Biomedical subjects

Janusz Pawliszyn

Publications and source records attributed to Janusz Pawliszyn.

At least 37 records · Page 2Linked to original sources

PubMed2006

Capillary isoelectric focusing coupled with dynamic imaging detection: A one-dimensional separation for two-dimensional protein characterization.

Here, we devised a novel approach for two-dimensional (2D) protein characterization using a single one-dimensional separation followed by a second characterization in the same instrument. The approach combines capillary isoelectric focusing (CIEF), which separates proteins according to pI, with dynamic imaging detection, which permits monitoring of protein diffusion in real time and thereby allows estimation of molecular weight from diffusion coefficient. Compared with classical 2D gel electrophoresis, the approach provided several significant advantages including speed and ease in operation and automation, while yielding comparable accuracy. The approach was applicable for protein samples of low to moderate complexity.

Cytochromes c↗

PubMed2006

Analytical microextraction: current status and future trends.

Analytical microextractions, defined as nonexhaustive sample preparation with a very small volume of extracting phase (microliter range or smaller) relative to the sample volume, represent an important development in the field of analytical chemistry. Analytes are extracted by a small volume of a solid or semi-solid polymeric material, as in solid-phase microextraction (SPME), or alternatively by a small volume of a liquid, as in liquid-phase microextraction (LPME). This paper gives an overview of the SPME and LPME techniques and discusses future trends. This includes a discussion of the different extraction formats available, commercial equipment, method transfer from traditional sample preparation methods to microextraction, and performance as well as robustness for the latter type of systems. In addition, the paper contains a unified approach to the understanding of extraction thermodynamics and kinetics applicable to both SPME and LPME.

Chemical Fractionation↗

PubMed2006

Field sampling with a polydimethylsiloxane thin-film.

In this research, field samplers are developed using polydimethylsiloxane (PDMS) thin-film as the extraction phase. This technique is based on a similar theory, the solid-phase microextraction (SPME) technique. More specifically, the development of the field sampler involves cutting a section of PDMS thin-film into a specific size and shape, and mounting it onto a stainless steel wire (the handle). The thin-film is then placed into a protective copper cage prior to deployment to prevent biofouling. Kinetic calibration or equilibrium calibration with the standards in the extraction phase is used to introduce an isotopically labeled internal standard for on-site calibration. The initial loading of the standard onto the thin-film and the amount of standard remaining on the thin-film are determined using gas chromatography-mass spectrometry and subsequently used to estimate the concentration of the target analytes. In addition, the field samplers are deployed in the field at two locations (the Meuse River in Eijsden, The Netherlands from April to May, 2005 and Hamilton Harbour located at the western tip of Lake Ontario, ON, Canada from September to December, 2006). Polycyclic aromatic hydrocarbons are identified, and concentrations of fluoranthene and pyrene are estimated in the low ng/L range. The results from both sampling sites are within the expected ranges for environmental samples. This polymeric extraction phase has a high surface-to-volume ratio compared with SPME, which results in higher sensitivity and mass uptake, leading to the detection of lower levels of analytes that many other techniques are unable to achieve.

Journal Article↗

PubMed2006

Online coupling of solid-phase microextraction and capillary electrophoresis.

Solid-phase microextraction (SPME) and capillary electrophoresis (CE) are two of the main inventions that shaped 20th Century analytical chemistry. SPME is an effective microscale sampling and sample preparation technique, and CE is a high-efficiency microanalytical method. Online coupling of SPME with CE can be a powerful combination because of the significant advantages of the two techniques. The progress in the development of online SPME-CE coupling is surveyed in this review. Problems encountered and solutions reported are highlighted.

Journal Article↗

PubMed2006

Determination of free concentration of Paclitaxel in liposome formulation.

PURPOSE: An important step in the development of liposome-based formulations is estimating the free drug concentration in the aqueous solution surrounding liposomes. This research presents a new method for determination of free concentrations, based on membrane-protected solid-phase microextraction (SPME). METHODS: For effective direct extraction of low molecular weight compounds from complex liquid samples, a hollow membrane was used to form a concentric sheath around a coated SPME fiber. The membrane blocked the access of large particles, like liposomes, to the coating surface, while target analytes with low molecular weight diffused through the membrane and reached the extraction phase. Quantification was conveniently performed by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry. RESULTS: The carbowax/templated resin SPME fiber was determined to be the most suitable for these assays, providing enough sensitivity when an extraction time of one hour was used. The free concentration of paclitaxel was found to be 0.36 microg/mL, significantly below the solubility limit of paclitaxel in water. CONCLUSION: The method was successfully applied for determining free paclitaxel in liposome formulations based on dioleyl-trimethyl-ammonium-propane, with good linearity over the range of concentrations of interest. The method was faster and more practical than equilibrium dialysis, as the SPME approach provided preconcentration and convenient delivery to the analytical system.

