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The kidney is the body's most important organ and the protein components in urine could be detected for diagnosing certain diseases. The amount of IgG protein in urine could be used to determine the degree of kidney function damage. IgG protein in human urine was detected by vertical flow paper-based microfluidic chip, double-antibody sandwich immunoreaction, and cell phone image processing. The results showed that using an IgG antibody concentration of 500 μg/mL and a gold standard antibody concentration of 100 μg/mL, the image signal showed a good linear relationship in the range of IgG concentration of 0.2-3.2 μg/mL, with R2=0.973 3 achieved. A complete set of detection devices were designed and the detection method showed good non-specificity.
Subject(s)
Humans , Microfluidics , Immunoglobulin G , Kidney , Microfluidic Analytical TechniquesABSTRACT
BACKGROUND@#Glyphosate and its salt formulations are nonselective herbicides that have been extensively used worldwide, both for residential and agricultural purposes. The possible carcinogenicity and teratogenicity of glyphosate remain to be elucidated. We developed a sensitive and high-throughput analytical method for urinary glyphosate using liquid chromatography-tandem mass spectrometry with the aim of contributing to glyphosate exposure assessment in epidemiological studies.@*METHODS@#After urine dilution (creatinine matching dilution to 0.05 g creatinine/L), glyphosate was extracted using two types of solid phase extraction columns (SCX and NH2) with automated sample preparation instruments. The eluate was dried and dissolved in the mobile phase, followed by liquid chromatography-tandem mass spectrometry analysis. The optimized method was applied to urine samples obtained from 54 Japanese adults and children.@*RESULTS@#The results from the validation study demonstrated good recoveries (91.0-99.6%), within- and between-run precisions (< 15%), low detection limits (0.1 μg/L), and lower limit of quantification (0.3 μg/L). The detection frequency and median concentration of the urinary glyphosate in Japanese subjects were 59% and 0.25 μg/L (0.34 μg/g creatinine).@*CONCLUSIONS@#Our reliable determination method was successful in measuring urinary glyphosate concentration. Moreover, this is the first biomonitoring report of urinary glyphosate levels in the Japanese general population.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Chromatography, Liquid/methods , Glycine/urine , Solid Phase Extraction/methods , Tandem Mass Spectrometry/methodsABSTRACT
BACKGROUND: Human urine-derived stem cells are newly discovered adult stem cells, characterized by rich sources, simple extraction, good proliferative ability and multi-directional differentiation potential. In recent years, human urine-derived stem cells have been used for the repair of neurological functions in urinary diseases, such as stress urinary incontinence and vesicoureteral reflux. OBJECTIVE: To explore the biological characteristics of human urine-derived stem cells and to study their repairing effect in a rat model of spinal cord injury. METHODS: Cell phenotypes of human urine-derived stem cells were detected using flow cytometry, and the immunohistochemical staining was used to identify neuron-like cells differentiated from human urine-derived mesenchymal stem cells. Then, an animal model of spinal cord injury at T9 segment was made by Allen method, and after modeling 24 Sprague-Dawley rats were assigned into spinal cord injury group or cell treatment group (n=12/group). In the cell treatment group, the model rats were injected 2 μL of 1.0×1011/L human urine-derived stem cells, while in the spinal cord injury group, the rats were administered the same volume of L-DMEM containing 10% fetal bovine serum. Basso, Beattie and Bresnahan scores were valued at 1, 10, 20, and 30 days after modeling. Spinal cord samples from all the rats were taken out at 30 days after modeling, and Luxol Fast Blue staining, microglia/macrophages staining and glial fibrillary acidic protein staining were used to value the injured area of the spinal cord and the fluorescence intensity of glial fibrillary acidic protein. RESULTS AND CONCLUSION: (1) Flow cytometry showed high expression on CD29 and CD90, and low expression on CD45 in human urine-derived mesenchymal stem cells. Moreover, human urine-derived mesenchymal stem cells could be induced to differentiating into neuron-like cells in vitro. (2) Basso, Beattie and Bresnahan scores showed no significant difference between the two groups at 1 and 10 days after modeling (P > 0.05), while, at 20 and 30 days after modeling, the scores in the cell treatment group were significantly higher than those in the spinal cord injury group (P < 0.05). (3) Luxol Fast Blue staining showed that the injured area of the spinal cord in the cell treatment group was markedly less than that in the spinal cord injury group (P < 0.05), and the glial fibrillary acidic protein showed lower fluorescence intensity in the cell treatment group than the spinal cord injury group (P < 0.05). To conclude, human urine-derived stem cells can differentiate into neuron-like cells and have therapeutic effects in the rat model of spinal cord injury.
