LC-MS/MS Method Development for Quantitative Analysis of Cyanogenic
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LC-MS/MS METHOD DEVELOPMENT FOR QUANTITATIVE ANALYSIS OF CYANOGENIC GLYCOSIDES IN ELDERBERRY AND LIPID PEROXIDATION PRODUCTS IN MICE _______________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ by MICHAEL KWAME APPENTENG Dr. C. Michael Greenlief, Dissertation Supervisor May 2021 © Copyright by Michael Kwame Appenteng 2021 All Rights Reserved The undersigned, appointed by the dean of the Graduate School at the University of Missouri-Columbia, have examined the dissertation entitled. LC-MS/MS Method Development for Quantitative Analysis of Cyanogenic Glycosides in Elderberry and Lipid Peroxidation Products in Mice presented by Michael Kwame Appenteng, a candidate for the degree of Doctor of Philosophy in Chemistry, and hereby certify that, in their opinion, it is worthy of acceptance. Professor C. Michael Greenlief Professor Silvia S. Jurisson Professor John D. Brockman Professor Chung-Ho Lin DEDICATION This dissertation is dedicated to my dear wife, Naa Adjorkor Sowah and delightful son, Frank Cudjoe Appenteng, for their love, unwavering support, and inspiration. Also, to my selfless parents, Frank Cudjoe and Comfort Asaku, who in little, never compromised on quality education. ACKNOWLEDGMENTS I have received a great deal of support and assistance throughout my research, dissertation writing and entire graduate school experience. I would first like to express my deepest appreciation to my advisor, Dr. C. Michael Greenlief for his unparalleled mentorship, patient and invaluable scientific insights and contributions to my work. Being part of the Greenlief research group has exposed me to great research projects, which have shaped me into an effective and efficient researcher. This has been a life changing experience and I am deeply indebted to him for his unwavering support and profound belief in me and my abilities. I would also like to thank the rest of my dissertation committee members (Silvia S. Jurisson, Chung-Ho Lin and John D. Brockman) who contributed immensely to the success of my research through their nurturing, constructive criticism and wise counsel. I am also very grateful to Renee D. JiJi who contribute immensely to my work as a committee member before leaving the University. I would also like to extend my gratitude to all members of the collaborative research team (Research Council Grant) especially Dr. Grace Sun, Dr. David Q. Beversdorf, Dr. Kevin L. Fritsche, Dr. Jiankun Cui, Li Runting and Kille Braina, for their rich insight and enthusiasm towards research. Special thanks to Dr. Andrew L. Thomas at the Division of Plant Sciences, Southwest Research Center, University of Missouri, Mt. Vernon, for providing different varieties of American elderberry samples at different growth stages for analysis. I would also like to specially acknowledge Dr. Brain Mooney and Dr. Pei Liu at the Gehrke Proteomics Center. I learned much as a graduate research assistant under their supervision. Their patience, clarity of direction and decisiveness in research work was second to none. Under their watch, I was nurtured to excel not only in team research but also as an independent thinker. I also want to acknowledge present and past members of the Greenlief research group especially Mitch Johnson and Bo Yang for ii their support and for enabling an intellectually stimulating environment during my research. Mitch Johnson was very instrumental in walking me through the basics of method development using the Xevo TQS-MS instrument. I cannot forget the assistance and input from undergrads like Ritter Krueger and Richard Bell who worked with me to optimize the picrate method. I am also very grateful to the Chemistry Department and Gehrke Proteomics Center for providing the necessary facilities, instruments, and equipment for my research. Notable mentions include Jerry Brightwell and Holly Oswald who provided all the needed help and support. To my parents Mr. Frank Cudjoe and Mrs. Comfort Asaku and all my siblings, I am extremely grateful for your continuous prayers, support, and love. To my spiritual and God parents, Reverend Mr. and Mrs. Acheampong and Dr. Mr. and Mrs. Appenteng respectively, I say, a big thank you for your prayers and wise counsel all these years. I cannot begin to express my immense appreciation to my lovely wife, Naa Adjorkor Sowah and son, Frank Cudjoe Appenteng. You always give me a reason to smile each morning and keep my focus amidst any challenges. I am extremely grateful to you. Finally, to Jehovah God, I say thank you so much for your grace, direction, good health, sound mind and the gift of life without which none of this success would be possible. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................ ii LIST OF ILLUSTRATIONS .......................................................................................... xi LIST OF TABLES ......................................................................................................... xvi ABSTRACT ................................................................................................................... xvii CHAPTER 1: Introduction .............................................................................................. 1 1.1 American Elderberry ................................................................................................. 1 1.2 Dietary Supplements ................................................................................................. 3 1.2.1 Botanical / Herbal Dietary Supplements .......................................................... 3 1.2.1.1 Herbal Supplements sales in US ........................................................... 4 1.3 Plant Metabolites ...................................................................................................... 6 1.3.1 Plant Secondary Metabolites or Bioactive Compounds ................................... 6 1.3.1.1 Phenolic compounds ............................................................................. 7 1.3.1.2 Cyanogenic glycosides ......................................................................... 9 1.4 Fatty Acids .............................................................................................................. 11 1.4.1 Essential Fatty Acids ...................................................................................... 12 1.4.1.1 Docosahexaenoic acid and Arachidonic Acid .................................... 13 1.4.2 Phospholipids ................................................................................................. 14 1.4.3 Lipids peroxidation products .......................................................................... 15 iv 1.5 Chromatography ..................................................................................................... 17 1.5.1 Chromatography Overview ............................................................................ 17 1.5.2 Gas Chromatography (GC) ............................................................................ 18 1.5.3 High-Performance Liquid Chromatography (HPLC) .................................... 18 1.5.4 Ultra-high Performance Liquid Chromatography (UHPLC) ......................... 18 1.6 Mass Spectrometry .................................................................................................. 19 1.6.1 Mass Spectrometry overview ......................................................................... 19 1.6.2 Inlet System .................................................................................................... 20 1.6.3 Ionization Sources .......................................................................................... 20 1.6.3.1 Electron Ionization (EI) ...................................................................... 20 1.6.3.2 Electrospray Ionization (ESI) ............................................................. 21 1.6.3.3 Atmospheric pressure chemical ionization ......................................... 22 1.6.4 Mass Analyzers .............................................................................................. 23 1.6.4.1 Quadrupole Mass Analyzers ............................................................... 23 1.6.5 Detectors ......................................................................................................... 24 1.6.5.1 Photomultiplier ................................................................................... 24 1.6.6 Tandem Mass Spectrometry ........................................................................... 25 1.6.6.1 Triple Quadrupole (QqQ) ................................................................... 25 1.6.6.2 Multiple Reaction Monitoring (MRM) ............................................... 26 1.6.7 Liquid Chromatography Mass Spectrometry (LC-MS) ................................. 27 v 1.7 References ............................................................................................................... 28 CHAPTER 2: Determining and mitigating the potential occurrence of cyanide in elderberries ...................................................................................................................... 42 2.1 Introduction ............................................................................................................