Przemyslaw Mielczarek

Plasma-based ambient ionization mass spectrometry in bioanalytical sciences

Plasma-based ambient ionization mass spectrometry techniques are gaining growing interest due to their specific features, such as the need for little or no sample preparation, its high analysis speed, and the ambient experimental conditions. Samples can be analyzed in gas, liquid, or solid forms. These techniques allow for a wide range of applications, like warfare agent detection, chemical reaction control, mass spectrometry imaging, polymer identification, and food safety monitoring, as well as applications in biomedical science, e.g., drug and pharmaceutical analysis, medical diagnostics, biochemical analysis, etc. Until now, the main drawback of plasma-based techniques is their quantitative aspect, but a lot of efforts have been done to improve this obstacle.

Gentamicin release from biodegradable poly-l-lactide based composites for novel intramedullary nails

One of the major problems in orthopedic surgery is infection associated with implantation. The treatment is a very difficult and long-term process. A solution to this issue can be the use of implants which additionally constitute an antibiotic carrier preventing the development of an infection. Prototypes of biodegradable intramedullary nails made of three different composites with a poly(L-lactide) matrix were designed. The nails served as gentamicin sulfate (GS) carrier - an antibiotic commonly used in the treatment of osteomyelitis. The matrix was reinforced with carbon fibers (CF), alginate fibers (Alg) and magnesium alloy wires (Mg), as well as modified with bioactive particles of tricalcium phosphate (TCP) in various systems. In this way, novel, multi-phase and multifunctional degradable intramedullary nails were obtained. The tests demonstrated strong dependence between the type of the modifying phase introduced into the composite, and the rate of drug release. Introduction of gentamicin into the nail structure strengthened and prolonged antibacterial activity of the nails.

Synthesis of metabolites of paracetamol and cocaine via photooxidation on TiO2 catalyzed by UV light

Prediction and synthesis of drug metabolites generated by photodegradation using TiO(2)/UV system in aqueous solution was performed to monitor illicit drugs and to support legal actions against drug dealers. Metabolism of paracetamol and cocaine applied as exemplary compounds was monitored by liquid chromatography mass spectrometry (LC-MS/MS) and direct analysis by electrospray ionization mass spectrometry (ESI-MS/MS). The experiments proved that simulated metabolic pathways of the drug samples are efficient and lead to the formation of products that are observed using living organisms or hepatocyte microsomal preparations. Routinely, TiO(2) nanopowders are used for complete degradation of unwanted waste to protect environment. We applied such system for prediction of potential metabolites of harmful substances, such as cocaine. The results demonstrate that TiO(2)/UV oxidative system can be an efficient, complementary method to the in vivo approaches to obtain important metabolites for further studies. Investigations using such methodology may be helpful for toxicologists providing a vital knowledge on metabolites derived from e.g. newly introduced cognitive enhancers (designer drugs), and home-made substances prepared from the over-the-counter tablets (legal highs).

Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme

The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

Electrochemical generation of selegiline metabolites coupled to mass spectrometry

The metabolic pathways of selegiline (a drug used for the treatment of early-stage Parkinsons disease) were analyzed by electrochemical oxidation with application of the flow electrochemical cell consisting of three electrodes (ROXY™, Antec, the Netherlands). Two types of working electrodes were applied: glassy carbon (GC) and boron-doped diamond (BDD). The potential applied at working electrode and composition of the solvent were optimized for the best conditions for oxidation and identification processes. All products were directly analyzed on-line by mass spectrometry. For further characterization of electrochemical oxidation products, the novel approach involving reversed phase chromatography linked to mass spectrometry with dielectric barrier discharge ionization (DBDI-MS) was used. In this manuscript, we report a novel technique for simulation of drug metabolism by electrochemical system (EC) connected to liquid chromatography (LC) and dielectric barrier discharge ionization (DBDI) mass spectrometry (MS) for direct on-line detection of electrochemical oxidation products. Here, we linked LC/DBDI-MS system with an electrochemical flow cell in order to study metabolic pathways via identification of drug metabolites generated electrochemically. The DBDI source has never been used before for identification of psychoactive metabolites generated in an electrochemical flow cell. Our knowledge on the biological background of xenobiotics metabolism and its influence on human body is constantly increasing, but still many mechanisms are not explained. Nowadays, metabolism of pharmaceuticals is mainly studied using liver cells prepared from animals or humans. Cytochrome P450, present in microsomes, is primarily responsible for oxidative metabolism of xenobiotics. It was also shown, that breakdown of popular medicines may be successfully simulated by electrochemistry under appropriate conditions. The presented experiments allow for comparison of these two entirely distinct techniques using selegiline as the model xenobiotic with well-described metabolic pathway in human body. The obtained results for selegiline oxidation show that it is possible to generate the most important selegiline metabolites present in human body - some of them with psychoactive properties, such as methamphetamine and amphetamine. These metabolites, serving as an evidence of the xenobiotic intake, can also be produced, among a larger group of metabolites, by incubation of selegiline with rat and human liver microsomes. The EC/LC/DBDI-MS system provides novel, promising platform for drugs screening of the phase I metabolism. The metabolites can be detected directly by MS or collected and separated by liquid chromatography.

