Jaroslav Blaško

Gas chromatography analysis of benzene, toluene, ethylbenzene and xylenes using newly designed needle trap device in aqueous samples

A newly designed needle trap device with Carbopack X as a sorbent material is used for sampling, preconcentration and injection of volatile analytes benzene, toluene, ethylbenzene and xylenes (BTEX) into gas chromatograph. The closed system of stripping the analytes from water samples was used. An injection port with a modified metal liner was used to desorb analytes trapped in needle trap device. The main advantage of needle trap device consists in the simple methodology and easiness and rapidity of the analysis. Needle trap device is suitable for sampling in field. The experimental parameters as breakthrough volume of stripping gas, linearity, repeatability and limit of detection (LOD) and quantification (LOQ) were investigated. LOD ranges from 0.05 to 0.07 microgL(-1) and relative standard deviation ranges from 0.5% to 11.6% at concentrations 5 and 0.1 microgL(-1), respectively.

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

Gas chromatography/mass spectrometry of oils and oil binders in paintings

A GC/MS procedure has been developed, optimized, and applied to characterization of oil binders in paintings. The procedure involves hydrolysis of lipids to fatty acids (FAs) and derivatization of FAs to fatty acid methyl esters (FAMEs) by a solution of sodium methanolate in methanol at an elevated temperature. FAMEs are analyzed by temperature-programed GC followed by full-scan MS. Old and dried samples are subjected to extraction of nonpolymerized FAMEs into dichloromethane prior to hydrolysis. The method provides a good repeatability of results and has been applied to the characterization of common plant oils used in paintings, to commercial oil and tempera paints, to model painting samples, and to samples taken from real paintings. The fresh oils and binders can readily be identified and characterized. The ratio of the methyl esters of palmitic and stearic acids can be used to characterize oil binders in old works of art.

Direct silylation of Trypanosoma brucei metabolites in aqueous samples and their GC-MS/MS analysis.

A simple two-step method for the derivatization of polar compounds (lactate, alanine, glycerol, succinate and glucose) using hexamethyldisilazane (HMDS) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was developed. This method allows direct derivatization of aqueous samples wihout sample pretreatment. The method was used for the analysis of the metabolites of the unicellular organism Trypanosoma brucei. The limits of detection by GC-MS/MS analysis were in the range of 0.02 mg L(-1) for glucose to 0.85 mg L(-1) for lactate.

Considerations on the determination of the limit of detection and the limit of quantification in one-dimensional and comprehensive two-dimensional gas chromatography

Methods based on the blank signal as proposed by IUPAC procedure and on the signal to noise ratio (S/N) as listed in the ISO-11843-1 norm for determination of the limit of detection (LOD) and quantitation (LOQ) in one-dimensional capillary gas chromatography (1D-GC) and comprehensive two-dimensional capillary gas chromatography (CG×GC) are described in detail and compared for both techniques. Flame ionization detection was applied and variables were the data acquisition frequency and, for CG×GC, also the modulation time. It has been stated that LOD and LOQ estimated according to IUPAC might be successfully used for 1D-GC-FID method. Moreover, LOD and LOQ decrease with decrease of data acquisition frequency (DAF). For GC×GC-FID, estimation of LOD by IUPAC gave poor reproducibility of results while for LOQ reproducibility was acceptable (within ±10% rel.). The LOD and LOQ determined by the S/N concept both for 1D-GC-FID and GC×GC-FID methods are ca. three times higher than those values estimated by the standard deviation of the blank. Since the distribution pattern of modulated peaks for any analyte separated by GC×GC is random and cannot be predicted, LOQ and LOD may vary within 30% for 3s modulation time. Concerning sensitivity, 1D-GC-FID at 2Hz and of GC×GC-FID at 50Hz shows a ca. 5 times enhancement of sensitivity in the modulated signal output.

GtrA Protein Rv3789 Is Required for Arabinosylation of Arabinogalactan in Mycobacterium tuberculosis.

