ABSTRACT
Biologically active compounds constitute a wide group of chemicals, therefore it is a big challenge to create sorbents that are sensitive as well as selective. Development of nanoporous sorbents based on conducting polymers has expanded the boundaries of detection and quantification. Additionally, electrochemical synthesis used to deposit polymeric coatings directly on solid supports makes it possible to control physico-chemical properties of such sorbents. Besides the development of new polymeric nanoporous materials, the question of selectivity needs to be addressed. One possibility, successfully adapted to solid-phase microextraction, is molecular imprinting. Coatings created with this technology allow obtaining higher selectivity, with sensitivity at a constantly high level. The main aim of this review is to present comprehensively the concept of nanoporous sorbents based on conducting polymers, possible coating methods with their characteristics, and their various applications. This article focuses on applications in environmental and biomedical analyses.
Subject(s)
Biomedical Research/methods , Environmental Monitoring/methods , Nanoparticles/chemistry , Polymers/chemistry , Solid Phase Microextraction/methods , Particle Size , Porosity , Solid Phase Microextraction/instrumentation , Surface PropertiesABSTRACT
BACKGROUND: Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. METHODS: To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. RESULTS: We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. CONCLUSIONS: This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.
ABSTRACT
Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery.
Subject(s)
Proteomics , Urine/chemistry , Chromatography, Liquid/methods , Freeze Drying , Mass Spectrometry/methods , Proteinuria/urine , Ultracentrifugation , UltrafiltrationABSTRACT
ABSTRACT: Polymeric polypyrrole and polythiophene solid phase microextraction (SPME) coatings were prepared using electropolymerization with a linear sweep voltammetry technique. Physicochemical properties were measured using different methods, in particular small angle X-ray scattering and scanning electron microscopy. By using innovative approaches for pore size measurement, we were able to calculate a maximum of the pore size range from 80 to 90 nm. Additionally, film thicknesses measured from 90 to 150 µm. Using scanning electron microscopy, we describe the characteristics of polymer growth on the support surface.
ABSTRACT
Pressure-assisted digestion of proteins, also known as pressure cycling technology (PCT), using a Barocycler NEP 2320 was compared with the conventional method using atmospheric pressure. Our objective was to demonstrate that PCT provides more controlled enzymatic digestion of proteins than prolonged digestion at atmospheric pressure ranging from 18 to 24 h. More controlled digestion would be beneficial for studies of highly posttranslationally modified protein such as histones. For the comparison of these two techniques, recombinant and native histone H4 were used as model proteins. PCT was optimized for pressure and time, and it was found to be most effective at 15 kpsi for 120 min of incubation. In conclusion, the PCT method was found to be much faster than using atmospheric pressure. PCT was also found to allow for unambiguous control of digestion parameters and to provide a high yield of sequence coverage compared with atmospheric pressure.
Subject(s)
Analytic Sample Preparation Methods/methods , Pressure , Proteomics/methods , Amino Acid Sequence , Chromatography, High Pressure Liquid , Chymotrypsin/metabolism , Histones/metabolism , Humans , Molecular Sequence DataABSTRACT
Extraction techniques for 4,4'-methylenebis(2-chloroaniline) (MOCA) in air samples and water solutions were developed and compared. Classic techniques for air sampling of MOCA were enhanced by incorporating a derivatization step (3,5-dinitrobenzoyl chloride solution in toluene), thus increasing the limit of detection and limit of quantification. Sampling of MOCA from water solution was performed using novel nanoporous polymeric (polypyrrole and polythiophene) fiber coatings and solid phase microextraction. Samples were analyzed by high-performance liquid chromatography coupled with a UV detector. Using the modified method for air sampling of MOCA, we found that the limit of detection was 7.90 ng m(-3) and the limit of quantification was 23.8 ng m(-3). In contrast, the limit of detection for MOCA in water samples was 11.26 ng mL(-1) (polypyrrole) and 84.62 ng mL(-1) (polythiophene) and the limit of quantification for MOCA was from 33.78 (polypyrrole) and 253.86 ng mL(-1) (polythiophene). Correlation coefficients were 0.9997 for air and 0.8790-0.9852 for water samples, respectively. The techniques presented provide alternative methods for the determination of MOCA in air samples and in water solutions that are more sensitive, quicker and less expensive than previously established procedures.