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Abstract The present study describes a method for simultaneous analysis of cocaine, benzoylecgonine and cocaethylene in urine samples. After solid phase extraction, Gas Chromatography-Mass Spectrometry was used for identification and quantification. The calibration curves were linear at 20 - 3000 ng/mL, r2 0.9997 for benzoylecgonine, 15 - 2000 ng/mL, r2 0.9985 and r2 0.9993 for cocaine and cocaethylene, respectively. Accuracy values: cocaine: 93,5 - 102,1%; benzoylecgonine: 97,5 - 104,8%; cocaethylene: 90,6 - 101,5%. Precision values: cocaine: C.V 5,4 - 14,6%; benzoylecgonine: C.V 7,8 - 12,3%; cocaethylene: C.V 5,9 - 12,3%. Detection and quantification limit values: cocaine and cocaethylene:10 ng/mL and 15 ng/mL, respectivalty; benzoylecgonine:15 ng/mL and 20 ng/mL, respectivaley. Recovery values: cocaine: 78,0 - 85,8%; benzoylecgonine: 74,0 - 79,8%; cocaethylene: 83,0 - 91,5%. The method described is advantageous compared to others, as it simultaneously detects the major analytes found in urine samples due to cocaine use and has been successfully validated.
Subject(s)
Humans , Male , Female , Urine , Cocaine/analysis , Validation Study , Gas Chromatography-Mass Spectrometry/methods , MethodsABSTRACT
Objective To study the effect of astragalus polysaccharides combined with hUSCs transplantation on type 2 diabetic rats. Methods Twenty-five SD rats were randomly selected into the normal control group, and the remaining 105 SD rats were used to establish type 2 diabetes model. The 100 rats successfully modeled were randomly divided into the diabetes group, astragalus polysaccharide treatment group, hUSCs treatment group, and astragalus polysaccharide+hUSCs treatment group, with 25 rats in each group. After 2 weeks of treatment, the FBG concentration, insulin and C-peptide concentrations, and body weight changes were measured in each group. The distribution and survival of PKH-26-labeled hUSCs in rat pancreatic tissue were observed by fluorescence microscopy. TUNEL method was used to detect the apoptosis of rat islet cells. Real-time quantitative PCR and Western Blot were used to detect the expression of TGF-β/Smad signaling pathway-related genes in rat pancreatic tissue. Results The FBG concentration of rats in the astragalus polysaccharide treatment group, hUSCs treatment group and astragalus polysaccharide +hUSCs treatment group were significantly decreased, and that in the combination treatment group was significantly lower those in the astragalus polysaccharide group and hUSCs group, and the differences were statistically significant ( all P<0 . 05 ) . Compared with the diabetic group , the insulin concentration, C-peptide concentration and body weight in the astragalus polysaccharide treatment group, hUSCs treatment group and combination treatment group rats were significantly increased, and those in the combination treatment group was significantly higher than those in the astragalus polysaccharide treatment group and in the hUSCs treatment group, the differences were statistically significant( all P<0.05). The results of fluorescence microscopy showed that the number of PKH-26 positive hUSCs in the combined treatment group was 74.64 ±9.75 in each high power field, which was significantly higher than that in the hUSCs treatment group (43.64±5.83), the difference was statistically significant (P<0.05). Compared with the diabetic group, the apoptotic rates of islet cells in the astragalus polysaccharide treatment group and the hUSCs treatment group were reduced, and the relative expressions levels of mRNA and protein of TGF-β1, Smad3, and Smad7 in the pancreatic tissue were also significantly reduced(all P<0.05). The reduction was more significant in the combination treatment group, and the differences were statistically significant (all P<0.05). Conclusions Astragalus polysaccharide combined with hUSCs transplantation can effectively reduce the FBG concentration, increase the concentration of insulin, C-peptide and body weight, reduce the apoptosis of pancreatic islet tissue, which may be related to the reduction of TGF-β/Smad in pancreatic tissue. Signaling pathways are involved in suppressing the inflammatory response.
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Objective@#To optimize the ion selective electrode method of measuring urinary fluorine in WS/T 30-1996.@*Methods@#The volume of 5 mol/L NaOH solution was fixed to confect the TISAB buffer solution. The samples were prepared by mixing 5 ml urine and equal volume of TISAB solution. Fluorine electrode and calomel electrode was used to test. When the potential value changed less than ±0.5 mV in one minute, it could be recorded. The standard curve method was selected as a quantitive method.@*Results@#The linear range of this method was 0.07~50.0 mg/L, E=238.96-57.50lgC, r=0.999 8. The LOD and LOQ were 0.02 mg/L and 0.07 mg/L (with 5 ml urine) , respectively. The RSD of intra -and inter-batch precision were 0.80%~2.82% and 2.17%~2.86%, respectively. The recovery rate was 102%~107%. The urine sample could be preserved stably for 14 days at room temperature, and for 30 days at 4 ℃ and -20 ℃.@*Conclusion@#In this method, the preparation process of TISAB buffer solution was simplified, and the linear range was expanded. It could meet the needs of occupational population detection.
