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Sucrose esters (SEs) are crucial tobacco smoke flavor precursors and play a significant role in tobacco's functionality. Due to their structural complexity, the separation and analysis of SEs in tobacco remain a major challenge, and massive structures of SEs have not yet been fully identified. In this study, the fractions enriched in SEs were obtained from oriental and flue-cured tobacco through a series of pretreatments, and two types of SEs (Types I and II) were distinguished by liquid chromatography-tandem mass spectrometry (LC-MSn ) analysis, with Type II SEs newly characterized in tobacco. Five groups of main SEs were further purified using preparative high-performance LC (HPLC) coupled to an evaporative light scattering detector, and their structures were characterized by nuclear magnetic resonance spectrometry techniques including 1 H, 13 C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. By combining LC-MSn and nuclear magnetic resonance spectrometry, the structures of eight SE isomers were finally proposed, of which four were newly identified. These findings further enhance the understanding of the structural diversity of SEs in tobacco, serving as a valuable reference for future research on the elucidation, synthesis, and metabolism of SEs.
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Ésteres , Sacarosa , Espectrometría de Masas , Cromatografía Liquida , Isomerismo , Cromatografía Líquida de Alta Presión/métodosRESUMEN
A novel infrared-assisted extraction coupled to headspace solid-phase microextraction followed by gas chromatography with mass spectrometry method has been developed for the rapid determination of the volatile components in tobacco. The optimal extraction conditions for maximizing the extraction efficiency were as follows: 65 µm polydimethylsiloxane-divinylbenzene fiber, extraction time of 20 min, infrared power of 175 W, and distance between the infrared lamp and the headspace vial of 2 cm. Under the optimum conditions, 50 components were found to exist in all ten tobacco samples from different geographical origins. Compared with conventional water-bath heating and nonheating extraction methods, the extraction efficiency of infrared-assisted extraction was greatly improved. Furthermore, multivariate analysis including principal component analysis, hierarchical cluster analysis, and similarity analysis were performed to evaluate the chemical information of these samples and divided them into three classifications, including rich, moderate, and fresh flavors. The above-mentioned classification results were consistent with the sensory evaluation, which was pivotal and meaningful for tobacco discrimination. As a simple, fast, cost-effective, and highly efficient method, the infrared-assisted extraction coupled to headspace solid-phase microextraction technique is powerful and promising for distinguishing the geographical origins of the tobacco samples coupled to suitable chemometrics.
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Nicotiana/química , Fitoquímicos/análisis , Compuestos Orgánicos Volátiles/análisis , Dimetilpolisiloxanos , Cromatografía de Gases y Espectrometría de Masas , Análisis Multivariante , Polivinilos , Microextracción en Fase SólidaRESUMEN
An ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction was first employed to determine the volatile components in tobacco samples. The method combined the advantages of ultrasound, microwave, and headspace solid-phase microextraction. The extraction, separation, and enrichment were performed in a single step, which could greatly simplify the operation and reduce the whole pretreatment time. In the developed method, several experimental parameters, such as fiber type, ultrasound power, and irradiation time, were optimized to improve sampling efficiency. Under the optimal conditions, there were 37, 36, 34, and 36 components identified in tobacco from Guizhou, Hunan, Yunnan, and Zimbabwe, respectively, including esters, heterocycles, alkanes, ketones, terpenoids, acids, phenols, and alcohols. The compound types were roughly the same while the contents were varied from different origins due to the disparity of their growing conditions, such as soil, water, and climate. In addition, the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method was compared with the microwave-assisted extraction coupled to headspace solid-phase microextraction and headspace solid-phase microextraction methods. More types of volatile components were obtained by using the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method, moreover, the contents were high. The results indicated that the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction technique was a simple, time-saving and highly efficient approach, which was especially suitable for analysis of the volatile components in tobacco.
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Cigarette smoke contains a large number of chemicals, including both flavor components and harmful substances. The mainstream smoke (MSS) generated by smoking is directly inhaled by individuals, making it crucial to establish an effective method for smoke detection and analysis. One promising technique for analyzing smoke is MPT-MS (Microwave plasma torch mass spectrometry). This approach offers several advantages in accurately detecting the composition of cigarette smoke. By combining MPT-MS with a smoke pumping device, we can achieve real-time online detection of smoke components. We successfully detected 22 flavor compounds present in the smoke. These compounds contribute to the distinct taste of cigarettes. Moreover, we identified 2 polycyclic aromatic hydrocarbons (PAHs) in the smoke. PAHs are known carcinogens and are of great concern in terms of their potential health risks. The successful detection and identification of flavor compounds and PAHs using our method confirm the online detection capability of MPT-MS. This approach provides an efficient and reliable means for analyzing the complex composition of cigarette smoke. By utilizing MPT-MS, we can gain valuable insights into the chemical composition of cigarette smoke and can inform the development of strategies and policies aimed at reducing the harmful effects of smoking and protecting public health.