Subject(s)
Air/analysis , Methylenebis(chloroaniline)/analysis , Methylenebis(chloroaniline)/isolation & purification , Solid Phase Microextraction/methods , Water/chemistry , Polymers/chemistry , Pyrroles/chemistry , Solutions , Solvents/chemistry , Thiophenes/chemistryABSTRACT
Lyme borreliosis (LB) is one of the most common tick-borne diseases in the northern hemisphere. It is a chronic inflammatory disease caused by the spirochaete Borrelia burgdorferi. In its early stages, pathological skin lesions, namely erythema chronicum migrans, appear. The lesions, usually localised at the site of the bite, may become visible from a few weeks up to 3 months after the infection. Predominant clinical symptoms of the disease also involve joint malfunctions and neurological or cardiac disorders. Lyme disease, in all its stages, may be successfully treated with antibiotics. The best results, however, are obtained in its early stages. In order to diagnose the disease, numerous medical or laboratory techniques have been developed. They are applied to confirm the presence of intact spirochaetes or spirochaete components such as DNA or proteins in tick vectors, reservoir hosts or patients. The methods used for the determination of LB biomarkers have also been reviewed. These biomarkers are formed during the lipid peroxidation process. The formation of peroxidation products generated by human organisms is directly associated with oxidative stress. Apart from aldehydes (malondialdehyde and 4-hydroxy-2-nonenal), many other unsaturated components such as isoprostenes and neuroprostane are obtained. The fast determination of these compounds in encephalic fluid, urine or plasma, especially in early stages of the disease, enables its treatment. Various analytical techniques which allow the determination of the aforementioned biomarkers have been reported. These include spectrophotometry as well as liquid and gas chromatography. The analytical procedure also requires the application of a derivatization step by the use of selected reagents.
Subject(s)
Biomarkers/analysis , Lyme Disease/diagnosis , Humans , Lyme Disease/microbiologyABSTRACT
The polypyrrole (PPy) and polythiophene (PTh) solid phase microextraction (SPME) coatings were obtained with the use of the electropolymerisation and linear sweep voltammetry. Such fibers were modified by an ozone treatment in a gaseous phase in the concentration of 2.1 ± 0.2 × 10(-5) mol dm(-3). Both kinds of fibers were applied in the microextraction of linezolid from standard solutions to compare the extraction efficiencies displayed by these sorption phases. In these investigations a better adsorption capacity was obtained for polypyrrole fibers and hence only these kinds of fibers were utilized in the measurements from human plasma. In all measurements the concentrations of the drugs were in the range from 1 to 20 µg ml(-1) (standard solutions) and 1 to 15 µg ml(-1) (human plasma). Before the measurements, an optimization of the desorption solution experiments was performed. The correlation coefficients (R) obtained in the standard solution and human plasma were in the range from 0.8399 to 0.9970. The relative standard deviations (RSDs) were in the range of 0.1-7.6%.
Subject(s)
Anti-Infective Agents/analysis , Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Solid Phase Microextraction/instrumentation , Acetamides/analysis , Humans , Linear Models , Linezolid , Models, Chemical , Oxazolidinones/analysis , Ozone , Polymers/chemistry , Pyrroles/chemistry , Reproducibility of Results , Thiophenes/chemistryABSTRACT
A novel sorbent in solid phase microextraction (SPME) method based on poly(3-alkylthiophenes) was used in the isolation of linezolid from human plasma samples following liquid chromatography determination. The effect of extraction time on the sorption capacity of the SPME process was studied and pointed at 10 min both for adsorption and desorption. Poly(3-methylthiophene) and poly(3-nonylthiophene) were applied for the extraction of linezolid from water solutions. In plasma samples, four coatings including polythiophene and poly(3-penthylthiophene) were investigated. With these measurements, correlation coefficients were calculated in the range from 0.9820 to 0.9995, and the relative standard deviations were below 15%. That allowed claiming that the synthesized and described materials can be successfully applied in the analysis of linezolid also from other matrices such as urine or blood.