Antineoplastic Agents↗

PubMed2005

Characterization of phospholipid-protein interactions by capillary isoelectric focusing with whole-column imaging detection.

The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid-protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid-protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (pI) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids-protein interactions by monitoring the protein cIEF profiles. Seven proteins with different pI and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid-protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC-protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids-protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid-protein interactions.

Animals↗

PubMed2005

High-performance SPME/AP MALDI system for high-throughput sampling and determination of peptides.

This paper presents the performance characteristics for a new multiplexed solid-phase microextraction/atmospheric pressure matrix-assisted laser desorption/ionization (SPME/AP MALDI) source configuration for a hybrid quadrupole-linear ion trap instrument. The results demonstrate that thorough optimization of parameters such as SPME coating material, optics configurations, extraction solvents, and fiber capacity provides dramatic sensitivity improvements (>1000x) over previous reports in the literature. The multiplexed SPME plate is capable of simultaneous extraction from 16 different wells on a multiwell plate, eliminating the need for extensive sample preparation. Subfemtomole sensitivity is demonstrated for peptide standards and protein digests with run-run reproducibility ranging from approximately 13 to 31%. This high-performance SPME/AP MALDI system shows potential for high-throughput extraction from biological samples.

Angiotensin I↗

PubMed2005

Kinetic calibration for automated headspace liquid-phase microextraction.

The kinetics of the absorption and desorption of analytes for headspace liquid-phase microextraction (HS-LPME) were studied. It was found that the desorption of analytes from the extraction phase into the sample matrix is isotropic to the absorption of the analytes from the sample matrix into the extraction phase under the same conditions. This therefore allows for the calibration of absorption using desorption. Calibration was accomplished by exposing the extraction phase, which contained a standard, to the sample matrix. The information from the desorption of the standard, such as time constant a, could be directly used to estimate the concentration of the target analyte in the sample matrix. This new kinetic calibration method for headspace LPME was successfully used to correct the matrix effects in the BTEX analysis of an orange juice sample. In this study, the headspace LPME techniques were successfully fully automated, for both static and dynamic methods, with the CTC CombiPal autosampler. All operations of headspace LPME, including sample transfer and agitation, filling of extraction solvent, exposing the solvent in the headspace, withdrawing the solvent to syringe and introducing the extraction phase into injector, were autoperformed by the CTC autosampler. The fully automated headspace LPME technique is more convenient and improved the precision and sensitivity of the method. This automated dynamic headspace LPME technique can be also used to obtain the distribution coefficient between the sample matrix (aqueous or another solution) and the extraction phase (1-octanol or another solvent). The distribution coefficient between 1-octanol and orange juice, at 25 degrees C, was obtained with this technique.

Adsorption↗

PubMed2005

Time-weighted average water sampling with a solid-phase microextraction device.

A fiber-in-needle SPME device was developed and investigated for time-weighted average water sampling. The device was designed so that the overall mass-transfer resistance is contained within the static water inside the needle, which ensures that mass uptake could be predicted with Fick's first law of diffusion and the sampling rate is less affected by water turbulence. The device possesses all of the advantages of commercialized devices, in addition to needle filling and replacement ease. Laboratory calibration with deployment of the device to a flow-through system demonstrated that there was a linear mass uptake for up to 12 days, and the linear range could be longer. PDMS coating is assumed to be a perfect zero sink for most polycyclic aromatic hydrocarbons, except naphthalene. The effect of water temperature was also investigated. Under normal field conditions, the change of mass uptake rate with temperature was negligible. To facilitate the convenience for long-term water sampling, a new standard aqueous generator was introduced. This study extended the application of SPME technology for long-term water sampling.

Journal Article↗

PubMed2005

Flow-through system for the generation of standard aqueous solution of polycyclic aromatic hydrocarbons.

