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Biomedical subjects

Janusz Pawliszyn

Publications and source records attributed to Janusz Pawliszyn.

At least 55 records · Page 3Linked to original sources
PubMed2005

Behaviors of the MS2 virus and related antibodies in capillary isoelectric focusing with whole-column imaging detection.

Capillary isoelectric focusing (CIEF) has potential importance for the study of viruses. CIEF with whole-column imaging detection (WCID) is a novel CIEF mode, providing the advantages of high resolution, high speed, and easy method development. To facilitate the application of CIEF-WCID to the immunoassay of viruses, a basic knowledge of related aspects is necessary. In this study, the MS2 bacteriophage was used as a virus model, and the behaviors of MS2 and related antibodies in CIEF were investigated with UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID. The adsorption of the virus and antibodies on the capillary wall was found to be the critical issue in method development. Addition of salt was found to be an effective way to reduce the adsorption and to improve peak shape. The formation of an immunocomplex, which forms the basis of an immunoassay, was monitored with CIEF-WCID. In comparison with UV-WCID, LIF-WCID was advantageous due to its higher detection sensitivity and the elimination of precipitation. Utilization of the noncovalent fluorescent dye, NanoOrange, was demonstrated to be a potential approach for the fluorescent labeling of the virus model and antibody and the associated immunocomplex. The change in microheterogeneity during the immune interactions at different ratios was also observed.

Adsorption↗
PubMed2005

Methyl benzoate as a marker for the detection of mold in indoor building materials.

A convenient analytical method to quantify volatile organic compounds (VOCs) emitted from various building materials has not been addressed yet. This work presents a new and rapid automated method using SPME combined with GC/MS. Methyl benzoate - as a metabolic biomarker for mold growth-was used to indicate VOCs and to determine and assess mold growth on damp samples. Gypsum board and wall-board paper were used as examples of common indoor building materials. Optimized extraction conditions were carried out manually, using a GC/flame ionization detector. Moldy samples were analyzed using an automated SPME-GC/MS analysis under optimized conditions. The amount of methyl benzoate emitted from the studied samples ranged from 32 to 46 ppb, where the density of the fungal biomass was found to be 8 x 10(4) cells/mL. A relationship between the amount of fungal biomass and the emitted concentration of methyl benzoate was found and assessed based upon cultured mold samples taken from indoor building sites. The analytical method shows promise for the compound methyl benzoate, which can easily be identified at low detection limits (LOD = 3 ppb) and good linearity (>0.988), and its extraction and detection can be accomplished cleanly by current extraction techniques. Results suggest that this method with easy sample preparation can be used for quantitation and, of importance, minimal matrix effects are observed.

Air Microbiology↗
PubMed2004

The transitional isoelectric focusing process.

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.

Journal Article↗
PubMed2004

Assay of stability, free and total concentration of chlorhexidine in saliva by solid phase microextraction.

This paper presents the development and application of a solid phase microextraction method for the full investigation of chlorhexidine interaction with saliva during a pharmacokinetic study: chemical stability, binding to proteins, free concentration, total concentration and kinetics of elimination after oral administration. Only 0.1 mL sample were needed for each time point and the concentration of salivary proteins was determined as well. It was shown that chlorhexidine remained stable in the oral cavity for at least 9 h and high concentrations of the drug (2 microg/mL total) were still present even 8 h after mouthrinsing. Supplementary facts were uncovered: while the total concentration followed first-order elimination kinetics, the free concentration remained almost constant for several hours; this showed that the oral cavity acted like a reservoir that slowly released the drug. It was also revealed that following oral administration of chlorhexidine, the normal composition of saliva changed for a few hours, probably as a physiological response to the bitter taste of the medicine. The method had a wide linear range (0.1-40 microg/mL free chlorhexidine) that was perfectly suitable for the study of chlorhexidine retention in the oral cavity. Separation and quantitation were achieved by liquid chromatography coupled to mass spectrometry; no interference from endogenous compounds was observed. This selective and sensitive solid-phase microextraction (SPME) approach for monitoring the free and total concentration of a drug, as well as the concentration of proteins that bind that drug, should prove to be more useful for pharmacokinetic studies than classic methods that only provide the total concentration as a final result.

Chlorhexidine↗
PubMed2004

Solid-phase microextraction field sampler.

To facilitate the use of solid-phase microextraction (SPME) for field sampling, a new field sampler was designed and tested. The sampler was versatile and user-friendly. The SPME fiber can be positioned precisely inside the needle for time-weighted average sampling or exposed completely outside the needle for grab sampling. The needle is protected within a shield at all times, hereby eliminating the risk of operator injury and fiber damage. A replaceable Teflon cap is used to seal the needle to preserve sample integrity. Factors that affect the preservation of sample integrity (sorbent efficiency, temperature, sealing materials) were studied. The use of a highly efficient sorbent for the fiber is recommended as the first choice for the preservation of sample integrity. Teflon was a good material for sealing the fiber needle, had little memory effect, and could be used repeatedly. To address adsorption of high boiling point compounds on fiber needles, several kinds of deactivated needles were evaluated. RSC-2 fiber needles were the more effective. A preliminary field sampling investigation demonstrated the validity of the new SPME device for field applications.

