ABSTRACT
For determination of selected carotenoids, various types of columns for high-performance liquid chromatography (HPLC) with different properties have been used. The characteristics of the laboratory-used packing material containing monomeric alkyl-bonded phases (C18, C30) and phenyl as well as phenyl-hexyl stationary phases were studied. The retention data of the examined compounds were used to determine the hydrophobicity and silanol activity of stationary phases applied in the study. The presence of the polar and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the stationary phase. Columns were compared according to methylene selectivity using a series of benzene homologues. The measurements were done using a methanol-water mobile phase. Knowledge of the properties of the applied stationary phase provided the possibility to predict the RP HPLC retention behaviours in analysis of carotenoids including lutein, lycopene and ß-carotene. The composition of the mobile phase, the addition of triethylamine and the type of stationary phase had been taken into account in designing the method of carotenoid identification. Also a monolithic column characterised by low hydrodynamic resistance, high porosity and high permeability was applied. The presented results show that the coverage density of the bonded ligands on silica gel packings and length of the linkage strongly influence the carotenoid retention behaviours. In our study, the highest retention parameters for lutein, lycopene and ß-carotene were observed for C30 and C18 stationary phase. This effect corresponds with pore size of column packing greater than 100 Å and carbon content higher than 11 %.
ABSTRACT
We analysed breath and inhaled room air samples from 39 healthy volunteers (28 non-smokers, 8 smokers and 3 ex-smokers) by SPME-GC-MS. Mixed expiratory and indoor air samples were collected in freshly cleaned Tedlar bags. Eighteen millilitres of each sample were transferred into sealed, evacuated glass vials, preconcentrated by solid-phase microextraction (SPME, carboxen/polydimethylsiloxane) and investigated by gas chromatography with mass spectrometric detection (GC-MS). For the unequivocal identification of potential marker compounds, pure calibration mixtures of reference compounds (depending on commercial availability) were prepared to determine the retention time and mass spectra with respect to our analytical setting. Applying the adapted SPME-GC/MS method with limit of detection in the high ppb range (0.05-15.00 ppb), we succeeded in identifying altogether 38 compounds with concentrations in exhaled breath being at least 50% higher than concentration in inhaled air. From these 38 compounds, 31 were identified not only by the spectral library match but also by retention time of standards. A comparison of retention times and spectrum obtained for standards and determined compounds was performed. We found hydrocarbons (isoprene, 2-pentene, 2-methyl-1-pentene, benzene, toluene, p-cymene, limonene, 2,4-dimethylheptane, n-butane), ketones (acetone, hydroxypropanone, methylvinyl ketone), ethers (dimethyl ether, 1,3-dioxolane), esters (ethyl acetate), aldehydes (propanal, hexanal, heptanal, acrolein) and alcohols (ethanol, 2-metoxyethanol, isopropyl alcohol, 2,2,3,3- tetramethylcyclopropanemethanol, 3,4-dimethylcyclohexanol). Proper identification of compounds in different cohorts of patients and volunteers is the base for further investigation of origin, biochemical background and distribution of potential breath biomarkers.