Biomedical subjects

J Pawliszyn

Publications and source records attributed to J Pawliszyn.

At least 55 records · Page 3Linked to original sources

PubMed2000

On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface.

A high performance liquid chromatography system, a sample preparation device, and an imaged capillary IEF (CIEF) instrument are integrated and multiplexed on-line. The system is equivalent to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), by transferring the principle of 2-D separation to the capillary format. High performance liquid chromatography (HPLC) provides protein separation based on size using a gel filtration chromatography (GFC) column. Each eluted protein is sampled and directed to a novel microdialysis hollow fiber membrane device, where simultaneous desalting and carrier ampholyte mixing occurs. The sample is then driven to the separation column in an on-line fashion, where CIEF takes place. The fluidic technology used by our 2-D system leads to natural automation. The coupling of the two techniques is simple. This is attributed to high speed and efficiency of the sample preparation device that acts as an interface between the two systems, as well as the speed and simplicity of our whole column absorption imaged CIEF instrument. To demonstrate the feasibility of this approach, the separation of a mixture of two model proteins is studied. Sample preparation and CIEF were complete in just 4-5 min, for each of the eluted proteins. Total analysis time is about 24 min. Three-dimensional data representations are constructed. Challenges and methods to further improve our instrument are discussed, and the design of an improved horseshoe-shaped sample preparation sample loop membrane interface is presented and characterized.

Animals↗

PubMed2000

Automated in-tube solid phase microextraction coupled with HPLC-ES-MS for the determination of catechins and caffeine in tea.

A polypyrrole (PPY) coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for catechins and caffeine. Compared with commercial capillaries that were currently used for in-tube solid phase microextraction (SPME), the PPY coated capillary showed better extraction efficiency for all of the compounds studied. Electrospray mass spectrometric (ES-MS) detection conditions were also investigated. After optimization of the extraction and detection conditions, a method for the sensitive and selective determination of catechins and caffeine was developed by coupling the PPY coated capillary in-tube SPME with HPLC-ES-MS. Catechins could be determined in both positive and negative ion detection modes. The detection limit (S/N = 3) for each of the studied catechins was < 0.5 ng mL-1. Caffeine could only be determined under positive ES-MS detection conditions and its detection limit was 0.01 ng mL-1. Caffeine and the five catechins in several tea samples were determined using the developed method. Small amounts of catechins were also detected in grape juice and wine samples.

Caffeine↗

PubMed2000

Theory of solid-phase microextraction

The main objective of this contribution is to describe the fundamental concepts associated with solid-phase microextraction (SPME). Theory provides insight when developing SPME methods and identifies parameters for rigorous control and optimization. A mathematical model has been developed to understand the principal processes of SPME by applying basic fundamental principles of thermodynamics and diffusion theory. The model assumes idealized conditions and is limited to air, liquid, or headspace above liquid sampling. Theory for ideal cases can be quite accurate for trace concentrations in simple matrices such as air or drinking water at ambient conditions when secondary factors such as thermal expansion of polymers and changes in diffusion coefficients because of solutes in polymers can be neglected. When conditions are more complex, theory for ideal cases still efficiently estimates general relationships between parameters.

Journal Article↗

PubMed1999

Automated in-tube solid-phase microextraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of beta-blockers and metabolites in urine and serum samples.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was evaluated for the determination of beta-blockers in urine and serum samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC/MS analyses of beta-blockers were initially performed by liquid injection onto a LC column. Nine beta-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]+ were observed for all beta-blockers. The beta-blockers were separated with a Hypersil BDS C18 column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. To optimize the extraction of beta-blockers, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30 microL of sample in 100 mM Tris-HCl (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary (Supelco, Bellefonte, PA). The beta-blockers extracted by the capillary were easily desorbed by mobile-phase flow, and carryover of beta-blockers was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of beta-blockers were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9982 (n = 18) and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This method was successfully applied to the analysis of biological samples without interference peaks. The recoveries of beta-blockers spiked into human urine and serum samples were above 84 and 71%, respectively. A serum sample from a patient administrated propranolol was analyzed using this method and both propranolol and its metabolites were detected.

