Stanovení acetylcholinu pomocí LC-MS ve vzorcích mozkových mikrodialyzátů LC-MS/MS

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Ivo Vrobel Supervisors: Prof. RNDr. Petr Solich, CSc; Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Eastern Finland in Kuopio Title of master's thesis: LC-MS/MS analysis of acetylcholine in brain microdialysis samples Novel fast and simple LC-MS/MS method of ACh quantification in brain microdialysis samples utilizing stable-isotope-labeled IS was developed. The chromatographic step is based on revered-phase mode of pentafluorophenylpropyl (PFPP) column. The satisfactory retention of ACh is achieved with highly aqueous mobile phase containing 0.05% of the ion-pairing agent TFA and 4% of ACN in 4 min analytical run. Ionization of ACh and IS with low background noise and tolerant towards use of TFA was performed with atmospheric pressure thermospray ionization (APTSI). The selectivity of ACh and IS detection was obtained by SRM modes of MS/MS in the linear ion trap mass analyzer. The performance of developed method was cross validated to the validated method used in the laboratory for ACh measurements. The set of microdialysis...Univerzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Katedra analytické chemie Kandidát: Ivo Vrobel Školitelé: Prof. RNDr. Petr Solich, CSc; Katedra analytické chemie, Farmaceutická fakulta, Univerzita Karlova v Praze Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Katedra farmaceutické chemie, Škola farmacie, Univerzita Východního Finska v Kuopiu Název diplomové práce: Stanovení acetylcholinu pomocí LC-MS/MS ve vzorcích mozkových mikrodialyzátů Byla vyvinuta nová rychlá a jednoduchá analytická metoda na stanovení acetylcholinu (ACh) ve vzorcích mozkových mikrodialyzátů založena na kapalinové chromatografii spojené s tandemovou hmotnostní spektrometrií (LC-MS/MS), využívající isotopicky značený vnitřní standard. Pentafluorofenylpropylová (PFPP) stacionární fáze spolu s mobilní fází složené z 0.05% vodného roztoku TFA a 4% ACN zabezpečuje dostatečnou retenci ACh a jeho separaci od anorganických solí. Ionizace ACh a vnitřního standardu, odolná vůči použití TFA, byla dosažena pomocí ionizace termosprejem za atmosférického tlaku (APTSI). Detekce ACh a vnitřního standardu byla docílena pomocí selektivního reakčního monitoringu (SRM) v lineární iontové pasti. Křížová validace byla provedena porovnáním nově vyvinuté metody s validovanou metodou rutinně používanou ke stanovení ACh. Soubor...Department of Analytical ChemistryKatedra analytické chemieFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Stanovení acetylcholinu pomocí LC-MS ve vzorcích mozkových mikrodialyzátů LC-MS/MS

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Ivo Vrobel Supervisors: Prof. RNDr. Petr Solich, CSc; Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Eastern Finland in Kuopio Title of master's thesis: LC-MS/MS analysis of acetylcholine in brain microdialysis samples Novel fast and simple LC-MS/MS method of ACh quantification in brain microdialysis samples utilizing stable-isotope-labeled IS was developed. The chromatographic step is based on revered-phase mode of pentafluorophenylpropyl (PFPP) column. The satisfactory retention of ACh is achieved with highly aqueous mobile phase containing 0.05% of the ion-pairing agent TFA and 4% of ACN in 4 min analytical run. Ionization of ACh and IS with low background noise and tolerant towards use of TFA was performed with atmospheric pressure thermospray ionization (APTSI). The selectivity of ACh and IS detection was obtained by SRM modes of MS/MS in the linear ion trap mass analyzer. The performance of developed method was cross validated to the validated method used in the laboratory for ACh measurements. The set of microdialysis..

Structural elucidation of novel biomarkers of known metabolic disorders based on multistage fragmentation mass spectra

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Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib

Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib

Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib