Marek Jackowski

Identification of volatile organic compounds secreted from cancer tissues and bacterial cultures

The early cancer diagnosis increases the possibility of total recovery. The infection of Helicobacter pylori is associated with gastric cancer, the second most common cancer in the world. The determination of volatile organic compounds (VOCs) excreted by stomach tissue and bacteria culture has been investigated. Solid-phase microextraction (SPME) was used for preconcentration and the determination was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The samples of tissue were taken from five patients (ten samples) with stomach cancer and normal (non-cancerous) segments from other parts of the stomach were used as a control. Eighteen compounds were identified in stomach tissue and seven of them were present both in healthy and cancer tissue. These compounds assumed to be endogenous and acetone ratio (AR) was calculated for ethanol, butane, carbon disulfide, 1-propanol, 2-butanone and 2-pentanone. The data shows that amount of 1-propanol and carbon disulfide in the gaseous composition is higher in cancer tissue than in normal tissue. Eight compounds were identified both in bacteria and tissue. These data suggest that bacteria present in the stomach might cause the increase in the concentration of 1-propanol and carbon disulfide in emission from cancer tissue.

Determination of volatile organic compounds in human breath for Helicobacter pylori detection by SPME‐GC/MS

Helicobacter pylori living in the human stomach release volatile organic compounds (VOCs) that can be detected in expired air. The aim of the study was the application of breath analysis for bacteria detection. It was accomplished by determination of VOCs characteristic for patients with H. pylori and the analysis of gases released by bacteria in suspension. Solid-phase microextraction was applied as a selective technique for preconcentration and isolation of analytes. Gas chromatography coupled with mass spectrometry was used for the separation and identification of volatile analytes in breath samples and bacterial headspace. For data calculation and processing, discriminant and factor analyses were used. Endogenous substances such as isobutane, 2-butanone and ethyl acetate were detected in the breath of persons with H. pylori in the stomach and in the gaseous mixture released by the bacteria strain but they were not identified in the breath of healthy volunteers. The canonical analysis of discrimination functions showed a strong difference between the three examined groups. Knowledge of substances emitted by H. pylori with the application of an optimized breath analysis method might become a very useful tool for noninvasive detection of this bacterium.

Preliminary study of volatile organic compounds from breath and stomach tissue by means of solid phase microextraction and gas chromatography-mass spectrometry.

The determination of volatile organic compounds (VOCs) in exhaled air and stomach tissue emission for the detection of cancer has been investigated. Solid phase microextraction (SPME) was used for sample preconcentration. The method presented in this paper showed satisfactory precision (RSD below 11%), linearity in the range of 2.8-136 ppb and limit of detection ranging from 0.6 to 2.1 ppb. The breath and emission from cancer tissue were collected from three patients with stomach cancer. Acetone, carbon disulfide, 2-propanol, ethyl alcohol and ethyl acetate were identified in breath and tissue samples. These compounds have been assumed as endogenous. Acetone ratio (AR) was calculated for carbon disulfide, 2-propanol and n-butane. The AR for carbon disulfide was found to be higher for normal tissue (20.64-44.95) than for emission from cancer tissue (2.01-18.20). A limitation of this study is that only a few clinical samples were investigated. These results should be evaluated as preliminary because of the small number of patients examined.

Development of novel molecularly imprinted solid-phase microextraction fibers and their application for the determination of antibiotic drugs in biological samples by SPME-LC/MSn

Novel molecularly imprinted polymer (MIP)-coated fibers for solid-phase microextraction (SPME) fibers were prepared by using linezolid as the template molecule. The characteristics and application of these fibers were investigated. The polypyrrole, polythiophene, and poly(3-methylthiophene) coatings were prepared in the electrochemical polymerization way. The molecularly imprinted SPME coatings display a high selectivity toward linezolid. Molecularly imprinted coatings showed a stable and reproducible response without any influence of interferents commonly existing in biological samples. High-performance liquid chromatography with spectroscopic UV and mass spectrometry (MS) detectors were used for the determination of selected antibiotic drugs (linezolid, daptomycin, amoxicillin). The isolation and preconcentration of selected antibiotic drugs from new types of biological samples (acellular and protein-free simulated body fluid) and human plasma samples were performed. The SPME MIP-coated fibers are suitable for the selective extraction of antibiotic drugs in biological samples.

Simultaneous determination of selected chemotherapeutics in human whole blood by molecularly imprinted polymers coated solid phase microextraction fibers and liquid chromatography-tandem mass spectrometry.

Development and validation of novel, general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of amoxicillin (AMOX), cefatoxime (CEF), ciprofloxacin (CIP), daptomycin (DAPTO), fluconazole (FLU), gentamicin (GEN), clindamycin (KLI), linezolid (LIN), metronidazole (MET), moxifloxacin (MOXI) in human whole blood are described. Samples were prepared on solid phase microextraction way with the use of polymeric sorption coatings with molecular imprints and analyzed using a gradient separation over an ACE C18-column (4.6mm×150mm, 3μm) with isocratic elution. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.1% formic acid or 5mM ammonium acetate) at a flow 0.4ml/min. The chromatographic run time was kept less than 9min. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 7.3%, while the accuracy was within ±8.4%. The mean recovery of all the analytes ranged from 65.0 to 83.0%. This method was successfully applied to clinical samples from patients with clinically diagnosed bacterial infections process.