Adrenergic beta-Antagonists↗

PubMed1999

Automated in-tube solid-phase microextraction-liquid chromatography-electrospray ionization mass spectrometry for the determination of ranitidine.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was evaluated for the determination of ranitidine. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated aspirate/dispense steps. In order to optimize the extraction of ranitidine, several in-tube SPME parameters such as capillary column stationary phase, extraction pH and number and volume of aspirate/dispense steps were investigated. The optimum extraction conditions for ranitidine from aqueous samples were 10 aspirate/dispense steps of 30 microliters of sample in 25 mM Tris-HCl (pH 8.5) with an Omegawax 250 capillary column (60 cm x 0.25 mm I.D., 0.25 micron film thickness). The ranitidine extracted on the capillary column was easily desorbed with methanol, and then transported to the Supelcosil LC-CN column with the mobile phase methanol-2-propanol-5 M ammonium acetate (50:50:1). The ranitidine eluted from the column was determined by ESI-MS in selected ion monitoring mode. In-tube SPME followed by LC-ESI-MS was performed automatically using the HP 1100 autosampler. Each analysis required 16 min, and carryover of ranitidine in this system was below 1%. The calibration curve of ranitidine in the range of 5-1000 ng/ml was linear with a correlation coefficient of 0.9997 (n = 24), and a detection limit at a signal-to-noise ratio of three was ca. 1.4 ng/ml. The within-day and between-day variations in ranitidine analysis were 2.5 and 6.2% (n = 5), respectively. This method was also applied for the analyses of tablet and urine samples.

Anti-Ulcer Agents↗

PubMed1999

Determination of lead in blood and urine by SPME/GC.

Lead is the most frequently quantitated toxic metal in biological matrixes. In this paper, a method is described for lead determination in whole blood and urine using solid-phase microextraction (SPME) gas chromatography. Lead ion is first derivatized with sodium tetraethylborate to form tetraethyllead, which is then extracted from the headspace over the sample by SPME. The analytical procedure was optimized for coating selection, pH, extraction time, and effect of salt. The relative standard deviation was less then 10% for both urine and blood samples. The limit of detection was 3 and 4 ppb; the limit of quantification is 5 and 10 ppb for urine and blood samples, respectively. Good linearity was found for both urine and blood samples when PDMS coating was used. The standard addition method was used for quantitation. Certified urine and blood samples were analyzed, and good accuracy was obtained.

Borates↗

PubMed1999

Effect of salt concentration on separation patterns in static capillary isoelectric focusing with imaging detection.

Salts introduced into protein samples have an impact on the pH gradient in free solution in isoelectric focusing (IEF), which is reflected by the separation pattern. In this study, samples containing different concentrations of phosphate-buffered saline (PBS) were focused in capillary format and detected in a real-time mode using an imaged capillary isoelectric focusing (CIEF) system at 280 nm. It was observed that salt compressed the pH gradient with a degree of 4.3% at a PBS concentration interval of 10 mM. As a result, the same sample components, therefore, were focused at different positions inside the capillary. Using two pI markers as the internal standards, the separation patterns in the presence of salts were corrected to the salt-free matrix by simply stretching the electropherograms. The stretched electropherograms of model samples, pI markers and myoglobin, demonstrated the feasibility of this correction. This simple method is promising for identifying proteins, which may exhibit different pI values after their mutation and stability process, when salt is present in the sample.

Electrophoresis, Capillary↗

PubMed1999

Time-weighted average sampling with solid-phase microextraction device: implications for enhanced personal exposure monitoring to airborne pollutants.

The solid-phase microextraction (SPME) device is used as a time-weighted average (TWA) sampler for gas-phase analytes by retracting the coated fiber a known distance into its needle housing during the sampling period. Unlike in conventional spot sampling with SPME, the TWA sampling approach does not allow the analytes to reach equilibrium with the fiber coating, but rather they diffuse through the opening in the needle to the location of the sorbent. The amount of analytes accumulated over time gives the measurement of the average concentration to which the device was exposed to. Depending on the sorbent used as the sink, TWA sampling for various analytes is possible with times ranging from 15 min to at least 16 h. Both the poly(dimethylsiloxane) (PDMS) and poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber coating phases were tested, with the latter employing on-fiber derivatization for reactive carbonyl compounds, e.g., formaldehyde. Described herein are the theoretical and practical considerations for using the SPME device as a TWA sampler.

Air Pollutants, Occupational↗

PubMed1999

Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides.

Whole column imaging detection has been developed for capillary isoelectric focusing (CIEF) of proteins and peptides. In this imaged C1EF technique, a solution of sample and ampholytes was introduced into a short (4-5 cm), internally coated capillary stabilized by a cartridge. After applying high DC voltage, the isoelectric focusing process takes place and the focused zones are monitored in a real-time mode using the imaging detectors developed. Three types of imaging detectors have been developed including refractive index gradient, laser-induced fluorescence (LIF), and absorption. Of these, absorption imaging detection is the most practical at the present time due to its quantitative ability and universal characteristics. Whole column imaging detection eliminates the mobilization step required for single point detection after the focusing process. Therefore, it provides a fast analysis speed (3-5 min for each sample), and avoids the disadvantages associated with the mobilization process, such as distortion of pH gradient and loss in resolution. In this paper, we review the methodology of imaged CIEF as well as progress in instrumental development, IEF performed on a microchip, and the application to protein and peptide analysis.

