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I ANALYSIS of L- CITRULLINE, L-ARGININE and L-GLUTAMIC

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I ANALYSIS of L- CITRULLINE, L-ARGININE and L-GLUTAMIC i ANALYSIS OF L- CITRULLINE, L-ARGININE AND L-GLUTAMIC ACID IN SELECTED FRUITS, VEGETABLES, SEEDS AND NUTS SOLD IN NAIROBI CITY COUNTY, KENYA PENINNAH MUENI MULWA (B.Ed. (Sc)) I56/29384/2014 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Chemistry) in the School of Pure and Applied Sciences, Kenyatta University NOVEMBER, 2019 ii DECLARATION I hereby declare that this thesis is my original work and has not been presented for award of a degree or examination at any other University Signature ……………………………….. Date ………………… PENINNAH MUENI MULWA DEPARTMENT OF CHEMISTRY SUPERVISORS We confirm that the work referred in this thesis was carried by the candidate with our approval as University supervisors Prof. Wilson Njue Signature …………………Date …………………….. Department of Chemistry Kenyatta University Dr. Margaret Mwihaki Ng’ang’a Signature ……..............Date ……………. Department of Chemistry Kenyatta University iii DEDICATION This work is dedicated to my beloved daughter, Blessing Mumo, my father Stephen Mulwa, my mother Agnes Mulwa, my sisters Tracy and Josphine for their encouragement, moral and spiritual support as i travelled through the research journey. iv ACKNOWLEDGEMENTS First, I would like to thank the Almighty God for granting me grace to carry out the research work successfully. I am indeed grateful and would like to express my sincere appreciation to my supervisors Prof. Wilson Njue and Dr. Margaret Mwihaki Ng’ang’a both of Department of Chemistry, Kenyatta University for their tireless effort and many hours of guidance throughout the course of study. I am highly indebted to technicians, John Kamathi of Jomo Kenyatta University of Agriculture and Technology (JKUAT) and Jane Mburu, Department of Chemistry, Kenyatta University (KU) for their technical support during the LC-MS analysis of the samples. I also sincerely thank academic staff and other technicians from Department of Chemistry, Kenyatta University for the support they accorded me during the study. I acknowledge the support of my employer, the Teachers’ Service Commission for granting me study leave to do my post-graduate studies. Finally, to my beloved daughter Blessing Mumo who innocently persevered my absence in the course of my study. v TABLE OF CONTENTS DECLARATION ..................................................................................................... ii DEDICATION ........................................................................................................ iii ACKNOWLEDGEMENTS .................................................................................... iv TABLE OF CONTENTS ........................................................................................ v LIST OF FIGURES ............................................................................................... vii LIST OF TABLES .................................................................................................. ix LIST OF APPENDICES ......................................................................................... x ABBREVIATIONS AND ACCRONYMS ............................................................. xi ABSTRACT ........................................................................................................... xii CHAPTER ONE ...................................................................................................... 1 INTRODUCTION ................................................................................................... 1 1.1 Background information of amino acids ............................................................... 1 1.2 Statement of the problem ..................................................................................... 3 1.3 Justification of the study ...................................................................................... 4 1.4 Hypotheses .......................................................................................................... 4 1.5 Objectives ............................................................................................................ 5 1.5.1 General objective .............................................................................................. 5 1.5.2 Specific objectives ............................................................................................ 5 1.6 Significance of the study ...................................................................................... 5 1.7 Scope and limitations of the study ........................................................................ 6 CHAPTER TWO ..................................................................................................... 7 LITERATURE REVIEW........................................................................................ 7 2.1 L-citrulline, L-arginine and L-glutamic acid......................................................... 7 2.2 Biosynthesis of L-glutamic acid, L-citrulline and L-arginine ................................ 8 2.3 Functions of L- citrulline in the human body ...................................................... 11 2.4 Functions of L-arginine in the human body ........................................................ 12 2.5 Functions of L-glutamic acid in the human body ................................................ 14 2.6 Identification and determination of levels of L-citrulline, L-arginine and L-glutamic acid ....................................................................................................... 15 2.7 Chromatographic separation .............................................................................. 15 2.7.1 Thin Layer Chromatography (TLC) ................................................................ 15 2.7.2 High Performance Thin Layer Chromatography (HPTLC) .............................. 16 vi 2.7.3 High Performance Liquid Chromatography (HPLC) ....................................... 16 2.7.4 Liquid Chromatography-Mass Spectrometry (LC-MS) .................................... 17 2.7.5 Gas Chromatography–Mass Spectrometry (GC-MS) ....................................... 18 2.8 Clean up of sample extracts ............................................................................... 19 CHAPTER THREE ............................................................................................... 20 MATERIALS AND METHODS ........................................................................... 20 3.1 Sampling ........................................................................................................... 20 3.2 Chemicals and reagents ...................................................................................... 20 3.3 Cleaning of apparatus ........................................................................................ 21 3.4 Instruments ........................................................................................................ 21 3.5 Experimental methods ....................................................................................... 21 3.5.1 Preparation of standard solutions of L-citrulline, L-arginine and L-glutamic acid ................................................................................................ 21 3.5.2. Preparation of fresh vegetable samples ........................................................... 22 3.5.3 Sample extraction ........................................................................................... 23 3.5.4 Sample clean up .............................................................................................. 24 3.5.5 TLC analysis of amino acids ........................................................................... 24 3.5.6 Analysis by LC-MS ........................................................................................ 25 3.6 LC-MS calibration ............................................................................................. 27 3.7 Data analysis...................................................................................................... 30 CHAPTER FOUR ................................................................................................. 31 RESULTS AND DISCUSSION ............................................................................. 31 4.1 Qualitative Thin Layer Chromatography analysis of L-arginine, L-citrulline and L-glutamic acid ....................................................................................................... 31 4.2 Mean levels of L-citrulline, L-arginine and L-glutamic acid (mg/100g) in selected fruits, vegetables, nuts and seeds.............................................................................. 37 CHAPTER FIVE ................................................................................................... 46 CONCLUSION AND RECOMMENDATIONS .................................................. 46 5.1 Conclusion ......................................................................................................... 46 5.2.1 Recommendations from this study .................................................................. 47 5.2.2 Recommendations for further study ................................................................ 47 REFERENCES ...................................................................................................... 48 APPENDICES ....................................................................................................... 55 vii LIST OF FIGURES Figure 1.1: Amino acid exists as a zwitterion in a physiological solution
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