Leukocytes and plasma samples from healthy controls (n=10), patients before treatment (n=9), treated with hydroxyurea (n=5), imatinib (n=24), dasatinib (n=8) and nilotinib (n=10) were prepared from whole blood and metabolites were extracted by the chloroform/methanol/water mixture. Analysis of 400 metabolites was performed using high performance liquid chromatography (Ultimate 3000, Dionex) coupled with tandem mass spectrometry (QTRAP 5500, AB Sciex). Metabolites were quantified using MultiQuant 3.0 software and statistically evaluated in R programme language with statistics packages. For metabolisation study plasma samples were taken from patients on treatment by imatinib (24h after dose of 400mg). Separation of imatinib metabolites was performed on Phenomenex Kinetex C18 column using UltiMate 3000 RS (Thermo Scientific) liquid chromatography. For detection in full scan mode and MS2 fragmentation experiments Orbitrap Elite mass spectrometer (Thermo Scientific) based on exact mass measurement was used. Data were evaluated using MetWorks 1.3 SP3 and Mass Frontier 7.0 software.
Total 170 metabolites were identified on the basis of its transition and retention time in all leukocyte and plasma samples. Significant changes in metabolic pathways were found. Both unsupervised and supervised statistic methods (PCA, PLS-DA, OPLS-DA, cluster analysis) separated all the studied groups. In plasma samples more than 90 potential metabolites in concentration range of 4 orders of magnitude were found. All metabolites were identified by exact m/z values and confirmed by MS2 and MS3 exact m/z fragmentation experiments. Changes in profiles of several metabolisations between patients were followed.
The work was supported by grant of IGA Ministry of Health, Czech Republic NT12218-4/2011.
Leukocytes and plasma samples from healthy controls (n=10), patients before treatment (n=9), treated with hydroxyurea (n=5), imatinib (n=24), dasatinib (n=8) and nilotinib (n=10) were prepared from whole blood and metabolites were extracted by the chloroform/methanol/water mixture. Analysis of 400 metabolites was performed using high performance liquid chromatography (Ultimate 3000, Dionex) coupled with tandem mass spectrometry (QTRAP 5500, AB Sciex). Metabolites were quantified using MultiQuant 3.0 software and statistically evaluated in R programme language with statistics packages. For metabolisation study plasma samples were taken from patients on treatment by imatinib (24h after dose of 400mg). Separation of imatinib metabolites was performed on Phenomenex Kinetex C18 column using UltiMate 3000 RS (Thermo Scientific) liquid chromatography. For detection in full scan mode and MS2 fragmentation experiments Orbitrap Elite mass spectrometer (Thermo Scientific) based on exact mass measurement was used. Data were evaluated using MetWorks 1.3 SP3 and Mass Frontier 7.0 software.
Total 170 metabolites were identified on the basis of its transition and retention time in all leukocyte and plasma samples. Significant changes in metabolic pathways were found. Both unsupervised and supervised statistic methods (PCA, PLS-DA, OPLS-DA, cluster analysis) separated all the studied groups. In plasma samples more than 90 potential metabolites in concentration range of 4 orders of magnitude were found. All metabolites were identified by exact m/z values and confirmed by MS2 and MS3 exact m/z fragmentation experiments. Changes in profiles of several metabolisations between patients were followed.
The work was supported by grant of IGA Ministry of Health, Czech Republic NT12218-4/2011.