31 research outputs found
Nontargeted Lipidomic Characterization of Porcine Organs Using Hydrophilic Interaction Liquid Chromatography and Off-Line Two-Dimensional Liquid Chromatography-Electrospray Ionization Mass Spectrometry
Abstract Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi-permeability and fluidity of membranes, signaling activity, anti-inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M-H] -in the negative-ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy-and hydroxy-fatty acyls. The detailed analysis of identified lipid classes using reversed-phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double-bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide
Analysis of natural mixtures of triacylglycerols by HPLC/MS technique
Analýza triacylglycerolů v rostlinných olejích pomocí RP-HLPC ve spojení s APCI/MS. Kvantitativní analýza triacylglycerolů pomocí UV detektoru, ELSD detektoru, APCI-MS a porovnání výsledků získaných těmito technikami.Katedra analytické chemieDokončená práce s úspěšnou obhajobo
Characterization of triacylglycerol composition in natural samples using HPLC/MS technique
Tato disertační práce se zabývá charakterizací směsí triacylglycerolů v rostlinných olejích. V první části práce jsou optimalizovány podmínky HPLC separace triacylglycerolů ve směsích. V druhé části jsou porovnávány běžné HPLC detektory pro analýzu triacylglycerolů. Třetí část je věnována HPLC/MS analýze řady důležitých rotlinných olejích v potravinářském a chemickém průmyslu.This dissertation is devoted to the characterization of triacylglycerols in plant oils. In the first part, the HPLC conditions are optimized for analysis of triacylglycerols in mixtures. The second part is devoted to the comparison of common HPLC detectors for analysis of triacylglycerols. The last part is devoted to the HPLC/MS characterization of triacylglycerols in important plant oils in nutrition and industry.Katedra analytické chemieDokončená práce s úspěšnou obhajobo
Relative quantitation of phosphatidylcholines with interfered masses of protonated and sodiated molecules by matrix-assisted laser desorption/ionization mass spectroscopy
This work presents a computational method for deconvolution of interfered peaks
of protonated and sodiated phosphatidylcholines (PCs) to solve the problem of
relative quantitation of these PCs. The method is based on the fact that adducts
of PCs with proton and sodium ion give unique characteristic peaks in tandem
mass spectra. The intensities of these peaks were used to find out contribution of
protonated and sodiated PCs to the formation of interfered peak in full-scan mass
spectrum and, further, to correlate peak intensities that correspond to PCs in fullscan
mass spectrum with PC’s concentrations. The model was calibrated on
synthesized PCs and verified for applicability in the range of Na+ ion
concentrations from trace to physiological one
Lipidomic analysis of porcine organs using hydrophilic interaction liquid chromatography and mass spectroscopy
The identification and quantitation of all lipids in complex biological tissues is the
first step towards the understanding how lipids function in a biological system and
the elucidation of the mechanism of lipid-related diseases including obesity,
atherosclerosis, cancer, cardiovascular diseases, etc. Our optimized nontargeted
method using hydrophilic interaction liquid chromatography (HILIC) coupled to
electrospray ionization mass spectrometry (ESI-MS) was used for the
characterization of the lipidome, mainly glycerophospholipids and sphingolipids
in porcine brain, heart, kidney, liver, lung, spinal cord, spleen and stomach.
Individual lipid classes are quantified based on the peak integration of individual
lipid classes separated in the HILIC mode multiplied by their response factors and
correlated by sphingosylethanolamine (d17:1/12:0) as a single internal standard.