Subject(s)
Acetamides/chemistry , Materials Testing , Oxazolidinones/chemistry , Oxazolone/analogs & derivatives , Polymers/chemistry , Solid Phase Microextraction/methods , Thiophenes/chemistry , Acetamides/blood , Adsorption , Chromatography, Liquid , Humans , Linezolid , Microscopy, Electron, Scanning , Molecular Structure , Oxazolidinones/blood , Oxazolone/blood , Oxazolone/chemistry , Stainless SteelABSTRACT
A new approach to the rapid determination of amoxicillin (AMO) in human plasma followed by solid phase microextraction (SPME) fiber coatings based on conducting polymers (polypyrrole and polythiophene) and high performance liquid chromatography (HPLC) has been described. The porous structures of the electrochemically deposited polymer coatings have been characterized by scanning electron microscopy (SEM). The experimental parameters relating to the extraction efficiency of the SPME fibers such as pH, extraction time and desorption conditions (solvents, time) were studied and selected. The SPME/HPLC-UV method was linear over a working range of 1-50 µg ml(-1). The inter-day accuracy (expressed as coefficients of variations, CVs) was less than 15% and precision (expressed as the relative standard deviations, RSDs) with percentage values was less than 5.9%. Amoxicillin was found to be stable in the human plasma at room temperature (20 °C) within 8 hours. The developed method was successfully applied to the analysis of real human plasma samples. The limit of detection and limit of quantification for amoxicillin in plasma were 1.21 µg ml(-1) and 3.48 µg ml(-1), respectively.
Subject(s)
Amoxicillin/blood , Anti-Bacterial Agents/blood , Chromatography, High Pressure Liquid/methods , Solid Phase Microextraction/methods , Amoxicillin/isolation & purification , Anti-Bacterial Agents/isolation & purification , Humans , Polymers/chemistry , Pyrroles/chemistry , Thiophenes/chemistryABSTRACT
Polythiophene (PTh) and polypyrrole (PPy) as sorbent phases for solid phase microextraction (SPME) were applied in order to extract the multi-resistant Staphylococcus aureus (MRSA) antibiotic drugs (linezolid and daptomycin) from whole blood followed by high performance liquid chromatography (HPLC) determination with UV detection. Relative standard deviations (RSDs) of in vitro and pseudo in vivo measurements performed in whole blood were in the range of 4.58-15.91% and 6.09-17.33% for linezolid and daptomycin, respectively. Determination coefficients (R(2)) were in range of 0.9884-0.9945 and 0.9807-0.9818 for linezolid and daptomycin, respectively. This study proved better adsorption capacity of PTh SPME coating compared to PPy coating for both, linezolid and daptomycin.
Subject(s)
Acetamides/blood , Anti-Bacterial Agents/blood , Chromatography, High Pressure Liquid/methods , Daptomycin/blood , Oxazolidinones/blood , Solid Phase Microextraction/methods , Humans , Linezolid , Polymers/chemistry , Pyrroles/chemistry , Thiophenes/chemistryABSTRACT
In this study, polypyrrole (PPy) and polythiophene (PTh) SPME coatings and their ability to extract selected adrenolytic drugs with different physico-chemical properties from standard solutions and human plasma samples were evaluated. In measurements metoprolol, oxprenolol, mexiletine, propranolol, and propaphenon were investigated. The main parameters such as extraction time, desorption conditions and pH influence were examined. Inter-day precisions were in range 0.1-2.0%, 1.1-2.9%, 1.3-2.6%, 0.1-2.6% and 0.3-2.1% for metoprolol, oxprenolol, mexiletine, propranolol and propaphenon, respectively. Accuracies were less than 15%, which was evaluated by analyzing preparation samples of five replicates. The method was successfully applied to human plasma samples spiked with selected adrenolytic drugs. The method was linear in the concentration range from 1 to 10microg/ml for all of studied adrenolytic drugs using human plasma samples. The PTh-SPME coating displayed higher extraction efficiency towards the target analytes in comparison to PPy-SPME. The reproducibility of the extraction using polypyrrole and polythiophene fibers was confirmed by variation coefficients lower than 8% and 3%, respectively.