Standard aqueous solution of organic pollutants is used for the development of analysis methods and novel water sampling devices. The use of solid-phase microextraction (SPME) and other novel technologies for research in water sampling and analysis requires systems for the reliable generation of standard aqueous solution. In this communication, a new flow-through system based on permeation is introduced for the generation of standard aqueous solution of polycyclic aromatic hydrocarbons (PAHs). The main difference between the new developed system and typical system is that the syringe pump was changed with a permeation generator. The generator-based permeation offers many advantages, including convenient, inexpensive, solvent-free, long lifetime and production of high concentrations of the target analytes. The observed change in the concentration of the six PAH compounds was less than 20% over 3 months.

Dialysis↗

PubMed2005

Characterization of bovine serum albumin-tryptophan interaction by capillary isoelectric focusing with whole column imaging detection.

Capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID) was explored for the characterization of bovine serum albumin (BSA)-tryptophan interaction, to further understand protein-drug interactions. The BSA-tryptophan interaction was dynamically monitored by cIEF-WCID, to provide the cIEF profiles of the BSA-tryptophan interaction system at different focusing times. Our study demonstrated that the cIEF behavior of BSA can serve as a probe into the study of BSA-tryptophan interaction, through monitoring the change in its cIEF profile when the interaction occurred. The study illustrated that the BSA peak split due to the BSA-tryptophan interaction, and the peak of BSA-tryptophan complex was clearly identified in the cIEF electropherograms. By comparing the cIEF profiles of BSA/L-tryptophan and BSA/D-tryptophan, respectively, our study demonstrated that BSA interacted with the enantiomers of tryptophan with a chiral recognition. L-Tryptophan demonstrated a very strong interaction with BSA, while D-tryptophan exhibited a much weaker interaction with BSA. The effects of the BSA concentration, the tryptophan concentration, the focusing time and the incubation time on the BSA-tryptophan interaction were investigated. This study offers a novel approach for the study of protein-drug interactions.

Animals↗

PubMed2005

Calibration of solid-phase micro-extraction for quantitative analysis by gas chromatography.

Calibration of SPME by the injection of liquid standard solution is a common method for SPME quantitative analysis by gas chromatography. The feasibility of this calibration method relies on the assumption that sample transfer efficiencies are the same for both the liquid injection and the SPME fiber injection. Sample transfer efficiencies for liquid injection and SPME fiber injection were studied in this paper. The results showed that the sample transfer efficiency for liquid injection was affected by several factors, such as the dimensions of the liner, the presence of the wool, and the temperature of the injector. The sample transfer efficiency for SPME fiber injection was affected by the cross-sectional area of the space between the column and the liner, the carrier gas flow rate, and the length of the column inside the liner. An equation was proposed to estimate the sample loss rate for SPME injection. It was found that the use of a direct injection (DI) liner and program temperature vaporizing provides high sample transfer efficiencies, for both liquid injection and SPME fiber injection. When a common SPME straight liner is used, large outer diameter (o.d.) pre-column will help to achieve high sample transfer efficiency.

Acenaphthenes↗

PubMed2005

Sampling and determination of volatile organic compounds with needle trap devices.

In this study, a sorbent was immobilized inside a needle resulting in the development of a needle trap (NT) device. This device was applied to extract organic components from gaseous samples and to introduce an enriched mixture into a conventional gas chromatography (GC) injector. Construction of this simple and integrated sampling/extraction/sample introduction device was optimized by considering different ways to immobilize a sorbent in the needle, packing single and multiple-layer sorbent beds, and applying different desorption strategies into the GC injector. A carrier gas system was modified to minimize the carryover for the needle trap with a sealed tip (NT-1), and a narrow-neckliner was used for the blunt-tip needle trap (NT-2). Breakthrough in the device was investigated by connecting two NT-2 devices in series. The needle trap performed very well as an exhaustive spot sampler, as well as in a time-weighted average (TWA) operation. The linear velocity of the mobile phase has no influence on the sampling rate of the needle trap. Validation results against the standard NIOSH 1501 method using charcoal tubes for indoor air surveys demonstrated good accuracy for the NT approach. The reproducibility of the NT-2 was about 1% for benzene. The detection limits for FID detection and for 25 ml gas sample were 0.23 ng/l, 2.10 ng/l and 1.12 ng/l for benzene, ethylbenzene and o-xylene, respectively.