Subsequently, relative abundances of deprotonated molecules [M-H]– in negativeion
ESI mass spectra are used for determination of lipid species concentration
inside individual classes. The approach using [M-H]– ions enables to obtain detailed information about phosphatidylethanolamines and their different forms
of fatty acyl linkage on the glycerol skeleton (plasmalogens and ether analogs),
phosphatidylinositols and hexosylceramides in studied organs. Our results
provide important knowledge about lipid representations in vital porcine organs
and can be applied for future metabolic studies on human to investigate serious
lipid-related human disorders
Estimation of stereospecific fatty acid distribution in vegetable oils from liquid chromatography data
The fatty acid distribution in triacylglycerols (TG), the major components of vegetable oils, affects the physical properties of oils and their physiological effects as components of the human diet. A computational scheme is proposed for estimating the stereospecific distribution of fatty acids in TG on the basis of the data from HPLC analysis, using a model for TG synthesis. A new model for the synthesis is proposed which, in contrast to the widely used model published independently by Vander Wal and Coleman, enables us to simulate the asymmetrical fatty acid distribution at the sn-1 and sn-3 stereochemical positions. The computational scheme combined with either literature models or the new model was validated by its application to several TG profiles and comparison of the resulting fatty acid distribution at stereochemical positions with experimental data from the literature. The stereospecific composition of TG was calculated using the new model for nine vegetable oils
Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry
Vysokoúčinná superkritická fluidní chromatografie ve spojení s hmotnostní spektrometrií s elektrosprejovou ionizací (UPHSFC/ESI-MS) je technika vhodná pro nepolární i polární látky, což je v lipidomické analýze velkou výhodou. Lipidy v krevní plasmě byly separovány do lipidových tříd pomoci kolony Acquity BEH UPC2 (100 mm × 3 mm, 1,7 µm) a gradientové eluce se složením mobilní fáze CO2 a směsi methanol – voda (99:1 v/v) obsahující 30 mM octan amonný. Během 6 minutové analýzy došlo k rozdělení lipidů do 9 tříd. Následná kvantifikace by měla sloužit ke srovnání lipidů v plazmě zdravých a nemocných jedinců.Ultrahigh-Performance Supercritical Fluid Chromatography with electrospray ionization mass spectrometry (UPHSFC/ESI-MS) is suitable for non-polar and polar substances, which is a great advantage in lipidomic analysis. Lipids in plasma were separated into lipid classes using an Acquity BEH UPC2 column (100 mm x 3 mm, 1,7 μm) and a gradient elution of the mobile phase of CO2 and methanol-water mixture (99:1 v/v) containing 30 mM ammonium acetate. During the 6 minute analysis lipids were divided into 9 lipid classes. Subsequent quantification should serve to compare lipids in the plasma of healthy and diseased individuals
Hydrofilní interakční chromatografie ve spojen s hmotnostní spektrometrií pro stanovení kyseliny fosfatidové, fosfatidylserinu a jejich lysoderivátů.
The goal of this work is a systematic optimization of hydrophilic interaction liquid chromatography (HILIC) separation of acidic lipid classes (namely phosphatidic acids-PA, lysophosphatidic acids-LPA, phosphatidylserines-PS and lysophosphatidylserines-LPS) and other lipid classes under mass spectrometry (MS) compatible conditions. The main parameters included in this optimization are the type of stationary phases used in HILIC, pH of the mobile phase, the type and concentration of mobile phase additives. Nine HILIC columns with different chemistries (unmodified silica, modified silica using diol, 2-picolylamine, diethylamine and 1-aminoanthracene and hydride silica) are compared with the emphasis on peak shapes of acidic lipid classes. The optimization of pH is correlated with the theoretical calculation of acidobasic equilibria of studied lipid classes. The final method using the hydride column, pH 4 adjusted by formic acid and the gradient of acetonitrile and 40 mmol/L of aqueous ammonium formate provides good peak shapes for all analyzed lipid classes including acidic lipids. This method is applied for the identification of lipids in real samples of porcine brain and kidney extracts.Cílem této práce byla optimalizace separace polárních lipidů zejména kyseliny fosfatidové, fosfatidylserinu a jejich lysoderivátů. Optimalizace byla provedena pomocí kapalinové chromatografie (HILIC) ve spojení s hmotnostní spektrometrií (ESI). Byly testovány kolony a vliv pH a koncentrace aditiv. Vyvinutá metoda byla použita pro analýzu mozku a ledvin prasete