Diagnostic Imaging↗

PubMed1999

Electrospray mass spectrometry of trimethyllead and triethyllead with in-tube solid phase microextraction sample introduction

A study of positive ionization electrospray mass spectrometry (ES-MS) was performed on trimethyllead (TML) and triethyllead (TEL). The system consisted of in-tube solid phase microextraction (SPME) coupled directly to an electrospray mass spectrometer. Fragmentation patterns of compounds were observed by applying different fragmentation voltages. High voltages produced sufficient fragmentation to elucidate the dissociation of the trialkyllead compounds. Electrospray mass spectrometry has been shown to be a suitable detection system for organolead speciation. Applying fragmentation energy programming, it might be possible to obtain in parallel the molecular and atomic signals of lead compounds. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗

PubMed1999

Determination of equilibrium constant of alkylbenzenes binding to bovine serum albumin by solid phase microextraction.

Solid phase microextraction (SPME) coupled with GC has been applied to study the binding properties between bovine serum albumin (BSA) and volatile organic compounds such as benzene, toluene, ethylbenzene, propylbenzene and butylbenzene. Their protein-ligand equilibrium constants have been determined. The measurement of free and bound ligand concentrations in the aqueous solution was based on the equilibrium among the analyte in the fiber coating (Cf), headspace (Ch) and aqueous solution (Cs). The work demonstrated that SPME is a simple and effective method in the study of protein binding to measure the freely dissolved analyte concentration as well as the equilibrium constant. The theoretical aspect of the SPME applied to the equilibrium constant measurement in two-phase (liquid sample-fiber coating) and three-phase (liquid sample-headspace-fiber coating) systems has been thoroughly discussed. The results demonstrated that the interpretation of the calibration data is crucial to the determination of freely dissolved analyte concentration and the equilibrium constant especially when the sample volume is small. The error in the experimental system is discussed. It is demonstrated in this study that for the three-phase system the amount of the analyte partitioned in the headspace could be ignored only in certain circumstances, where the Henry's law constant and the ratio between headspace volume and sample volume are sufficiently small.

Animals↗

PubMed1998

Recent developments in capillary isoelectric focusing with whole-column imaging detection.

Capillary isoelectric focusing (CIEF) is a high resolution technique for protein separation. The on-column single point detector requires a mobilization step which lengthens the analysis time and causes an uneven resolution along the separation column. The real time and whole column imaging detection has been developed for performing CIEF without mobilization. Three types of imaging detection systems have been developed: optical absorption, refractive index gradient, and laser induced fluorescence. This technique provides a fast analysis speed (about 6 min) and a good resolution of 0.03 pH unit level. Using the absorption imaging detector, ampholyte-free IEF in tapered capillary is being demonstrated, which eliminates the interference of the expensive carrier ampholytes for protein detection in UV region. Recent advancements in this imaged CIEF technique as well as its applications are reviewed.

Electrophoresis, Capillary↗

PubMed1997

Method optimization for the analysis of amphetamines in urine by solid-phase microextraction.

Solid-phase microextraction is under investigation in many laboratories for its usefulness in the analysis of an ever widening variety of compounds. As new classes of compounds are investigated and new challenges arise, the methods are adapted to accommodate them. Polar semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamine and methamphetamine were selected as semivolatiles exhibiting these limitations, and methods to optimize their analyses were investigated. Amphetamines are frequently monitored in very complex matrixes. Headspace methods minimize interactions between the sample and the fiber and have proven useful for these analyses. Several areas of experimental design were considered in the process of method optimization. These included matrix modification by heating, stirring, methanol content, addition of salt, and pH buffering. It was found that these amphetamines could be reliably analyzed using modified sample conditions, with excellent sensitivity, limits of detection, and method linearity. Clinical urine samples were successfully analyzed and gave clean chromatograms with no interfering peaks. Finally, the method developed was found to be useful for the analysis of narcotic analgesics. In the future, it is hoped that the method can be used to develop a general screen for a wide range of drugs of abuse.

Amphetamines↗

PubMed1997

Solid-phase microextraction for the analysis of human breath.

