In-quest of Biomarkers For Alzheimer’s Disease And Pharmacokinetic Profile of Anticancer Agents Using Lc-ms In Human Plasma

In this dissertation work, a semi-quantitative assay using a lipidomics approach and an absolute quantitative assay using liquid chromatography-mass spectrometry techniques were developed (LC-MS). In the lipidomics approach, ultra-high pressure liquid chromatography in tandem with quadrupole time of flight (UHPLC-QTOF) mass spectrometry was used to profile, compare and quantitate the human plasma lipids in Alzheimer’s disease subjects (AD) and normal cognitive controls (NCCs). The purpose of this study is to identify potential plasma lipid markers of AD and to explore the relationships between AD and lipid pathways in humans by using bioinformatic tools. The plasma samples of study subjects were first spiked with a mix of 12 synthetic-lipid internal standards, then total lipids were extracted using a modified Blight-Dyer method and fractionated into phospholipids (PL) and neutral lipids (NL) using the aminopropyl cartridge. The UHPLC-QTOF-MS/MS data were processed and analyzed with corrections of retention time and mass shifts. Molecular features were extracted for lipid identifications based on mass-to-charge ratios, isotopic patterns, adducts, and charge states. Venn diagrams were plotted to group the common and the unique features of lipids between AD patients and NCCs. The common significant molecular features between these two study groups were analyzed using principal component analysis (PCA), partial least vii squares-discriminant analysis (PLS-DA), and non-parametric Wilcoxon rank-sum t-test with false discovery rate calculated p-values. Quantitative lipidomics was performed on the twenty-eight identified significant common lipids. Our results indicate these significant common lipids between AD patients and NCCs were belong to glycerophospholipids, glycerolipids, and sphingolipids. Gene-lipid centric pathway analysis was performed on these significant lipids to obtain the implicated pathways in AD and to understand it’s relation to AD. In absolute quantitation work, an assay using a liquid chromatography system in tandem with triple quadrupole mass spectrometry (LC-QqQ) was developed and validated to measure the O6Benzylguanine (O6BG) and its metabolite, 8-oxo-O6Benzylguanine (8-oxoO6BG) in human plasma. O6BG and 8-oxo-O6BG along with the analog internal standard, pCl-O6BG, were extracted from alkalinized human plasma by liquid-liquid extraction (LLE) using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 mL/min. Quantification was performed using multiple-reaction-monitoring (MRM) mode with positive ion-spray ionization. The linear calibration ranges of the method for O6BG and 8-oxo-O6BG were 1.25 to 250 ng/mL and 5.00 to 1.00 x 103 ng/mL respectively with acceptable assay accuracy, precision, recovery and matrix factor. The method was validated as per the Food and Drug Administration (FDA) guidelines and was applied to the measurement of O6BG and 8-oxo-O6BG in patient plasma samples from the prior phase I clinical trial

Evaluation of cetane values of glycerolipids extracted from algae Scenedesmus dimorphus grown in various salinity concentrations using gas chromatography and mass spectrometry (GC-MS)

Algae\u27s ability to store lipids, renewability, and potentially safer for the environment has made it a promising alternative fuel source. An industry rating for a biofuel\u27s potential is the cetane value, which is a measure of a fuel\u27s quality related to various glycerolipid concentrations. Growing conditions will affect lipid profile in algae, thereby affecting the cetane value. This project will attempt to identify changes in the centane value of the algae Scenedesmus dimorphus grown in various salinity concentrations. Scenedesmus dimorphus is the algae chosen for this experiment because of its ability to rapidly grow under harsh conditions. In this experiment the growth conditions were controlled in bioreactors and shaker baths. Total lipids were extracted from dry mass algae with the Bligh-Dyer method, which allows for the extraction of the glycerolipids with the solid phase extraction method. Upon the final extraction, a transesterification reaction is carried out in order to convert the glycerolipids into FAME (fatty acid methyl esters), which allows the GC- MS (gas chromatography and mass spectrometry) instrument to better quantify the lipid concentration.https://engagedscholarship.csuohio.edu/u_poster_2017/1044/thumbnail.jp

Glycerolipid Analysis of Adaptation to Saline Changes in the Culture Conditions of Algae, Scenedesmus dimorphus, by GC-MS