Adsorption↗
PubMed2004

On-line monitoring of breath by membrane extraction with sorbent interface coupled with CO2 sensor.

In order to establish a simple and reproducible sampling device and method, which is crucial for a wide application of breath analysis, the original membrane extraction with sorbent interface (MESI) system was improved by coupling with a palm-size CO2 sensor. Variations in analyte concentrations due to mass losses and different breathing patterns were normalized by simultaneously measuring the partial pressure of CO2 and the concentrations of target analytes in the breath sampled. Analyte concentrations can then be expressed normalized to CO2 as in the alveolar air. The MESI system was applied to study light hydrocarbons such as methane and ethane, which are difficult to analyse by other methods. A systematic study of breakthrough, which relates to the sorbent capacity and is characteristic of the analytical efficiency, was performed through the effects of analyte concentration, trap temperature, sample humidity and extraction time on breakthrough. Continuous on-line monitoring of breath methane and ethane was carried out under the optimum operation conditions based on the breakthrough study.

Breath Tests↗
PubMed2004

Kinetics and the on-site application of standards in a solid-phase microextraction fiber.

The kinetics of the desorption of analytes from a SPME fiber into an agitated sample matrix was studied, and a theoretical model was proposed to describe the dynamic desorption process, based on the steady-state diffusion of analytes in the extraction phase and in the boundary layer. It was found that the desorption of analytes from a SPME fiber into an agitated sampling matrix is isotropic to the absorption of the analytes onto the SPME fiber from the sample matrix under the same agitation conditions, and this allows for the calibration of absorption using desorption. The calibration was accomplished by exposing a SPME fiber, preloaded with a standard, to an agitated sample matrix, during which desorption of the standard and absorption of analytes occurred simultaneously. When the standard was the isotopically labeled analogue of the target analyte, the information from the desorption process, i.e., time constant a, could be directly used for estimating the concentration of the target analyte. When the standard varied from the target analyte, the mass-transfer coefficient of the analyte could be extrapolated from that of the standard. These predictions agree well with experimental results. This approach facilitates the full integration of sampling, sample preparation, and sample introduction, especially for on-site or in vivo investigations, where the addition of standards to the sample matrix, or control of the velocity of the sample matrix, is very difficult.

Journal Article↗
PubMed2004

Buffer composition changes in background electrolyte during electrophoretic run in capillary zone electrophoresis.

The electrophoretic behaviors of different analytes in capillary zone electrophoresis were studied by the Whole Column Imaging Detection (WCID). For capillary zone electrophoresis (CZE) in conventional buffer systems, non-constant sample plug movement characterized by progressive decrease of peak migration velocity was observed. The appropriate velocity decrease was correlated with a degree of ionization of the analyzed ion, thus the effect observed could be explained by fast buffer composition change resulting in the development of a non-linear pH gradient. To visualize the appropriate pH gradient, the concentration profile of initially uniformly distributed amphoteric substances was also monitored. The evolution of the concentration profile exhibited very complex dynamics. In addition, it was found that the nature of the electrode solutions strongly affect changes in the background electrolyte. In the case of traditional background electrolytes with an acid-base pair for electrode solutions a non-uniform ampholyte concentration developed quickly, leading finally to a quasi-stationary profile similar to those typical of IEF. Possible approaches to suppress a negative impact of the background electrolyte composition changes during electrophoretic run on CE-separation are presented herein. In particular it was observed that zwitterionic buffers are able to withstand prolonged electrolysis much better compared to traditional buffers.

Journal Article↗
PubMed2004

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry.

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.

Animals↗
PubMed2004

Full automation of derivatization--solid-phase microextraction-gas chromatography-mass spectrometry with a dual-arm system for the determination of organometallic compounds in aqueous samples.

The determination of organometallic compounds in aqueous samples by in-vial derivatization and headspace solid-phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS) has been fully automated using a Twin PAL dual-arm robotic system. Linearity, accuracy, sensitivity for a series of n-methyl, n-ethyl, and n-phenyl metal substituted chloride compounds of tin, lead, and mercury were investigated. The automated method was compared to similar manual methods and improved precision, speed and throughput was achieved. By originally programming the Twin PAL dual-arm system with the supplier's software (Cycle Composer, Version 1.5.0) the arms on the robot were only able to work in sequence. However, in order to have a flexible system and exploit time efficiently the robotic arms must work simultaneously. This was accomplished by programming the robot with the new software package called Cruise Control 4-2 for Twin PALs. Compared to Cycle Composer, Cruise Control 4-2 enhanced the speed and throughput of the automated system further. In addition, with a built-in crash prevention protocol and an improved user interface a more user-friendly system was obtained.

Automation↗
PubMed2004

Air sampling of aromatic hydrocarbons in the presence of ozone by solid-phase microextraction.