Alkanes↗

PubMed2005

Equilibrium in-fibre standardisation technique for solid-phase microextraction.

This note describes a fundamental investigation into solid-phase microextraction (SPME) using a standard loaded into the fibre coating as a means of internal standardisation for the analysis of samples contained in vials. The loading of reproducible amounts of standards into a non-porous SPME fiber was investigated. It was found that spiking low milligram quantities of standards such as benzene, toluene, ethylbenzene, xylenes (BTEX) and/or naphthalene into a few grams of pump oil sealed in a 20 mL vial provided an excellent standard generator. A single solution allowed over a hundred standard loadings with a reproducibility of <4% R.S.D. When a fiber, loaded with the standard(s) was introduced into a sample vial, extraction of analytes into the fiber and desorption of the standard(s) into the sample matrix occur simultaneously. Quantification was then based on the equilibrium distribution of the standards and the analytes between the fibre coating and the sample matrix in the vial. A comparison of equilibration profiles obtained using traditional internal standardisation and the in-fibre approach generally showed the same equilibration behaviour. The developed method was successfully used to correct for matrix effects in the BTEX analysis of a wine sample.

Automation↗

PubMed2005

Determination of low-molecular mass aldehydes by automated headspace solid-phase microextraction with in-fibre derivatisation.

Headspace solid-phase microextraction (HS-SPME) analysis of low-molecular mass (C1-C10) aldehydes in aqueous solutions was investigated, using pentafluorophenylhydrazine (PFPH) and o-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) as in-fibre derivatisation reagents. Analysis of the derivatives was achieved, using GC-flame ionisation detection (FID). A comparison of the two reagents showed that PFBHA was superior to PFPH under the investigated conditions. Fundamental studies of the PFBHA and PFPH reactions showed that the kinetics of the process was limited by the mass transport rate of the analytes to the fibre. The developed PFBHA method gave detection limits in the low to sub-microgram per litre range for most of the aldehydes tested. The method was applied successfully to the analysis of particleboard, wine and fish samples.

Aldehydes↗

PubMed2005

Approaches for coupling solid-phase microextraction to nanospray.

Biocompatible solid-phase microextraction (SPME) devices were prepared using two restricted access materials (RAM) as the SPME coating. The restricted access materials were immobilized on steel and platinum wires. The selective coating eliminated most of the matrix interference, which allowed the coupling to mass spectrometry without further purification. The SPME devices were interfaced to mass spectrometry by electronanospray. Several experimental set-ups are described and discussed herein. For the in situ extraction of peptides from the tryptic digests, trypsin was immobilized both on steel wires and on the inside wall of a vial. The devices were incubated together with the RAM-SPME devices and a protein (casein) solution. After the protein digestion, the resulting peptides were analyzed by SPME/nanospray. The vial approach provided the best results; up to eight peptides could be identified which corresponds to a sequence coverage of 58%. The limit of detection of SPME/nanospray for the extraction of peptides from an aqueous solution was about 50 fmol/mL. The results demonstrate that the direct coupling of SPME to nanospray can reduce analysis time and is an attractive alternative to conventional approaches like Zip-Tip purification.

Amino Acid Sequence↗

PubMed2005

Applications of capillary isoelectric focusing with liquid-core waveguide laser-induced fluorescence whole-column imaging detection.

Capillary isoelectric focusing (CIEF) with liquid-core waveguide (LCW) laser-induced fluorescence (LIF) whole-column imaging detection (WCID) is a recently developed high-resolution, high-sensitivity, and high-speed analytical tool for protein analysis. Several potential applications of this system were demonstrated in this study. First, this system was employed to separate naturally fluorescent phycobiliproteins. Second, denaturing CIEF was suggested to study the conformational and chemical microheterogeneity and to characterize proteins with identical pI values. Third, a modified noncovalent fluorescent labeling procedure was presented, which allows the simple and effective labeling of proteins, antibodies, and viruses with reduced multiple labeling and preserved activity. Finally, extracellular proteins were suggested as signaling biomarkers for evaluation of cell viability. The separation of cyanobacteria and their extracellular phycoerythrins was demonstrated. The effectiveness of CIEF-LCW-LIF-WCID for the analysis of proteins, antibodies, viruses, and cells has been illustrated.

Antibodies↗

PubMed2005

Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis.

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.

Fluorescence↗