Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.
Title of diploma work: The possibilities of re-socialization of the condemned in duration of sentence through the programs of treating The diploma work pays attention to the possibilities and limits of the programs of treating, which are applied in prisons Prague ? Pankrac and Prague ? Ruzyně. The basic theme of this work is the research question: ?Are the programs of treating, which are applied, sufficient? Do they serve the purpose in re-socialization of the condemned? Are the manual activities of bigger importance than the educational activities in the process of re-socialization?? The employees of both prisons have been asked these questions. The work is divided into two parts.The first part is the theoretical part. Its introduction chapter is focused on the beginnings of prison system in the world, the history of prison system in our country, on defining the terms, on legal aspects of Prison Service of the Czech Republic, on issues related to the program of treating and its activities, on introduction of the prisons Prague ? Pankrac and Prague ? Ruzyně, both on general and concrete levels. The end of the theoretical part is represented by essential information on prisons Prague ? Pankrac and Prague ? Ruzyně.The second part is empirical. It contains methodology and justification of proceedings of quality research, information on data gathering and on respondents. Furthermore, it contains hypothesis delineation, the main research, theoretical comparison of opinions of the specialists of prisons Prague ? Pankrac and Prague ? Ruzyně, and the analysis and hypothesis verification.
Micro-SPE in pipette tips (μ-SPE-PT) with particle sorbent has never been used in small-molecule drug analysis. Methodology & results: μ-SPE-PT was used for the extraction of statins from biological materials followed by UHPLC-MS/MS. The commercial and homemade μ-SPE-PT tips filled with particle sorbent were compared. While the homemade tips enabled direct serum sample loading into the sorbent, protein precipitation (PP) had to be implemented before μ-SPE-PT procedure using commercial tips. Three μ-SPE-PT methods were developed and validated: method A: μ-SPE-PT with homemade tips; method B: PP + μ-SPE-PT with homemade tips; and method C: PP + μ-SPE-PT with commercial tips. Method A enabled a simple high-throughput approach (48 samples in 90 min) compared with methods B and C that required three-times longer time. However, PP increased the recoveries of protein-bound analytes and extracts purity in methods B and C. The matrix effects without internal standards correction for method C were significantly higher than those for the methods A and B.Compared with commercial tips, homemade tips filled with particles were found to be more suitable for drug analysis. Commercial tips tested in this study were found challenging but the conditions under which they could be applicable were also defined.