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Mass spectrometric analysis of non-volatile salts containing samples remains challenging due to salt-induced ion suppression and contamination. This challenge is even more pronounced for a liquid chromatography-mass spectrometry analysis, where the accumulation of salts in the transmission system poses an ongoing problem. In this study, a novel thermal assisted recrystallization ionization mass spectrometry (TARI-MS) device was developed to achieve efficient on-line desalting and prolonged analysis of saline samples. The core component of this device was a heated plate positioned between the electrospray unit and the MS inlet. The desalting mechanism was demonstrated as the spontaneous separation of target molecules from salts during the "crystallization" process. After optimization, the angle between the nebulizer and the heated plate was 45°; the distance between the front end of the heated plate and the MS inlet was 2 mm; the distance between the front edge of the heated plate and the center of the sample spray projected onto the heating plate was 3 mm; the distance between the emitter of nebulizer and the heated plate was 3 mm. TARI-MS realized direct analysis of eight drugs dissolved in eight commonly used non-volatile salts solutions (up to 0.5 mol/L). The high sensitivity, repeatability, linearity, accuracy, and intra- and inter-day precision of TARI-MS confirm its reliability as a robust tool for the analysis of saline samples. Furthermore, TARI-MS allowed continuous analysis of salty eluates of LC for up to nearly 1 h without maintenance and verified the feasibility of LC-MS analysis through detecting a five-drug mixture and a crude aripiprazole product. Finally, six impurities in the crude aripiprazole product were successfully detected by LC-TARI-MS. The established method holds promise for applications across academic and pharmaceutical domains.
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Tobacco, as an important cash crop and model plant, has been the subject of various types of research. The quality of flue-cured tobacco products depends on the compound collection of tobacco leaves, including pigments, carbohydrates, amino acids, polyphenols, and alkaloid aroma precursors. The present study investigates tobacco seedling organs (leaf, stem, and root) with the assistance of label-free proteomic technology and untargeted metabonomic technology. We analyzed 4992 proteins and 298 metabolites obtained in the leaf, stem, and root groups and found that there were significant differences in both primary and secondary metabolism processes involved in aroma precursor biosynthesis, such as carbohydrate metabolism, energy metabolism, and amino acid biosynthesis, and phenylpropanoid, flavonoid, and alkaloid biosynthesis. The findings showed that the contents of alkaloid metabolites such as nornicotine, anatabine, anatalline, and myosmine were significantly higher in tobacco roots than in leaves and stems at the seedling stage.
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Tobacco has a high economic value as the largest cash crop worldwide. The quality of flue-cured tobacco is closely related to the overall status of compounds in fresh tobacco leaves, and the aroma precursor plays a key role in the aroma quality of flue-cured tobacco. The untargeted metabolomics and label-free quantitative proteomics analysis of tobacco leaves in three growth stages (root stretching, prosperous growth, and maturation) retrieved 243 metabolites and 4313 proteins (944 differentially expressed proteins), which showed that carbohydrate, amino acid, and fatty acid metabolism varies among the three growth stages. Also, the most of amino acids, organic acids, fatty acids, and polyphenols reduced in the vegetative growth stage, while increased in the reproductive growth stage. On the other hand, alkaloids such as nicotine, nornicotine, and anatabine increased continuously in tobacco leaves during the three growth stages. This study helps us understand the growth and development characteristics of Yun87 flue-cured tobacco in the field before harvest, and it provides a certain omics basis for the industrial crop flue-cured tobacco.