UNLABELLED Mycobacterium tuberculosis possesses a thick and highly hydrophobic cell wall principally composed of a mycolyl-arabinogalactan-peptidoglycan complex, which is critical for survival and virulence. DprE1 is a well-characterized component of decaprenyl-phospho-ribose epimerase, which produces decaprenyl-phospho-arabinose (DPA) for the biosynthesis of mycobacterial arabinans. Upstream of dprE1 lies rv3789, which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides. We demonstrate that rv3789 and dprE1 are cotranscribed from a common transcription start site situated 64 bp upstream of rv3789. Topology mapping revealed four transmembrane domains in Rv3789 and a cytoplasmic C terminus consistent with structural models built using analysis of sequence coevolution. To investigate its role, we generated an unmarked rv3789 deletion mutant in M. tuberculosis. The mutant was characterized by impaired growth and abnormal cell morphology, since the cells were shorter and more swollen than wild-type cells. This phenotype likely stems from the decreased incorporation of arabinan into arabinogalactan and was accompanied by an accumulation of DPA. A role for Rv3789 in arabinan biosynthesis was further supported by its interaction with the priming arabinosyltransferase AftA, as demonstrated by a two-hybrid approach. Taken together, the data suggest that Rv3789 does not act as a DPA flippase but, rather, recruits AftA for arabinogalactan biosynthesis. IMPORTANCE Upstream of the essential dprE1 gene, encoding a key enzyme of the decaprenyl phospho-arabinose (DPA) pathway, lies rv3789, coding for a short transmembrane protein of unknown function. In this study, we demonstrated that rv3789 and dprE1 are cotranscribed from a common transcription start site located 64 bp upstream of rv3789 in M. tuberculosis. Furthermore, the deletion of rv3789 led to a reduction in arabinan content and to an accumulation of DPA, confirming that Rv3789 plays a role in arabinan biosynthesis. Topology mapping, structural modeling, and protein interaction studies suggest that Rv3789 acts as an anchor protein recruiting AftA, the first arabinosyl transferase. This investigation provides deeper insight into the mechanism of arabinan biosynthesis in mycobacteria.

The analysis of linear and monomethylalkanes in exhaled breath samples by GC × GC-FID and GC–MS/MS

A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6C19n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC-MS/MS and GC×GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC-MS/MS detection limits were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The GC×GC-FID detection limits ranged from 19 pg for n-dodecane to 110 pg for 3-methyloctane.

Equivalent chain lengths of all C4–C23 saturated monomethyl branched fatty acid methyl esters on methylsilicone OV-1 stationary phase

Isomer mixtures of monomethyl branched saturated C7-C23 fatty acid methyl esters (FAME) were prepared by performing a methylene insertion reaction to the straight chain FAME and this study model was completed by using commercially available standards of C4-C7 FAME. The equivalent chain lengths (ECL) of all 220 C4-C23 monomethyl branched FAME on OV-1 stationary phase were measured, achieving an average repeatability of ±0.0004 ECL units. The monomethyl branched FAME was identified by GC on the basis of regularity of the fractional chain lengths (FCL) dependence on the number of carbon atoms (C(z)) of individual homologous series of methyl 2-, 3-, …, 21-FAME. The prediction of retention of the first homologues, having the new position of methyl group beginning at higher carbon atoms number, and analogously for the second, third, fourth, and other members of the homologous series, allowed the dependence FCL=f(C(z)) for the first and subsequent members of beginning homologous of monomethyl derivatives of FAME. The identification was confirmed by mass spectrometry. All of the methyl isomers of FAME, which could not be completely separated by gas chromatography due to having a methyl group in surroundings of the middle of the carbon chain, were resolved by mass spectrometry using deconvolution in a SIM-mode. Measured gas chromatographic and mass spectrometric data were applied for identification of the monomethyl branched saturated FAME in tongue coating.

Determination of sevoflurane and its metabolite hexafluoroisopropanol by direct injection of human plasma into gas chromatography-tandem mass spectrometry.

The developed method for trace analysis of volatile components in plasma allows direct injection of up to 150 samples to the GC-MS/MS system without injector cleaning. This method requires no modification of plasma and the working environment does not interfere with the determination of these analytes. The method allows simultaneous quantification of non-polar sevoflurane and its polar metabolite hexafluoroisopropanol (free, unconjugated form). It is characterized by high repeatability and sensitivity with the detection limit of 0.009 mg L(-1) for sevoflurane and 0.018 mg L(-1) for hexafluoroisopropanol and the linear range 0.050-150 mg L(-1). The method was used to determine the concentration of sevoflurane and hexafluoroisopropanol in plasma samples of 7 patients undergoing general anesthesia with sevoflurane. The average concentration of sevoflurane and free hexafluoroisopropanol was 57.2 mg L(-1) and 0.39 mg L(-1), respectively. The method can be applied for clinical monitoring, as well as for analytical toxicology.

Effects of 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitor pravastatin on membrane lipids and membrane associated functions of Methanothermobacter thermautotrophicus

The role of archaeal membrane and its lipid constituents was investigated in bioenergetic functions of Methanothermobacter thermautotrophicus. The effects were determined of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, pravastatin, on lipid composition, and its impact on some bioenergetic functions of treated cells. Pravastatin remarkably inhibited the growth of M. thermautotrophicus. On membrane level, pravastatin treatment modulated the composition of the mixture of squalene and hydrosqualene derivatives as well as the activities of ATPase, A1Ao-ATP synthase and Na+/H+ antiporter. SDS-PAGE of chloroform-methanol extracts of membranes from control and pravastatin-treated cells revealed changes in the amount of AtpK proteolipids, which suggests that pravastatin modifies cell-membrane composition, hereby modulating the properties of some membrane-bound enzymes participating in energy transformation in methanoarchaea.