Effects of ozone on air sampling of standard gas mixtures of aromatic hydrocarbons were tested using solid-phase microextraction (SPME). Standard concentrations of ozone ranging from 10 ppb (v/v) to 6400 ppm (v/v) were generated using an in-house built ozone generator based on corona discharge. Effects of temperature, discharge voltage, and oxygen flow on the ozone generation were tested. The working dc voltage had the greatest effect on generated ozone concentration and was proportional to the ozone concentration. Generation temperature and oxygen flow rate were inversely proportional to ozone concentrations. Produced ozone was mixed with standard benzene, toluene, ethylbenzene, and xylenes (BTEX) gas at less than 100 ppb (v/v). Air samples were collected with poly(dimethylsiloxane) (PDMS) 100 microm SPME fibers and analyzed by gas chromatography (GC)-flame ionization detection (FID) and GC-MS. Significant reductions of BTEX concentrations were observed. In addition, some products of BTEX-ozone-oxygen reactions were identified. SPME worked well as a rapid sampler for BTEX and BTEX-ozone-oxygen reaction products. No significant deterioration of the PDMS coating and no significant reduction of absorption capacity were observed after repeated exposure to ozone.

Air↗
PubMed2004

System for the generation of standard gas mixtures of volatile and semi-volatile organic compounds for calibrations of solid-phase microextraction and other sampling devices.

Standard gases are used for quality control and quality assurance, development of analysis methods and novel air sampling devices. The use of solid-phase microextraction (SPME) and other novel technologies for research in the area of air sampling and analysis requires systems/devices for reliable standard gas generation and sampling. In this paper we describe a new gas standard generating system for volatile organic compounds (VOCs) and semi-VOCs that was designed, built, and tested to facilitate fundamental and applications research with SPME. The system provided for the generation of a wide range of VOC/semi-VOC concentrations and mixing various standard gases, estimation of detection limits, testing the effects of sampling time, air temperature and relative humidity, testing the effects of air velocity and ozone on sampling/extractions. The system can be also used for calibrations of analytical instrumentation, quality control and quality assurance checks, and cross-validations of SPME with/and other sampling techniques.

Calibration↗
PubMed2004

Isoelectric point determination of norovirus virus-like particles by capillary isoelectric focusing with whole column imaging detection.

A capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) method was used to determine the isoelectric point (pI) of norovirus virus-like particles (VLPs). The VLPs were produced from noroviruses that represented the two genogroups, genogroup I (Funabashi, Seto, and Norwalk) and genogroup II (Hawaii, Kashiwa, and Narita). Using the imaged CIEF-WCID detection technique, separation was accomplished using a short (4-5 cm) internally coated capillary (100-microm diameter) and a whole-column optical absorption imaging detector operated at 280 nm. CIEF-WCID experiments showed the similarity of the pI values of VLPs from genogroups I and II, with pI values of 5.9, 5.9, 6.0, and 6.0 for Funabashi, Norwalk, Seto, and Hawaii. The two other VLPs displayed pI values of 5.5 (Kashiwa) and 6.9 (Narita). The VLP peaks were shown to be reproducibility resolved. CIEF-WCID shows great promise for norovirus detection in public health, clinical, and food safety applications, as CIEF-WCID overcomes several limitations of the currently used genetic and immunological methods.

Electrophoresis, Capillary↗
PubMed2004

High-resolution computer simulation of the dynamics of isoelectric focusing of proteins.

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

Computer Simulation↗
PubMed2004

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection.

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.

Animals↗
PubMed2004

Whole-column fluorescence-imaged capillary electrophoresis.

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.

Electrophoresis, Capillary↗
PubMed2004

Solid-phase microextraction combined with surface-enhanced laser desorption/ionization introduction for ion mobility spectrometry and mass spectrometry using polypyrrole coatings.

The successful application of polypyrrole (PPY) solid-phase microextraction (SPME) coatings as both an extraction phase and a surface to enhance laser desorption/ionization (SELDI) of analytes is reported. This SPME/SELDI fiber integrates sample preparation and sample introduction on the tip of a coated optical fiber, as well as acting as the transmission medium for the UV laser light. Using ion mobility spectrometry (IMS) detection, the signal intensity was examined as a function of extraction surface area and concentration of analyte. The linear relationship between concentration and signal intensity shows potential applicability of this detection method for quantitative analysis. Extraction time profiles for the fiber, using tetraoctylammonium bromide as test analyte, illustrated that equilibrium can be reached in less than one minute. To investigate the performance of the PPY coating, the laser desorption profile was studied. The fiber was also tested using a quadrupole time-of-flight (Q-TOF) mass spectrometer with leucine enkephalin as test analyte. Since no matrix was used, mass spectra free from matrix background were obtained. This novel SPME/SELDI fiber is easy to manufacture, and is suitable for studying low-mass analytes because of the intrinsic low background. These findings suggest that other types of conductive polymers could also be used as an extraction phase and surface to enhance laser desorption/ionization in mass spectrometry.

Journal Article↗