Desorption electrospray ionization-based imaging of interaction between vascular graft and human body

The desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) is known as a fast and convenient MS-based method for lipid imaging in various biological materials. Here, we applied this technique to visualize lipid distribution in a vascular graft removed from a patients body. This is a good example of the DESI system capabilities toward imaging of interaction between artificial material and living tissues. Detailed analysis allowed for visualization of the spatial distribution of selected lipids in this implanted, artificial material. Not only DESI-MSI allowed visualization of lipid distribution in the investigated material but also enabled identification of the detected molecular species using MS/MS. Here, this technique was successfully used to evaluate the saturation and spatial distribution of endogenous lipids in the artificial vascular graft. Unambiguous identification of the lipids was done with the aid of fragmentation procedure. We also showed that various lipids localize preferably in graft material or internal plaque existing inside the graft.

Electrochemical simulation of cocaine metabolism-a step toward predictive toxicology for drugs of abuse.

Knowledge of the metabolic pathways and biotransformation of the most popular drugs, such as cocaine, amphetamine, morphine and others, is crucial for the elucidation of their possible toxicity and mechanism of action in the human body. In vitro studies on metabolism are mainly based on the incubation of drugs with liver cell homogenate and utilizing living animals. These methods need to be followed by isolation and detection of metabolic products, which makes these techniques time-consuming and technically demanding. We show here that the oxidative metabolism that occurs in the liver cells and is mainly caused by cytochrome P450 can be successfully mimicked with the electrochemical system (EC) connected on-line with electrospray ionization mass spectrometry. Cocaine was chosen as a model drug for these studies and was analyzed with a previously described system under various conditions using the boron-doped diamond working electrode. The results were compared with the number of metabolites generated by a standard procedure based on the reaction with the rat liver microsomes. Two electrochemical products of cocaine oxidation were created, of which one was a natural metabolite of cocaine in the human body—norcocaine. The EC provides a promising platform for the screening of the addictive drug phase I metabolism. The metabolites can be directly analyzed by mass spectrometry or collected and separated by liquid chromatography. No liver cell homogenate or microsome is necessary to generate these metabolites, which simplifies separation of the mixtures and reduces time and costs of all experiments.

The Forty-Sixth Euro Congress on Drug Synthesis and Analysis: Snapshot †

The 46th EuroCongress on Drug Synthesis and Analysis (ECDSA-2017) was arranged within the celebration of the 65th Anniversary of the Faculty of Pharmacy at Comenius University in Bratislava, Slovakia from 5–8 September 2017 to get together specialists in medicinal chemistry, organic synthesis, pharmaceutical analysis, screening of bioactive compounds, pharmacology and drug formulations; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topic of the conference, “Drug Synthesis and Analysis,” meant that the symposium welcomed all pharmacists and/or researchers (chemists, analysts, biologists) and students interested in scientific work dealing with investigations of biologically active compounds as potential drugs. The authors of this manuscript were plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

Online and Offline Sample Fractionation

Fractionation of complex biological samples is necessary to separate a vast number of proteins that need to be separated before they enter further analytical procedures. Several strategies were described in this chapter, including on-line and off-line protocols, stationary phases and mobile phases, their features and drawbacks, and compatibility with mass spectrometry measurements.

Technical note: Air compared to nitrogen as nebulizing and drying gases for electrospray ionization mass spectrometry

In the present study we tested the application of compressed air instead of pure nitrogen as the nebulizing and drying gas, and its influence on the quality of electrospray ionization (ESI) mass spectra. The intensities of the signals corresponding to protonated molecules were significantly (twice) higher when air was used. Inspection of signal-to-noise (S/N) ratios revealed that, in both cases, sensitivity was comparable. A higher ion abundance after the application of compressed air was followed by a higher background. Another potential risk of using air in the ESI source is the possibility for sample oxidation due to the presence of oxygen. To test this, we selected five easily oxidizing compounds to verify their susceptibility to oxidation. In particular, the presence of methionine was of interest. For all the compounds studied, no oxidation was observed. Amodiaquine oxidizes spontaneously in water solutions and its oxidized form can be detected a few hours after preparation. Direct comparison of the spectra where nitrogen was used with the corresponding spectra obtained when air was applied did not show significant differences. The only distinction was slightly different patterns of adducts when air was used. The difference concerns acetonitrile, which forms higher signals when air is the nebulizing gas. It is also important that the replacement of nitrogen with air does not affect quantitative data. The prepared calibration curves also visualize an intensity twice as high (independent of concentration within tested range) of the signal where air was applied. We have used our system continuously for three months with air as the nebulizing and drying gas and have not noticed any unexpected signal deterioration caused by additional source contamination from the air. Moreover, compressed air is much cheaper and easily available using oil-free compressors or pumps.