Robert Jirásko

Recent developments in liquid chromatography-mass spectrometry and related techniques.

This review summarizes the state-of-art in liquid chromatography-mass spectrometry (LC-MS) and related techniques with the main focus on recent developments in the last decade. LC-MS records an enormous growth in recent years due to the application potential in analytical chemistry, biochemistry, pharmaceutical analysis, clinical analysis and many other fields, where the qualitative and quantitative characterization of complex organic, bioorganic and organometallic mixtures is needed. Beginners and moderately experienced LC-MS users may be confused by the number of different LC-MS systems on the market, therefore an actual overview of mass spectrometers designed for the LC-MS configuration and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) from main manufacturers is compiled here together with an independent assessment of their advantages and limitations. Current trends in terms of mass analyzers, ionization techniques, fast LC-MS, LC-MALDI-MS, ion mobility spectrometry used in LC-MS, quantitation issues specific to MS and emerging mass spectrometric approaches complementary to LC-MS are discussed as well.

Basic rules for the interpretation of atmospheric pressure ionization mass spectra of small molecules

This review summarizes the basic rules for the interpretation of atmospheric pressure ionization (API) mass spectra of small molecules written with the style primarily intended for beginners and low-experienced researchers with the mass spectra interpretation. The first and basic step in any interpretation of mass spectra is always the determination of molecular weight, which is relatively easy in case of soft ionization techniques due to the limited extend of fragmentation and the prevailing presence of (de)protonated molecules in the full scan mass spectra. These [M+H](+) and [M-H](-) ions are often accompanied by low abundant molecular adducts, which can be used as the supplementary information for the unambiguous determination of molecular weights. In certain cases, adduct ions may dominate the spectra. The subsequent interpretation of full scan and tandem mass spectra is more complicated due to a high number of possible functional groups, structural subunits and their combinations resulting in numerous competitive fragmentation pathways. Typical neutral losses and the effect of individual functional groups on the fragmentation are discussed in detail and illustrated with selected examples. Modern mass analyzers have powerful features for the structural elucidation, for example high resolving power, high mass accuracy, multistage tandem mass spectrometry, dedicated softwares for the interpretation of mass spectra and prediction of their fragmentation. Background information on differences among individual ionization techniques suitable for the HPLC-MS coupling and basic types of mass analyzers with consequences for the data interpretation is briefly discussed as well. Selected examples illustrate that the right optimization of chromatographic separation and the use of other than mass spectrometric detectors can bring valuable complementary information.

Structural analysis of organometallic compounds with soft ionization mass spectrometry

The analysis of organometallic compounds with mass spectrometry has some special features in comparison with organic and bioorganic compounds. The first step is the choice of a suitable ionization technique, where the electrospray ionization is certainly the best possibility for most classes of organometallic compounds and metal complexes. Some ionization mechanisms of organometallic compounds are comparable to organic molecules, such as protonation/deprotonation, and adduct formation with sodium or potassium ions; however, in many cases, different mechanisms and their combinations complicate the spectra interpretation. Organometallics frequently undergo various types of adduct and polymerization reactions that result in significantly higher masses observed in the spectra in comparison to molecular weights of studied compounds. Metal elements typically have more natural isotopes than common organic elements, which cause characteristic wide distributions of isotopic peaks; for example, tin has ten natural isotopes. The isotopic pattern can be used for the identification of the type and number of metal elements in particular ions. The ionization and fragmentation behavior also depend on the type of metal atom; therefore, our discussion of mass spectra interpretation is divided according to the different type of organometallic compounds. Among various types of mass spectrometers available on the market, trap-based analyzers (linear or spherical ion-traps, Orbitrap) are suitable to study complex fragmentation pathways of organometallic ions and their adducts, whereas high-resolution and high-mass accuracy analyzers (time-of-flight-based analyzers, or Fourier transform-based analyzers-Orbitrap or ion cyclotron resonance mass spectrometers) provide accurate masses applicable for the determination of the elemental composition of individual ions.

Identification of new phase II metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using high-performance liquid chromatography electrospray ionization tandem mass spectrometry

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called alpha- and beta-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MS(n) data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H](+) and [M-H](-) ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.

Investigation of the metabolism of monepantel in ovine hepatocytes by UHPLC/MS/MS

Monepantel (MOP) belongs to a new class of anthelmintic drugs known as aminoacetonitrile derivatives. It was approved for use in veterinary practice in Czech Republic in 2011. So far, biotransformation and transport of MOP in target animals have been studied insufficiently, although the study of metabolic pathways of anthelmintics is very important for the efficacy of safety of therapy and evaluation of the risk of drug–drug interactions. The aim of this study was to identify MOP metabolites and to suggest the metabolic pathways of MOP in sheep. For this purpose, primary culture of ovine hepatocytes was used as a model in vitro system. After incubation, medium samples and homogenates of hepatocytes were extracted separately using solid-phase extraction. Analysis was performed using a hybrid quadrupole-time-of-flight analyzer with respect to high mass accuracy measurements in full scan and tandem mass spectra for the confirmation of an elemental composition. The obtained results revealed S-oxidation to sulfoxide and sulfone and arene hydroxylation as MOP phase I biotransformations. From phase II metabolites, MOP glucuronides, sulfates, and acetylcysteine conjugates were found. Based on the obtained results, a scheme of the metabolic pathway of MOP in sheep has been proposed.

Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics.

The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant). H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed. The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.

Biotransformation of benzimidazole anthelmintics in reed (Phragmites australis) as a potential tool for their detoxification in environment.

Benzimidazole anthelmintics, the drugs against parasitic worms, are widely used in human as well as veterinary medicine. Following excretion, these substances may persist in the environment and impact non-target organisms. In order to test phytoremediation as a possible tool for detoxification of anthelmintics in environment, the biotransformation pathways of albendazole (ABZ) and flubendazole (FLU) were studied in reed (Phragmites australis) in vitro. Reed cells were able to uptake and biotransform both anthelmintics. Ten ABZ metabolites and five FLU metabolites were found. Some atypical biotransformation reactions (formation of glucosylglucosides, acetylglucosides and xylosylglucosides), which have not been described previously, were identified. Based on the obtained results, the schemes of metabolic pathways of ABZ and FLU in reed were proposed. Most of ABZ and FLU metabolites can be considered as anthelmintically less active; therefore uptake and biotransformation of these anthelmintics by reed could be useful for decrease of their toxicity in environment.

Metabolic pathways of anthelmintic drug monepantel in sheep and in its parasite (Haemonchus contortus).

Monepantel (MOP) is a new anthelmintic drug intended for the treatment and control of gastrointestinal roundworms (nematodes) infection and associated disease in sheep. The aim of our study was to find out metabolic pathways of MOP in sheep in vivo and in its parasite Haemonchus contortus ex vivo. MOP biotransformation in two H. contortus strains with different sensitivity to anthelmintics was also compared. Ultra high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) technique is used for the identification of MOP metabolites in ovine urine, faeces, and nematodes. MOP biotransformation study in sheep in vivo led to the identification of 13 MOP metabolites; 7 of them have not been described previously in in vitro study. The study of MOP biotransformation in H. contorus ex vivo reveals four MOP metabolites. The nitrile hydrolysis as a new biotransformation pathway in helminths ex vivo is reported here for the first time. Unlike sheep, H. contorus nematodes are not able to metabolize MOP via phase II biotransformation. Nematodes of resistant White river (WR) strain form more types of MOP metabolites than nematodes of sensitive inbred susceptible Edinburgh (ISE) strain. Based on obtained results, schemes of metabolic pathways of MOP in sheep and nematodes are proposed.

Synthesis, crystal structures, cytotoxicity and qualitative structure–activity relationship (QSAR) of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes, nBu2Sn(L)2

A series of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes has been synthesized and characterized by (1)H-, (13)C-, (119)Sn NMR, ESI-MS (electrospray ionization mass spectrometry), IR and (119m)Sn Mössbauer spectroscopic techniques in combination with elemental analyses. The structures of four di-n-butyltin(IV) complexes, viz., (n)Bu(2)Sn(L(3))(2) (3), (n)Bu(2)Sn(L(4))(2) (4), (n)Bu(2)Sn(L(5))(2) (5) and (n)Bu(2)Sn(L(7))(2).0.5C(6)H(6) (7) (LH=5-[(E)-2-(aryl)-1-diazenyl)quinolin-8-ol) were determined by single crystal X-ray diffraction. In general, the complexes were found to adopt a distorted cis-octahedral arrangement around the tin atom. These complexes retain their solid-state structure in non-coordinating solvent as evidenced by (119)Sn and (13)C NMR spectroscopic results. The in vitro cytotoxicity of di-n-butyltin(IV) complexes (3-8) is reported against seven well characterized human tumour cell lines. The basicity of the two quinolinolato donor N and O atoms of the ligands are discussed in relation to the cytotoxicity data.

NCN-chelated organoantimony(III) and organobismuth(III) phosphates: synthesis and solid-state and solution structures.

.Organoantimony(III) and organobismuth(III) phosphates (LM)(3)(PO(4))(2) [M = Sb (3) and Bi (4)], containing the NCN-chelating ligand L [L = 2,6-(CH(2)NMe(2))(2)C(6)H(3)], were prepared by the simple treatment of parent oxides 1 and 2 with H(3)PO(4). Both compounds were characterized by elemental analysis, electrospray ionization mass spectrometry, and IR and NMR spectroscopy and in the case of 3 by X-ray diffraction techniques. Compound 3 has an interesting behavior in solution, i.e., the formation of two possible conformational isomers, which was studied by (1)H, (13)C, and (31)P NMR spectroscopy.