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A method was developed for the determination of four kinds of bisphenolic and halogenated bisphenolic compounds including bisphenol F, bisphenol A, tetrachlorobisphenol A, tetrabromobisphenol A in human urine using high performance liquid chromatography-tandem mass spectrometry. The analytes was extracted by solid phase extraction. The separation of the analytes was achieved on an Atlantis T3 column (3. 0×150 mm, 3 μm) gradient eluted with the mobile phase of acetonitrile and water at the rate of 250 μL/min, and detected by an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring negative mode. The quantification was carried out by matrix-matched calibration curve. The average recoveries at 3 spiked levels were 86%-118%, with intra-day precision of 2 . 6%-17 . 0% and inter-day precision of 3. 2%-18. 0%. The limits of detection of four analytes (S/N=3) were 0. 01-0. 25 μg/L. The method was applied to the analysis of 200 human urines samples and the results showed that the method was simple, sensitive and reliable.
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A novel method for the simultaneous quantification of seven metabolites of polycyclic aromatic hydrocarbon in human urine was developed using online solid phase extraction-HPLC with double ternary liquid chromatography system combined with fluorescence detector. The target compounds were online concentrated on the Turboflow Cyclone solid phase extraction column at first, then transferred by the six-way valve to the Hypersil Green PAH column for separation with acetonitrile and water as mobile phase at a flow rate of 1. 0 mL/min and at 35 ℃. A single sample analysis cycle took only 20 min. Under the optimized chromatographic conditions, the method showed good linear relationship ( r≥0. 999 ) in the range of 5-2000 ng/L or 50-20000 ng/L. The LODs were 0. 5-15 ng/L, and the recoveries were 80. 7%-110. 7%. The proposed method was successfully applied in the detection of metabolites of polycyclic aromatic hydrocarbons in urine from several smokers and non-smokers. The concentrations of 2-hydroxynaphthalene, 1-hydroxynaphthalene, 2-hydroxyfluorene, 2-hydroxyphenanthrene, 4-hydroxyphenanthrene and 6-hydroxychrysene in the smokers urine were much higher than that in non-smokers.
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A method was developed for the determination of N-acetyl-S-( N-methylcarbamoyl ) cysteine ( AMCC) in human urine by online solid-phase extraction ( SPE )-high performance liquid chromatography ( HPLC ) . The separation of AMCC from the urine matrix was performed on AmoniPac PA Solid phase Extraction ( SPE ) column with 5 mmol/L KH2 PO4 as the mobile phase by left pump. Then the time was controlled to switch the valve to make only the section of sample containing AMCC transferred into the analytic column-Acclaim PAⅡ C18 . The determination was performed using gradient elution of 0. 1% H3 PO4 (containing 5% acetonitrile) and acetonitrile by right pump. The results showed that AMCC present good linear correlation in the range of 1 . 0-100 mg/L with a correlation coefficient of above 0 . 999 , the quantitation limit of the method was 0. 2 mg/L (with the sample inject volume =10 μL), the recoveries of spiked samples were in the range of 82 . 9%-85 . 9%, and the relative standard deviation ( n=6 ) of retention time and peak area were 0. 2% and 4. 0% respectively. Compared with offline SPE-HPLC, the proposed method was convenient, environmentally friendly, efficient and stable, and feasible for the detection of AMCC in 7 human urine samples.
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A simple and environmentally friendly microextraction technique was used for determination of chlorpheniramine (CPM), an antihistamine drug, in human urine samples using dispersive liquid-liquid microextraction (DLLME) followed by high performance liquid chromatography with diode array detection (HPLC-DAD). In this extraction technique, an appropriate mixture of acetonitrile (disperser solvent) and carbon tetrachloride (extraction solvent) was rapidly injected into the urine sample containing the target analyte. Tiny droplets of extractant were formed and dispersed into the sample solution and then sedimented at the bottom of the conical test tube by centrifugation. Under optimal conditions, the calibration curve was linear in the range of 0.055-5.5 µg mL-1, with a detection limit of 16.5 ng mL-1. This proposed method was successfully applied to the analysis of real urine samples. Low consumption of toxic organic solvents, simplicity of operation, low cost and acceptable figures of merit are the main advantages of the proposed technique.