Solid-phase microextraction (SPME) has been applied to the quantitative determination of ethanol, acetone, and isoprene in human breath. The method involves extraction and preconcentration with a fused silica fiber coated with a polymeric stationary phase, desorption at 200 degrees C, and assay by gas chromatography/mass spectrometry. Three different fiber coatings have been evaluated with regard to sensitivity, linear range, precision, and detection limits. Typical RSD values in the range 2%-6% could be obtained, depending on the fiber coating and the compound investigated. The calibration curves for the compounds are reproducible and linear over the concentration ranges found in human breath samples. The method is capable of detecting concentrations of acetone and isoprene reported for healthy subjects. The influence of temperature and humidity on the extraction process has been studied in detail. A linear relationship between log K versus 1/T allows the calibration of the method for any given temperature. The device is portable, economical, and easy to use in patient sampling.

Acetone↗

PubMed1997

Effect of sample volume on quantitative analysis by solid-phase microextraction. Part 1. Theoretical considerations.

This paper discusses the effect of sample volume on the amount of analyte extracted from a sample by solid-phase microextraction (SPME) in two-phase (sample-fiber coating) and three-phase (sample-headspace-fiber coating) systems. Up-to-date knowledge is summarized, and new concepts are introduced. The effect of sample volume on quantification and precision of results can be neglected only in rare cases. The minimum sample volume which ensures that the amount extracted, n, is lower than 1% of the initial amount of the analyte present in the sample, as well as the volume for which exactly half of the initial amount of the analyte is extracted, have been calculated for both two- and three-phase systems. It is critical that the volumes of samples and standards are the same during analysis by SPME. Extraction kinetics in headspace analysis is dependent on the headspace capacity. If it is sufficiently large, the analyte is extracted almost exclusively from the gaseous phase, and equilibration can be very fast. On the other hand, this causes a significant loss of sensitivity. The effect of sample volume on the determination of the value of the partition coefficient, K, is also discussed. If the change in concentration of the analyte in the sample at equilibrium is not taken into account, erroneous results are obtained. Even when a proper procedure is used, there are practical limitations to the accuracy of the K value determination. Large sample volumes should always be used for K value determination, as they enable broader ranges of K values to be covered with good accuracy.

Chemical Phenomena↗

PubMed1996

Analysis of pesticides in environmental water samples by solid-phase micro-extraction-high-performance liquid chromatography.

Solid-phase micro-extraction (SPME) followed by high-performance liquid chromatography (LC) has been applied to analyze pesticides in water samples. A device interfaces SPME to the LC injection process by solvent extracting analytes from the fiber and then introducing the solvent into the LC injector. LC analysis with UV detection was performed first with a conventional column (4.6 mm I.D.). To enhance efficiency of SPME, three extraction conditions, stirring, temperature and salt concentration, were optimized. Subsequently, semi-microcolumn LC (1.5 mm I.D.) was evaluated for this method, giving lower detection limits and less solvent consumption. Detection limits were within Japanese regulatory limits for drinking water. The SPME-LC method was applied to real-world environmental water samples.

Acetonitriles↗

PubMed1996

Capillary-zone electrophoresis in agarose gels using absorption imaging detection.

A simple home-built electrophoretic unit and a commercially available charge-coupled device (CCD) camera with image acquisition and analysis software were used to study the separation process in zone electrophoresis experiments in 4 cm long, round capillaries (inside diameter 0.2 mm). Several capillaries could be investigated simultaneously. The absorption imaging system was used not only to follow the course of the separation but also to study the interaction between biologically active substances (proteins, detergents, enzymes and-substrate). Since the system allows visual on-line observation of the separation one can rapidly decide when the analysis is finished, which often shortens the analysis time. The electrophoresis method presented is suitable also for preparative runs, since direct visualizations of a solute zone allows it to be excised and then used for further studies.

Electrophoresis, Agar Gel↗

PubMed1995

Protein analysis by isoelectric focusing in a capillary array with an absorption imaging detector.

Isoelectric focusing (IEF) was successfully performed in capillary arrays with up to four capillaries. Separated proteins in the capillary array were detected by an UV absorption imaging detector. The whole analysis time for all samples in the capillary array was only 3 min due to the real-time imaging detector. The instrument was applied to analyse several protein samples including different human hemoglobin variants, myoglobin, transferrin, carbonic anhydrase and a monoclonal antibody to fluorescein. Because of good reproducibility of the focused pattern, unknown samples can be run simultaneously with a standard in the multichannel instrument and the components of unknown samples can be identified by comparing their zone positions to those of the standard. Minor components can be determined by the instrument in the presence of major components with 100 times higher concentrations in human hemoglobin samples. This instrument could be a powerful analytical tool for clinical analysis and for quality control in pharmaceutical companies.

Antibodies, Monoclonal↗