Since fossil fuels are decreasing over time an alternative energy source will be soon required. The algae, Scenedesmus dimorphus, grows in freshwater and is known for its fast growth of glycerolipid content which is used for biodiesel production. After the algae is grown in optimal conditions, the released fatty acids and glycerolipids are transformed into fatty acid methyl esters (FAMEs) which are used as biodiesel. The FAMEs were quantitatively determined by gas chromatography mass spectrometry (GC-MS) to determine the total glycerolipid content in the different algae samples. The samples that were analyzed include freshwater controls and saline adapted samples. Analysis also included using a calibration curve with calibrators ranging from 0.500 to 1,000 μM. In the calibration curve and algae samples, a heavy isotope internal standard of C16:0-d-2 was used to determine the accuracy of the results. Results: total percent glycerolipid content in each sample ranged from 2.95-8.5%. The lower range of results could be due to the control 2-L bottle which had no CO2since it did not have optimal growth conditions, and the bioreactor control was low possibly due to low light intensity. However, the 1.005 TSG for the 2-L bottle was similar to the controls which proves that adaptation is successful. Also, the bioreactor control was lower than the 1.005 TSG saline sample in the bioreactor which shows that increasing the salt concentration and controlling the environment is useful for saline adaptation.https://engagedscholarship.csuohio.edu/u_poster_2016/1036/thumbnail.jp

IN-QUEST OF BIOMARKERS FOR ALZHEIMER’S DISEASE AND PHARMACOKINETIC PROFILE OF ANTICANCER AGENTS USING LC-MS IN HUMAN PLASMA

In this dissertation work, a semi-quantitative assay using a lipidomics approach and an absolute quantitative assay using liquid chromatography-mass spectrometry techniques were developed (LC-MS). In the lipidomics approach, ultra-high pressure liquid chromatography in tandem with quadrupole time of flight (UHPLC-QTOF) mass spectrometry was used to profile, compare and quantitate the human plasma lipids in Alzheimer’s disease subjects (AD) and normal cognitive controls (NCCs). The purpose of this study is to identify potential plasma lipid markers of AD and to explore the relationships between AD and lipid pathways in humans by using bioinformatic tools. The plasma samples of study subjects were first spiked with a mix of 12 synthetic-lipid internal standards, then total lipids were extracted using a modified Blight-Dyer method and fractionated into phospholipids (PL) and neutral lipids (NL) using the aminopropyl cartridge. The UHPLC-QTOF-MS/MS data were processed and analyzed with corrections of retention time and mass shifts. Molecular features were extracted for lipid identifications based on mass-to-charge ratios, isotopic patterns, adducts, and charge states. Venn diagrams were plotted to group the common and the unique features of lipids between AD patients and NCCs. The common significant molecular features between these two study groups were analyzed using principal component analysis (PCA), partial least vii squares-discriminant analysis (PLS-DA), and non-parametric Wilcoxon rank-sum t-test with false discovery rate calculated p-values. Quantitative lipidomics was performed on the twenty-eight identified significant common lipids. Our results indicate these significant common lipids between AD patients and NCCs were belong to glycerophospholipids, glycerolipids, and sphingolipids. Gene-lipid centric pathway analysis was performed on these significant lipids to obtain the implicated pathways in AD and to understand it’s relation to AD. In absolute quantitation work, an assay using a liquid chromatography system in tandem with triple quadrupole mass spectrometry (LC-QqQ) was developed and validated to measure the O6Benzylguanine (O6BG) and its metabolite, 8-oxo-O6Benzylguanine (8-oxoO6BG) in human plasma. O6BG and 8-oxo-O6BG along with the analog internal standard, pCl-O6BG, were extracted from alkalinized human plasma by liquid-liquid extraction (LLE) using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 mL/min. Quantification was performed using multiple-reaction-monitoring (MRM) mode with positive ion-spray ionization. The linear calibration ranges of the method for O6BG and 8-oxo-O6BG were 1.25 to 250 ng/mL and 5.00 to 1.00 x 103 ng/mL respectively with acceptable assay accuracy, precision, recovery and matrix factor. The method was validated as per the Food and Drug Administration (FDA) guidelines and was applied to the measurement of O6BG and 8-oxo-O6BG in patient plasma samples from the prior phase I clinical trial

Evaluation of cetane values of glycerolipids extracted from algae Scenedesmus dimorphus grown in various salinity concentrations using gas chromatography and mass spectrometry (GC-MS)

Algae\u27s ability to store lipids, renewability, and potentially safer for the environment has made it a promising alternative fuel source. An industry rating for a biofuel\u27s potential is the cetane value, which is a measure of a fuel\u27s quality related to various glycerolipid concentrations. Growing conditions will affect lipid profile in algae, thereby affecting the cetane value. This project will attempt to identify changes in the centane value of the algae Scenedesmus dimorphus grown in various salinity concentrations. Scenedesmus dimorphus is the algae chosen for this experiment because of its ability to rapidly grow under harsh conditions. In this experiment the growth conditions were controlled in bioreactors and shaker baths. Total lipids were extracted from dry mass algae with the Bligh-Dyer method, which allows for the extraction of the glycerolipids with the solid phase extraction method. Upon the final extraction, a transesterification reaction is carried out in order to convert the glycerolipids into FAME (fatty acid methyl esters), which allows the GC- MS (gas chromatography and mass spectrometry) instrument to better quantify the lipid concentration.https://engagedscholarship.csuohio.edu/u_poster_2017/1044/thumbnail.jp