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Localization of double and triple donds positions in fatty acids methylesters by HPLC/APCI-MS2
Horká, Petra ; Vrkoslav, Vladimír ; Cvačka, Josef
Poloha a počet dvojných vazeb v alifatickém řetězci ovlivňuje fyzikální, chemické a biologické vlastnosti lipidů, avšak jednoznačné určení poloh těchto násobných vazeb je problematické. V roce 2012 byla publikována metoda kapalinové chromatografie s chemickou ionizací za atmosférického tlaku s hmotnostní detekcí (HPLC/APCI-MS) [1] vhodná pro analýzu polohy dvojných vazeb využívající tvorby molekulových aduktů [M + C3H5N]+. při APCI za přítomnosti acetonitrilu. Methylestery mastných kyselin (FAMEs) byly analyzovány HPLC/APCI-MS2 se 100% acetonitrilem jako mobilní fází. Při APCI docházelo v plynné fázi k tvorbě reaktivních částic, vznikajících ze složek mobilní fáze, které se vázaly na násobné vazby v alifatickém řetězci FAMEs za vzniku aduktu [M + C3H5N]+.. Kolizně indukovanou disociací (CID) aduktu [M + C3H5N]+., se získaly diagnostické fragmenty určující polohu (polohy) násobných vazeb. Zmíněná metoda byla evaluována pro běžně analyzované polynenasycené FAMEs s tradiční polohu dvojných vazeb (methylene-interrupted).
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Structural characterization of fatty acids with triple bond and unusual double bond positions by HPLC/APCI-MS2
Horká, Petra ; Vrkoslav, Vladimír ; Cvačka, Josef
Fatty acid methyl esters (FAMEs) were analyzed by reversed-phase HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry. Acetonitrile was used as mobile phase. The gas-phase reactions of acetonitrile and unsaturated FAMEs in the APCI source provided [M + C3H5N]+• adducts. Subsequent MS/MS analysis of these ions gave diagnostic fragments. The formation and fragmentation of the acetonitrile-related adducts were utilized for the structural characterization of FAME with isolated double bonds in (Z)-9,19-hexacosadienoic acid methyl ester, FAME with conjugated double bonds in (Z)-9,11-octadecadienoic acid methyl ester and FAME with a triple bond in 9-octadecynoic acid methyl ester.
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Characterization of lipids with four ester bonds in vernix caseosa by HPLC / MS
Tomášková, Sabina ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
Diploma thesis is devoted to the analysis of triacylglycerol estolides (TG-est) by HPLC/MS. These lipids contain four ester bonds. TG-est are part of some vegetable oils and in mammals function as a storage form of metabolically important fatty acid estolides. The aim of this thesis was to prove the existence of TG-est in vernix caseosa and explore the possibilities of analysis in this material. Standards of TG-est were studied with using high-resolution and tandem mass spectrometry. Was suggested and optimized HPLC method for TG-est in a reverse nonaqueous system with the use of column Kinetex C18 and gradient elution with acetonitrile and 2-propanol. This method was used for analysis vernix caseosa fraction, and it was shown that TG-est are present in the sample. Attempts to simplify the lipid sample before analysis HPLC/MS by TLC fractionation were not very successful.
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Separation and identification of lipopeptides by chromatography and mass spectrometry
Pravdová, Adéla ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
5 Abstract Protein lipidation occupies an important place in the post-translational modification group. It plays an important role in the processes of cell differentiation or synaptic transmission. Changes in the presence of lipidated proteins may in some cases indicate the appearance of human diseases such as Huntington's disease, schizophrenia, or cancer. For this reason, their analysis has become desirable, but also problematic due to its specific characteristics. This work is focused on the development of conditions for measurement of lipopeptides produced by cleavage of proteins containing lipomodification. Two separation procedures were tested, in which the aim of work was to enrich the lipopeptides and remove as many other molecules as possible from the samples. The first test method - phase interface separation, using water and chloroform, was able to concentrate the lipopeptides on the phase interface and separate some of the added unmodified peptides from the mixture. The second method of separation was a solid phase using a C18 stationary phase. As a more suitable solid phase extraction variant, elution with solutions containing acetonitrile with trifluoroacetic acid was chosen as compared to solutions containing isopropyl alcohol. With the modified elution procedure, it was possible to separate...
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Temperature-programed micro-HPLC analysis of fatty acid methyl esters with APCI-MS detection
Vrkoslav, Vladimír ; Rumlová, Barbora ; Cvačka, Josef
HPLC/APCI-MS analysis at microliters-per-minute flow rates was optimized for separation of fatty acid methyl esters (FAMEs). HPLC C18 column with an internal diameter of 0.3 mm and isocratic elution using 99.9 % acetonitrile and 0.1% formic acid were employed. Standard APCI ion source was suitable for detection of FAMEs at 10 μl/min flow rate with the detection limit of micrograms-per-milliliter. APCI-MS spectra with predominant [M + H]+ molecular adducts were observed. The main advantage of micro-flow measurements is the possibility of using a temperature gradient, which significantly reduces retention times of FAMEs with longer aliphatic chain. The significant reduction of solvent consumption is also an important economic and environmental advantage. The positions of double bonds in FAME chains were established using acetonitrile-related adducts and tandem mass spectrometry. The optimized method was applied for analysis of FAMEs in triacylglycerol fraction of black currant seeds oil.
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