Utilizou-se uma técnica de microextração simples e ambientalmente amigável para a determinação de clorfeniramina (CPM), anti-histamínico, em amostras de urina humana, utilizando a microextração dispersiva líquido-líquido (DLLME), seguida por cromatografia líquida de alta eficiência com detecção por arranjo de diodos (HPLC-DAD). Nesse método de extração, mistura apropriada de acetonitrila (solvente dispersor) e tetracloreto de carbono (solvente de extração) foi injetada rapidamente na amostra de urina contendo o analito alvo. As pequenas gotículas de agente de extração foram formadas e dispersas na solução da amostra e, em seguida, sedimentadas no fundo do tubo cônico de ensaio por centrifugação. Em condições ótimas, a curva de calibração foi linear no intervalo entre 0,055 e 5,5 µg mL-1, com limite de detecção de 16,5 ng mL-1. O método proposto foi aplicado com sucesso na análise de amostras de urina reais. Baixo consumo de solventes orgânicos tóxicos, simplicidade de operação, baixo custo e figuras de mérito aceitáveis são as principais vantagens do método sugerido.
Subject(s)
Chlorpheniramine/analysis , Chromatography, Liquid/methods , Urine Specimen Collection , Liquid Phase Microextraction/methods , /analysis , Histamine Antagonists/analysisABSTRACT
A simple method has been proposed for the determination of clozapine (CLZ) and chlorpromazine (CPZ) in human urine by dispersive liquid-liquid microextraction (DLLME) in combination with high-performance liquid chromatography-ultraviolet detector (HPLC-UV).All important variables influencing the extraction efficiency,such as pH,types of the extraction solvent and the disperser solvent and their volume,ionic strength and centrifugation time were investigated and optimized.Under the optimal conditions,the limit of detection (LODs) and quantification (LOQs) of the method were 13 and 39 ng/mL for CLZ,and 2 and 6 ng/mL for CPZ,respectively The relative standard deviations (RSDs) of the targets were less than 5.1％ (C=0.100 μg/mL,n=9).Good linear behaviors over the tested concentration ranges were obtained with the values of R2＞0.999 for the targets.The absolute extraction efficiencies of CLZ and CPZ from the spiked blank urine samples were 98.3％ and 97.8％,respectively.The applicability of the technique was validated by analyzing urine samples and the mean recoveries for spiked urine samples ranged from 93.3％ to 105.0％.The method was successfully applied for the determination of CLZ and CPZ in real human urine.
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Objecave To develop a sensitive and specific LC-MS/MS method for determination of testolactone in human urine.Methods A C_(18 )column(2.1×50mm,3.5μm) was used.The mobile phase Was a mixture of acetonitrile and the buffer solution(ammonium acetate-water solution adjusted with formic acid to pH 3.5)at a flow rate of 0.5ml/min.A mass spectrometer equipped with electrospray ionization source was used as a detector and operated in the positive mode.In multiple reaction monitoring(MRM)mode,the ion transitions of m/z 301→121 and m/z 301→25 was used to qualify and quantify the testolactone,respectively.Results Chromatograms showed no endogenous interfering peaks with the urine blank sample.Each analysis was completed within 7min The calibration wag linear in the concentration range within 0.1～50μg/ml.The intra-batch and inter-batch RSD were less than 10%.The recovery rate of the extraction was about 60%.Conclusions The method is proved to meet the requirements of WADA and be suitable for routine screening.
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OBJECTIVE:To presume the structures and determine the contents of two metabolites of omeprazole in human urine after extraction and purification.METHODS:24 healthy volunteers were assigned to receive single oral dose of 40mg omepazole capsules,whose urinary samples collected within 12 hours after administration were extracted and concentrated with diethyl ether,and separated by HPLC.The relative purified metabolites were detected by mass spectrum,the structures of which were presumed and the contents were computed.RESULTS: The isolated 2 metabolites of omeprazole were presumed to be pyridine 5'— or 3'— methyl oxidation-generated hydroxy sulfone metabolite and 5'—methy hydroxylation thioether metabolite in pyridine ring,and the contents of which were 96.54% and 97.26%,respectively.CONCLUSION:The metabolites of omeprazole isolated from urinary samples by the method mentioned above were of high purity.
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AIM To study antitumor activity of the extract denominated as UAP(Uric Antitumor Peptides isolated and purified from normal human urine). MOTHODS Proliferation inhibition assay was examined by cell counting and MTT method. Experiments in vivo were tested by treatment on tumor mice and acute toxicity on healthy mice. RESULTS UAP exerted strong dose dependent inhibition on the proliferation of three tumor cell lines, but not human normal leucocytes. Treatments on tumor mice show that UAP effectively inhibited tumor growth of mice HAC hepatoma, mice Lewis lung carcinoma and mice S 180 sarcoma with apparently dose dependent effect. Acute toxicity proved that UAP had very low acute toxicity in mice with LD 50 = 2 137 13 mg?kg -1 . CONCLUSION UAP has strong antitumor activity in vitro and in vivo. This provides a promising alternative antitumor treatment.