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BACKGROUND: Complex chronic diseases such as rheumatoid arthritis have become a major challenge in medicine and for the pharmaceutical industry. New impulses for drug development are needed. OBJECTIVE: : A systems biology approach is explored to find subtypes of rheumatoid arthritis patients enabling a development towards more personalized medicine. METHODS: Blood samples of 33 rheumatoid arthritis (RA) patients and 16 healthy volunteers were collected. The RA patients were diagnosed according to Chinese medicine (CM) theory and divided into 2 groups, the RA Heat and RA Cold group. CD4 T-cells were used for a total gene expression analysis. Metabolite profiles were measured in plasma using gas chromatography/mass spectrometry. Multivariate statistics was employed to find potential biomarkers for the RA Heat and RA Cold phenotype. A comprehensive biologic interpretation of the results is discussed. RESULTS: : The genomics and metabolomics analysis showed statistically relevant different gene expression and metabolite profiles between healthy controls and RA patients as well as between the RA Heat and RA Cold group. Differences were found in the regulation of apoptosis. In the RA Heat group caspase 8 activated apoptosis seems to be stimulated while in the RA Cold group apoptosis seems to be suppressed through the Nrf2 pathway. CONCLUSIONS: RA patients could be divided in 2 groups according to CM theory. Molecular differences between the RA Cold and RA Heat groups were found which suggest differences in apoptotic activity. Subgrouping of patients according to CM diagnosis has the potential to provide opportunities for better treatment outcomes by targeting Western or CM treatment to specific groups of patients.
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Artritis Reumatoide/clasificación , Artritis Reumatoide/terapia , Medicina Tradicional China/métodos , Biología de Sistemas/métodos , Adulto , Apoptosis , Artralgia/patología , Artralgia/fisiopatología , Artralgia/terapia , Artritis Reumatoide/sangre , Biomarcadores/sangre , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Caspasa 8/sangre , Frío , Femenino , Calor , Humanos , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/sangreRESUMEN
This chapter illustrates the usefulness of capillary electrophoresis (CE) for the detection of gene mutation, i.e., point mutation, methylation, and microsatellite analysis. In order to provide a general description of the main results and challenges in the field, some relevant applications and reviews on CE of gene mutation are tabulated. Furthermore, some detailed experimental procedures are shown. Several CE methods of gene mutation detection were developed including the following: (1) single-strand conformation polymorphism with capillary electrophoresis; (2) SNaPshot analysis; (3) constant denaturant capillary electrophoresis; (4) microsatellite analysis; and (5) methylation analysis.
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Electroforesis Capilar/métodos , Mutación/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Metilación de ADN , Exones/genética , Humanos , Repeticiones de Microsatélite/genética , Homólogo 1 de la Proteína MutL , Neoplasias/genética , Proteínas Nucleares/genética , Desnaturalización de Ácido Nucleico , Polimorfismo de Nucleótido Simple/genética , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Temperatura , Proteína p53 Supresora de Tumor/genética , Proteínas ras/genéticaRESUMEN
Two mutants of E. coli with deletion of sdhAB and ackA-pta genes respectively and their wild-type strains were subjected to gas chromatography-flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) metabolomics analysis. Intracellular metabolites of the three strains were profiled by GC-FID firstly. Methodological evaluation of the employed platform indicated that the limit of detection ranges were from 0.2 to 12.5 ng for some representative metabolites and the corresponding recoveries were varied from 68.7 to 122.7%. Secondly, multivariable data analysis was applied to the acquired data sets. As expected, the three phenotypes could be easily differentiated, and the perturbed metabolite pools in the genetically modified strains were screened. Lastly, the metabolites playing key roles in the differentiation were further identified by GC-MS. It was confirmed that succinic acid and aspartic acid were similarly affected in the modified strains. But proline content was altered contrarily. Additionally, deletion of sdhAB gene also affected the growth property of relevant mutant greatly. The potential mechanism was postulated accordingly.
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Cromatografía de Gases/métodos , Biología Computacional/métodos , Escherichia coli/clasificación , Escherichia coli/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolismo , Escherichia coli/genética , FenotipoRESUMEN
"Metabonomics" method requires the development of rapid, advanced analytical tools and GC will play an important role for its special advantage. In this study we show the application of GC-based metabonomics to investigate the control and type 2 diabetes (DM2) patients by urinary organic acids metabolic profile. After peak matching, multivariate statistical analysis methods: principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used. The results showed that there was a relationship between organic acids metabolic profiles and DM2, and PLS-DA can distinguish the DM2 patients from the control. Five organic acids as potential biomarkers were identified.