A new approach for antibiotic drugs determination in human plasma by liquid chromatography–mass spectrometry

Sensitive and selective analytical procedures based on high performance liquid chromatography with mass spectrometric detection were developed for the determination of linezolid (LIN) and amoxicillin (AMOX) in human plasma samples. Samples were prepared by applying protein precipitation (PP), solid phase extraction (SPE), and microextraction in packed syringe (MEPS). The analytical separation was carried out using reversed phase liquid chromatography in isocratic mode. All analytes were monitored by mass spectrometry (MS) detection in the product ion mode and the method was validated covering the corresponding therapeutic range of 1-30 μg/mL and 1-50 μg/mL for LIN and AMOX respectively. The assay was linear over AMOX and LIN concentration ranges. The method provided good validation data: accuracy (102.9% (LIN), 100.9% (AMOX)), limit of detection (0.1407 ng/mL (LIN); 0.1341 ng/mL (AMOX); quantification (0.3814 ng/mL (LIN), 0.4249 ng/mL (AMOX)) and acceptable stability within 24h in the auto-sampler. Three different methods were compared as regards precision, accuracy, recovery and matrix effects. The proposed methods offer a fast and simple way to determine selected antibiotic drugs in human plasma that could be applied in pharmacokinetic studies.

Differentiation of Staphylococcus aureus strains by CE, zeta potential and coagulase gene polymorphism

Staphylococcus aureus is a common cause of infection in both hospitals and the community, and it is becoming increasingly virulent and resistant to antibiotics. Possibilities of fast, sensitive and cheap determination of these pathogenic bacteria are extremely important in antimicrobial therapy. In the present study, CE with chemically modified capillary and zeta potential measurements were used for differentiation of three different clinical strains of S. aureus. The data presented in this contribution suggested that electrophoretic behavior and the values of zeta potential should be very useful in distinguishing between closely related strains, which exhibited coagulase gene/protein polymorphism. Understanding the differences between S. aureus strains could help to improve our knowledge about S. aureus pathogenecity and to monitor for and respond to emergence of more virulent strains.

Pharmacokinetic study of amoxicillin in human plasma by solid-phase microextraction followed by high-performance liquid chromatography-triple quadrupole mass spectrometry.

Sensitive and selective analytical procedures based on high-performance liquid chromatography with mass spectrometric detection were developed for the determination of amoxicillin in human plasma samples. Samples were prepared by applying in-house manufactured molecularly imprinted solid-phase microextraction probes. The detection of target compounds was performed in multiple reaction monitoring mode. The multiple reaction monitoring detection was operated in the positive electrospray ionization mode using the transitions of m/z 366 ([M + H](+) ) → 349 for amoxicillin and m/z 390 ([M + H](+) ) → 372 for gemifloxacin. The method was validated with precision within 15% relative standard deviation and accuracy within 15% relative error. The method was successfully applied to study of the pharmacokinetics of amoxicillin in human plasma after oral administration of amoxicillin.

Combination of capillary electrophoresis, PCR and physiological assays in differentiation of clinical strains of Staphylococcus aureus.

Fast, sensitive and cheap determination of pathogenic bacteria is extremely important in many branches, for example biotechnology, quality control, analysis of samples and antimicrobial therapy. The development and application of analytical techniques in practice could provide new possibilities in this regard. The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality. Rapid and sensitive determination is therefore very important. In the present study, novel methods, based on capillary zone electrophoresis and (as confirmation of these results) molecular analysis of a part of the coag gene, were developed for identification and differentiation of three S. aureus strains. The electrophoretic measurements rely on the differential mobility of bacteria in the fused silica capillary under the direct current electric field. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the S. aureus gene were amplified using the PCR technique followed by restriction enzyme digestion and analysis of restriction fragment length polymorphism patterns as well as sequencing. Finally, the results of electrophoretic measurements with molecular analysis were compared.

Antimicrobial properties of biosynthesized silver nanoparticles studied by flow cytometry and related techniques.

This work reports the effect of silver bionanoparticles (Bio(AgNPs) synthesized by Actinobacteria CGG 11n on selected Gram (+) and Gram (–) bacteria. Flow cytometry, classical antibiogram method and fluorescent microscopy approach was used for evaluation of antimicrobial activity of Bio(AgNPs) and their combination with antibiotics. Furthermore, the performed research specified the capacity of flow cytometry method as an alternative to the standard ones and as a complementary method to electromigration techniques. The study showed antibacterial activity of both BioAgNPs and the combination of antibiotics/BioAgNPs against all the tested bacteria strains in comparison with a diffusion, dilution and bioautographic methods. The synergistic effect of antibiotics/BioAgNPs combination (e.g. kanamycin, ampicillin, neomycin and streptomycin) was found to be more notable against Pseudomonas aeruginosa representing a prototype of multi‐drug resistant “superbugs” for which effective therapeutic options are very limited.