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Low-cost microextraction and preconcentration procedures for biomedical applications
Vašátko, Jan ; Diviš, Pavel (referee) ; Dvořák, Miloš (advisor)
This thesis focuses on low-cost microextraction techniques and their application for purification and preconcentration of biological samples, specifically on the experimental study of supported liquid membrane (SLM) extraction. The described microextraction technique uses commercially available filtration plates as the extraction units and allows the extraction of basic drugs from biological samples of urine and blood (in the form of dried blood spots). The experimental part includes the optimization of microextraction conditions of basic drugs from real samples through a SLM coupled in-line to lab-made capillary electrophoresis. The basic optimization of microextraction conditions involved selecting the appropriate organic phase for membrane impregnation (1:1 mixture of ENB and DHE), appropriate agitation speed for sample convection during extraction (1000 rpm), and optimal ratio of donor to acceptor volumes for high preconcentration of the analytes (400:15 µL). After basic optimization, the effect of donor alkalization with NaOH on extraction recovery (ER) was investigated. For all matrices used (saline solution, undiluted human urine samples, human capillary blood eluted from dry blood spots with deionized water), the highest ER values were achieved using a neutral donor and an acidic acceptor. The extraction time (60 minutes) was optimized based on the time profile of the microextraction for 120 minutes. This optimized microextraction method is suitable for the determination of basic drugs in real matrices with sufficient sample clean-up, preconcentration and ER values.
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Application of capillary electrophoresis for dried blood spots analysis
Ryšavá, Lenka ; Tůma,, Petr (referee) ; Petr,, Jan (referee) ; Kubáň, Pavel (advisor)
Blood samples are normally analyzed in the form of plasma or serum. As an alternative for blood sampling and storage, dry blood spots (DBS) have been increasingly used in recent years. In the actual dissertation thesis, DBS are processed and analyzed by capillary electrophoresis (CE), which has been very rarely used for DBS analysis in the past. However, CE offers several features, given by the recent development of this technique, not available for standard analytical methods. Direct coupling of microextraction techniques to CE and simultaneous determination of analytes present in complex matrices leads to minimization or elimination of deficiencies in DBS analysis, for example the effect of sample matrix, hematocrit effect, and inhomogeneous distribution of analytes in DBS. In the simplest possible arrangement, sample treatment, injection, and analysis of DBS samples are achieved exclusively by a single commercial CE instrument. Application of newly developed fully soluble materials for DBS sampling offers better extraction efficiency and more accurate quantitation. The developed concepts include new methods for efficient treatment of DBS samples and their direct analysis without the need for operator intervention and they provide sufficient selectivity and sensitivity for the determination of important analytes not only in DBSs but also in other complex samples.
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Pathology and physiology of de novo purine synthesis.
Krijt, Matyáš ; Zikánová, Marie (advisor) ; Šebesta, Ivan (referee) ; Čajka, Tomáš (referee)
Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...
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Low-cost microextraction and preconcentration procedures for biomedical applications
Vašátko, Jan ; Diviš, Pavel (referee) ; Dvořák, Miloš (advisor)
This thesis focuses on low-cost microextraction techniques and their application for purification and preconcentration of biological samples, specifically on the experimental study of supported liquid membrane (SLM) extraction. The described microextraction technique uses commercially available filtration plates as the extraction units and allows the extraction of basic drugs from biological samples of urine and blood (in the form of dried blood spots). The experimental part includes the optimization of microextraction conditions of basic drugs from real samples through a SLM coupled in-line to lab-made capillary electrophoresis. The basic optimization of microextraction conditions involved selecting the appropriate organic phase for membrane impregnation (1:1 mixture of ENB and DHE), appropriate agitation speed for sample convection during extraction (1000 rpm), and optimal ratio of donor to acceptor volumes for high preconcentration of the analytes (400:15 µL). After basic optimization, the effect of donor alkalization with NaOH on extraction recovery (ER) was investigated. For all matrices used (saline solution, undiluted human urine samples, human capillary blood eluted from dry blood spots with deionized water), the highest ER values were achieved using a neutral donor and an acidic acceptor. The extraction time (60 minutes) was optimized based on the time profile of the microextraction for 120 minutes. This optimized microextraction method is suitable for the determination of basic drugs in real matrices with sufficient sample clean-up, preconcentration and ER values.
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Hollow fibre-liquid phase microextraction of basic drugs from dried dried blood spots
Dvořák, Miloš ; Miková, B. ; Šlampová, Andrea ; Kubáň, Pavel
Hollow fibre-liquid phase microextraction (HF-LPME) was applied for rapid and efficient extraction of basic drugs from dried blood spots (DBS). Basic drugs from DBS were first extracted into 10 mM NaOH solution, which acted as the HF-LPME donor solution, and simultaneously the neutralized drugs were transferred across the HF and preconcentrated in 10 mM HCl acceptor solution inside the HF lumen. Mixture (1:1, v:v) of 1-ethyl-2-nitrobenzene and dihexyl ether was used for impregnation of the porous HF membrane. Analytes in the acceptor solution were injected directly from the HF into capillary electrophoresis instrument for further separation, detection and quantification. Repeatability of the hyphenated analytical system ranged from 9.8 to 13.8 % (RSD) and enrichment factors of 52 – 78 were obtained for model basic drugs spiked to DBS extracts at a concentration level of 0.1 mg/L.
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