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Ácidos/orina , Cromatografía de Gases/métodos , Diabetes Mellitus Tipo 2/orina , Compuestos Orgánicos/orina , Adulto , Algoritmos , Estudios de Casos y Controles , Cromatografía de Gases y Espectrometría de Masas , Humanos , Persona de Mediana Edad , Análisis MultivarianteRESUMEN
Metabolomics is a new branch of systems biology exerting its influence in many aspects. In order to appraise the effects of antibiotics on central carbon metabolism, a CE based method was set up. With this platform, we estimated the organic acid metabolite pools' fluctuation of Escherichia coli and Pseudomonas aeruginosa cultured under 11 different antibiotics' stimuli. Multivariate data analysis showed that different antibiotics had clustered distributions for each strain and could be easily distinguished. Genetic, metabolic and antibiotic mechanism differences could also be deduced by the aid of further correlation analysis. For P. aeruginosa, even synergy action amid antibiotics could be ascertained.
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Antibacterianos/farmacología , Ciclo del Ácido Cítrico , Electroforesis Capilar/métodos , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/clasificación , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Modelos Biológicos , Análisis Multivariante , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especificidad de la EspecieRESUMEN
OBJECTIVE: To assess the therapeutic effect of losartan on type 2 diabetes mellitus (DM2) with gas chromatography (GC)-based metabonomics. METHODS: DM2 patients were dosed with losartan (100 mg/d) and urines were collected at week 8 and 12. The biochemical criteria (blood pressure, urinary albumen, urinary 8-hydroxy-2'-deoxyguanosine and blood creatinine) were analyzed. Urine samples were derivatived and analyzed by GC. Multivariate metabonomics analysis was performed after peak alignment. RESULTS: After 8-12 weeks, losartan showed little curative effect and no remarked changes of biochemical criteria were observed. However, metabonomics analysis revealed that some biomarkers such as glucitol and inositol changed. CONCLUSION: GC-based metabonomics analysis enables the rapid identification of metabolic differences and provides information concerning therapeutic effect of losartan.
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Cromatografía de Gases/métodos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Losartán/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina , Albuminuria/orina , Biomarcadores/sangre , Biomarcadores/química , Biomarcadores/orina , Creatinina/sangre , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Monitoreo de Drogas , Humanos , Inositol/química , Metaboloma/efectos de los fármacos , Sorbitol/químicaRESUMEN
A simple, rapid and low-cost method using capillary electrophoresis coupled with field-amplified sample stacking and electrochemical detection was developed for the separation and determination of monoamines. In this present work, a systematic study of the parameters (pH value and concentration of electrophoretic buffer, composition of sample solvent, injection voltage and time) affecting separation and on-line concentration of monoamines has been performed enabling the detection sensitivity of these monoamines to be improved by 5,000 times compared with the conventional electrokinetic injection. This developed method was applied to the direct analysis of these monoamines in human urine without off-line sample preconcentration. Due to the requirement for urine dilution to minimize the detrimental effects of high salt on analyte stacking, the real sensitivity improvement is about 50-fold when applying the optimized method to urine samples. In order to quantitate these monoamines accurately, internal standard calibration curves were constructed with standard monoamines in presence of salt with similar concentration as in human urine. In the method validation, the calibration curves were linear over a range of 1.0 x 10(-9) to 2.5 x 10(-8) mol/L for each monoamine and the limits of detection (signal to noise ratio of 3) for these monoamines were in the sub-nmol/L concentration range (6.0 x 10(-10) mol/L).
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Catecolaminas/orina , Electroforesis Capilar/métodos , Serotonina/orina , Dopamina/orina , Electroquímica/métodos , Epinefrina/orina , Humanos , Concentración de Iones de Hidrógeno , Masculino , Norepinefrina/orina , Reproducibilidad de los ResultadosRESUMEN
Pre-processing of near-infrared (NIR) spectral data has become a necessary part of chemometrics modeling and is widely used in many practical applications. The objective of the pre-processing is to remove physical phenomena in the spectra in order to improve subsequent qualitative or quantitative analysis. Herein, a localized version of standard normal variate (SNV) is proposed, in which the correction parameters are estimated from local spectral areas. The method of determining the optimal spectral segmentation is also presented. Compared with full range methods, the local method demonstrates advantages in spectral linearity correction, model interpretation and prediction accuracy. Several benchmark NIR data sets were studied in our experiments; the proposed method achieved comparable performance against proven full range methods, with the reduction of prediction errors being statistically significant in many cases.