HILIC/MS analysis of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipidclasses
Lipidomika je obor zabývající se studiem biochemických drah lipidů v biologických systémech. Cílem lipidomiky je pochopení funkce lipidů v biologickém metabolismu, vysvětlení mechanismu vzniku a průběhu některých metabolických chorob. Cílem této práce byla optimalizace separace co největšího počtu tříd lipidů s využitím kapalinové chromatografie hydrofilních interakcí (HILIC) za podmínek vhodných pro spojení s hmotnostní spektrometrií. Zvláštní důraz byl při optimalizaci HILIC podmínek kladen na ty třídy polárních lipidů, které se dosud touto technikou nedařilo analyzovat, fosfatidylseriny, kyseliny fosfatidové a jejich lysoderiváty. V této práci byly studovány vlivy typu náplně kolony, složení mobilní fáze, aditiva, pH mobilní fáze, teploty kolony a sklonu gradientu na účinnost separace. Největší vliv na separaci těchto tříd lipidů má typ náplně kolony a pH mobilní fáze. Jednotlivé třídy lipidů byly identifikovány díky spojení s hmotnostní spektrometrií (MS) s využitím ionizace elektrosprejem v kladném i záporném módu záznamu iontů. Optimalizovaná metoda byla použita pro charakterizaci jednotlivých lipidů v extraktu prasečího mozku a ledviny.Lipidomics is a discipline dealing with studies of biochemical pathways of lipids in biological systems. The goal of lipidomics is to understand the function of lipids in biological metabolism, to explain the mechanism of the development and progress of some metabolic diseases. The aim of this work was the optimization of hydrophilic interaction liquid chromatography (HILIC) separation of the greatest number of classes of lipids using conditions compatible with the mass spectrometry. A special attention of this optimization has been paid to lipid classes not yet analyzed by HILIC so far, i.e. phosphatidylserines, phosphatidic acids and their lyso-derivatives. Effects of stationary phases, the composition of mobile phase, additives, pH value of the mobile phase, separation temperature and the gradient steepnesshave been studied to achieve a betterseparation efficiency. The highest impact on the separation of lipid classes hadthe type of stationary phase and pH value of the mobile phase. All lipid classes were identified by using MS in connection with the electrospray ionization in positive-ion and negative-ion mode.The optimized method was used to describe particular lipids in extracts of porcine brain and kidney
Stanovení regioisomerů trigliceridů pomocí MDS
RATIONALE: Triacylglycerols (TG) contain three fatty acyls attached to the glycerol backbone in stereochemically numbered positions sn-1, 2 and 3. Isobaric TG with exchanged fatty acyl chains in positions sn-1/3 vs. sn-2 are referred to as regioisomers and the determination of their regioisomeric ratios is important for nutrition purposes. METHODS: Differential mobility spectrometry (DMS) coupled to electrospray ionization mass spectrometry (ESI-MS) is applied for the separation of simple unsaturated TG regioisomers extracted from porcine adipose tissue using their silver-ion molecular adducts. RESULTS: Four pairs of TG regioisomers containing combinations of unsaturated and saturated fatty acyl chains are successfully separated using DMS with 1-butanol or 1-propanol as the chemical modifier. Various experimental parameters are carefully optimized, such as the separation and compensation voltages applied to DMS electrodes, the type and flow rate of chemical modifier and the dwell time of analyte ions in the DMS cell. The optimized DMS approach is used for the characterization of TG regioisomers in less than one minute, compared to tens of minutes typical for silver-ion or reversed-phase high-performance liquid chromatography/mass spectrometry approaches. CONCLUSIONS: The application of this method for the characterization of TG regioisomers in porcine adipose tissue shows the method suitability for analyses of other animal fats.Triglyceridy obsahují tři mastné kyseliny které jsou spojené pře glycerolový řetězec. Pro separaci jednoduchých nenasycených TG regioisomerů získaných z prasečí tukové tkáně je využita DMS spojená s ESI-MS. Byly optimalizovány parametery DMS. Metoda je vhodná pro analýzu jiných živočišných tuků