Subject(s)
Adrenergic Antagonists/blood , Chromatography, High Pressure Liquid/methods , Polymers/chemistry , Pyrroles/chemistry , Solid Phase Microextraction/methods , Thiophenes/chemistry , Calibration , Humans , Hydrogen-Ion Concentration , Linear Models , Propanolamines/blood , Reproducibility of ResultsABSTRACT
Simple or even rapid bioanalytical methods are rare, since they generally involve complicated, time-consuming sample preparation from the biological matrices like LLE or SPE. SPME provides a promising approach to overcome these limitations. The full potential of this innovative technique for medical diagnostics, pharmacotherapy or biochemistry has not been tapped yet. In-house manufactured SPME probes with polypyrrole (PPy) coating were evaluated using three antibiotics of high clinical relevance - linezolid, daptomycin, and moxifloxacin - from PBS, plasma, and whole blood. The PPy coating was characterised by scanning electron microscopy. Influences of pH, inorganic salt, and blood anticoagulants were studied for optimum performance. Extraction yields were determined from stagnant media as well as re-circulating human blood using the heart-and-lung machine model system. The PPy-SPME fibres showed high extraction yields, particularly regarding linezolid. The reproducibility of the method was optimised to achieve RSDs of 9% or 17% and 7% for SPME from stagnant or re-circulating blood using fresh and re-used fibres, respectively. The PPy-SPME approach was demonstrated to meet the requirements of therapeutic monitoring of the drugs tested, even from re-circulating blood at physiological flow rates. SPME represents a rapid and simple dual-step procedure with potency to significantly reduce the effort and expenditure of complicated sample preparations in biomedical analysis.
Subject(s)
Anti-Bacterial Agents/analysis , Polymers/chemistry , Pyrroles/chemistry , Solid Phase Microextraction/methods , Acetamides/analysis , Acetamides/blood , Acetamides/isolation & purification , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/isolation & purification , Anticoagulants/chemistry , Aza Compounds/analysis , Aza Compounds/blood , Aza Compounds/isolation & purification , Chromatography, High Pressure Liquid , Daptomycin/analysis , Daptomycin/blood , Daptomycin/isolation & purification , Fluoroquinolones , Humans , Hydrogen-Ion Concentration , Linezolid , Moxifloxacin , Oxazolidinones/analysis , Oxazolidinones/blood , Oxazolidinones/isolation & purification , Quinolines/analysis , Quinolines/blood , Quinolines/isolation & purification , Salts/chemistryABSTRACT
Five adrenolytic drugs have been analyzed by liquid chromatography-mass spectrometry (LC-MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5-13%, however RSD values for the drugs in human plasma were 2.5-4.5%. Using LC-MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11-0.18 and 0.39-0.54 ng mL(-1), respectively, for the five drugs.
Subject(s)
Adrenergic Antagonists/blood , Chromatography, High Pressure Liquid/methods , Solid Phase Microextraction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Humans , Limit of DetectionABSTRACT
The aim of this study was the preparation of polypyrrole (PPy) fibers for solid phase microextraction (SPME). PPy coatings were obtained during the electrochemical polymerization process. The utility of various metal wires (Fe, Cu, Ag, Cu/Ag, kanthal and medical stainless steel) as a support for polymers was compared. Various experimental conditions of the synthesis process such as scan rate, voltage limits and number of scans and deposition time were applied. The average polymer thickness was in the range of 7-125 microm and its weight was in the scope of 0.65-5.6 mg. Different techniques, mainly elemental analysis, Fourier transform infrared spectroscopy, microscopy, and chromatography were performed for the characterization of obtained fibers with microporous structure. The extraction efficiency of cardiovascular drugs (metoprolol, propranolol, oxprenolol, propafenone and mexiletine) by means of fibers was tested. The concentration of mentioned compounds in standard solution was in the span of 10-150 ng/mL. LC-MS was employed for determination of drugs in desorption solution. LODs varied from 0.013 to 1.51 ng/mL for metoprolol and mexiletine respectively. The repeatability of extraction was obtained with the RSD values lower than 10%.