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Algoritmos , Carne/análisis , Preparaciones Farmacéuticas/análisis , Espectroscopía Infrarroja Corta/métodos , Bases de Datos Factuales , Espectroscopía Infrarroja Corta/normas , Comprimidos/análisisRESUMEN
The major components of the plant curcuma longa are the curcuminoids that include curcumin, demethoxycurcumin and bisdemethoxycurcumin. It has been reported the curcuminoids have some important activities. A new CZE method with diode array detection has been developed for the separation and determination of the curcumin, demethoxycurcumin and bisdemethoxycurcumin. Three curcuminoids could be readily separated within 7 min with a 15 mM sodium tetraborate buffer containing 10% methanol (v/v) at pH 10.8, 25 kV and 30 degrees C. The method has been validated and shows good performance with respect to selectivity, reproducibility, linearity, limits of detection and recovery. The proposed method was successfully applied to determine the curcuminoids in urine.
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Curcumina/aislamiento & purificación , Electroforesis Capilar/métodos , Tampones (Química) , Calibración , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The crude methanol extract of roots of Lithospermum erythrorhizon was subjected to successive chromatographic fractionation which afforded two new dimeric naphthoquinone derivatives shikometabolin E (2) and shikometabolin F (3) as well as one known compound shikometabolin A (1). The structures of compounds 1-3 were elucidated by using UV, MS, 1D and 2D NMR spectroscopic analysis. The two new dimeric naphthoquinone derivatives showed significant neuraminidase inhibitory activities.
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Lithospermum/química , Naftoquinonas/química , Neuraminidasa/antagonistas & inhibidores , Raíces de Plantas/química , Inhibidores Enzimáticos/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Naftoquinonas/aislamiento & purificaciónRESUMEN
The cryIAc and sck genes were introduced to the rice for the purpose of improving the insect resistance. Metabolic profiles of wild and transgenic rice were compared to assess the unintended effects related to gene modification. Wild samples with different sowing dates or sites were also examined to determine the environmental effects on metabolites. The polar compounds of grains were extracted, trimethylsilylated and analyzed by gas chromatography-flame ionization detection (GC-FID). Partial least squares-discriminant analysis (PLS-DA) and principal component analysis (PCA) were applied to differentiate transgenic and wild rice grains. The significantly distinguishable metabolites were picked out, and then identified by gas chromatography-mass spectrometry (GC-MS). It was found that both the environment and gene manipulation had remarkable impacts on the contents of glycerol-3-phosphate, citric acid, linoleic acid, oleic acid, hexadecanoic acid, 2,3-dihydroxypropyl ester, sucrose, 9-octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester and so on. Sucrose, mannitol and glutamic acid had a significant increase in transgenic grains in contrast to those in non-genetically modified (GM) rice.
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Proteínas Bacterianas/metabolismo , Endotoxinas/metabolismo , Ionización de Llama/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Proteínas Hemolisinas/metabolismo , Metaboloma , Oryza/química , Proteínas de Plantas/metabolismo , Inhibidores de Tripsina/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Inhibidores de Tripsina/genéticaRESUMEN
The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.
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Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Electroforesis Capilar/métodos , Látex , Staphylococcus aureus/aislamiento & purificación , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Heces/microbiología , Humanos , Látex/química , Infecciones Estafilocócicas/diagnóstico , Staphylococcus aureus/inmunologíaRESUMEN
A method based on micellar electrokinetic capillary chromatography with electrochemical detection (MEKC-EC) was developed for the simultaneous determination of norepinephrine (NE) and dopamine (DA) in Chinese herbal plant extracts of Portulaca oleracea L. The effects of several factors such as the acidity and concentration of running buffer, sodium dodecylsulfate (SDS) concentration and detection potential were investigated to acquire the optimum conditions. The two analytes could be well separated within 12 min in a 64 cm capillary at the separation voltage of 25 kV in a 10 mmol/L phosphate buffer (pH 6.23) containing 10 mmol/L SDS. The excellent linearity was obtained in the concentration range from 1.0 x 10(-6) to 5.0 x 10(-4) mol/L. The detection limits of concentration were 4.2 x 10(-7) mol/L for NE and 8.7 x 10(-7) mol/L for DA and those of quantity were 0.41 fmol for NE and 1.45 fmol for DA at a signal-to-noise ratio of 3. This method was successfully used in the analysis of Portulaca oleracea L. without derivatization procedure, and real average contents of NE and DA in Portulaca oleracea L. were 0.015% and 0.20%, respectively.