, 50:50 (v/v) com vazão de 1,0 mL.min-1. A detecção foi realizada empregando fluorescência em λex= 280 nm e λem= 310 nm para o etinilestradiol e na região de UV em 200 nm para a drospirenona. O etinilestradiol e a drospirenona tiveram tempo de retenção de 4,0 e 5,7 min, respectivamente. O método foi validado de acordo com as diretrizes da USP 34. O método proposto apresentou vantagens sobre os relatados na literatura e pode ser considerado adequado para o controle de qualidade do etinilestradiol e da drospirenona em comprimidos revestidos.
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Chromatography, High Pressure Liquid , Contraceptives, Oral/analysis , Ethinyl Estradiol/pharmacokinetics , Tablets, Enteric-Coated , FluorescenceABSTRACT
Microwave-assisted extraction was optimized with response surface methodology for HPLC-fluorescence determination of puerarin and daidzein in Radix Puerariae thomsonii.The optimized extraction procedure was achieved by soaking the sample with 70% methanol（1∶15,v/v）for 30 min,and then microwave irradiation for 11 min at a power of 600 W.Coupling the extraction process with HPLC-fluorescence presented good recovery,satisfactory precision,and good linear relation.Compared with a method from the Chinese Pharmacopoeia,the proposed method enables higher extraction efficiency and more accurate analytical results.It can be of potential value in quality assessment of Radix Puerariae thomsonii medicinal materials.
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status.Conclusion HPV6 and HPV-16 were the most two popular HPV types in the whole population,while HPV-16 was the most common type in CIN2+ population.HPV-16 seroprevalence increased with severity of cervical intraepithelial neoplasia.
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To investigate the disposition and tissue distribution of ML12 after intravenous (iv) ad- ministration in rats, the compound in plasma or in tissue was extracted into ethyl acetate under basic condition and was determined by HPLC after extracted by dilute sulfuric acid. Excitation wavelength and emission wavelength of fluorescence detection were 278 nm and 307 nm, respectively. The data were processed with the software 3P97 to calculate the main pharmaceutical parameters of ML12, At dose of 5 and 10 mg/kg, the elimination of the drug from plasma was found to be kinetically linear, but when the dosage was 20 mg/kg, a non-linear feature was observed. The highest level of MLI2 was found in the kidney. Distribution of MLI2 after iv administration was extensive and the concentration-time profile was found to he fitted to an open two-compartment model.
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OBJECTIVE:To establish a HPLC-fluorescence detection method for the determiniation of the serum concentration of metoprolol.METHODS:The determination was performed on Symmetry C18 column,and the mobile phase consisted of acetonitrile-water-triethylamine -phosphoric acid(110∶390∶2.5∶1.6) at a flow rate of 0.8 mL?min-1.The excitation wavelength was 265 nm and the emission wavelength 298 nm,and the sample size was 20 ?L.RESULTS:Good linearity was obtained for metoprolol over the range of 2.0～100.0 ?g?L-1 with correlation coefficient r=0.999 6.At low,medium and high concentrations,the average recoveries of metoprolol were 99.73%,98.21% and 99.38%,respectively.The intra-day RSD were 2.89%,2.36% and 1.32%,respectively,and the inter-day RSD were 3.73%,3.03% and 2.25% respectively.The lowest detectable limit was 1.0 ?g?L-1.CONCLUSION:This method is precise,accurate,specific,simple yet with high recovery,and it is applicable for clinical monitoring of blood concentration and pharmacokinetic study.
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OBJECTIVE:To determine plasma concentration of irbesartan by HPLC with fluorescence detection.METHO-DS:Samples were separated on Diamonsil C18 with mobile phase consisted of 0.02mol?L-1 KH2PO4-acetonitrile(50∶50) at a flow rate of 1.0mL?min-1.The fluorescence detector was set at an excitation wavelength of 250nm and an emission waveleng-th of 375nm.The column temperature was 40℃.RESULTS:The linear range for irbesartan was from 1.0 to 1000ng?mL-1 (r=0.999 8).The mean relative recoveries of irbesartan at low,middle and high concentrations(2.0,50.0,and 500.00ng? mL-1) were 97.4%,106.1% and 101.6%,respectively.Both intra-day and inter-day RSD were below 10%.CONCLUSION:The method is simple,rapid,accurate and specific,and it can be used for the plasma concentration determination and pharmacokinetic study of irbesartan.
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Objective A method was developed for the simultaneous determination of tryptophan(Trp) and its metabolites kynurenine(Kyn) and kynurenic acid(Kyna) by high performance liquid chromatography with fluorescence detection(HPLC-FD),and testing serum levels of Trp metabolites in systemic lupus erythematosus(SLE) patients.Methods Serum samples were deproteinized by equal volume of 0.624 mol/L perchloric acid.The analytical column was Hypersil C18 column,and the mobile phase was 0.20 mol/L zinc acetate,8.3 mmol/L acetic acid,and 2.5% acetonitrile;flow rate was 1.5 ml/min.The excitation and emission wave length of fluorescence detector were 365 nm and 480 nm in 0~11 min,344 nm and 404 nm in 11~15.5 min,254 nm and 404 nm in 15.5~20 min,respectively.Results The linear range of Trp was 0.610~196 ?mol/L,the detection limit was 0.005 ?mol/L,and the average recovery was 103.71%.The linear range of Kyn was 0.049~98 ?mol/L,the detection limit 0.025 ?mol/L,and the average recovery was 97.45%.The linear range of Kyna was 1.050~1047 ?mol/L,the detection limit was 0.050 nmol/L,and the average recovery was 100.60%.Inter-and intra-day precisions were both less than 5%.Phenylalanine,tyrosine,and 5-hydroxytryptamine had no interference.The assay was employed to analyze serum samples of SLE patients.The result showed significant difference in Trp,Kyn,and Kyna content,Kyn/Trp ratio between SLE patients and control group.Conclusions A new method was established for simultaneous determination of Trp,Kyn,and Kyna in serum.The method is simple,fast,sensitive,specific,and suitable for applicability to clinical measurement.