UNIVERSITY OF GRANADA FACULTY OF SCIENCES Department of Analytical Chemistry Research Group FQM-297 “Environmental, Biochemical and Foodstuff Analytical Control” Functional Food Research and Development Center (CIDAF) DOCTORAL THESIS CHARACTERIZATION OF PHENOLIC COMPOUNDS IN HIGHLY-CONSUMED VEGETABLE MATRICES BY USING ADVANCED ANALYTICAL TECHNIQUES CARACTERIZACIÓN DE COMPUESTOS FENÓLICOS EN MATRICES VEGETALES MEDIANTE TÉCNICAS ANALTICALAS AVANZADAS Presented by IBRAHIM M. ABU REIDAH Submitted for a Doctoral degree in Chemistry GRANADA, 2013 Editor: Editorial de la Universidad de Granada Autor: Ibrahim M. Abu Reidah D.L.: GR 1899-2013 ISBN: 978-84-9028-591-6 This doctoral thesis has been conducted through financing from the Ministry of Foregin Affairs of Spain & The Spanish Agency Of International Cooperation for Development (MAEC-AECID) scholarship and funds from the Research Group FQM-297 “Environmental, Biochemical and Foodstuff Analytical Control” (Department of Analytical Chemistry, University of Granada) and Functional Food Research and Development Center (CIDAF) from different projects, contracts and grants from the central and autonomic administrations and research plan of the University of Granada. CHARACTERIZATION OF PHENOLIC COMPOUNDS IN HIGHLY-CONSUMED VEGETABLE MATRICES BY USING ADVANCED ANALYTICAL TECHNIQUES By IBRAHIM M. ABU REIDAH Granada, 2013 Signed by Dr. Alberto Fernández-Gutiérrez Full Professor of the Department of Analytical Chemistry Faculty of Sciences, University of Granada Signed by Dr. Antonio Segura Carretero Full Professor of the Department of Analytical Chemistry Faculty of Sciences, University of Granada Signed by Dr. David Arráez-Román Assistant Professor of the Department of Analytical Chemistry Faculty of Sciences, University of Granada Submitted for a Doctoral Degree in Chemistry Signed by Ibrahim M. Abu Reidah ALBERTO FERNÁNDEZ GUTIÉRREZ, Full Professor of University of Granada, Department of Analytical Chemistry, Head of Research Group FQM-297 “Environmental, Biochemical, and Foodstuff Analytical Control” and Director of CIDAF, Certifies: That the work presented in this doctoral thesis entitled “CHARACTERIZATION OF PHENOLIC COMPOUNDS IN HIGHLY- CONSUMED VEGETABLE MATRICES BY USING ADVANCED ANALYTICAL TECHNIQUES” has been carried out under my direction and also under the supervision of both Dr. Antonio Segura Carretero and Dr. David Arráez-Román in the laboratories of the Department of Analytical Chemistry and Functional Food Research and Development Center (CIDAF). He demonstrates all requirements of eligibility to obtain the Doctoral Degree in Chemistry from the University of Granada. Granada, March 2013 Acknowledgments Acknowledgments In the name of Allah, Praise be to him & Peace be upon his Prophet Mohammad First, I would like to give a heartfelt, special thanks to my thesis directors: to Dr. Alberto Fernández Gutiérrez, Dr. Antonio Segura Carretero for having given me the opportunity to work within the FQM 297 and at CIDAF Groups, and to earning my Ph.D. In fact, you have been always motivating, encouraging, and enlightening. Also, I’d thank Dr. David Arráez Román for his continuous assistance and perspective remarks. Dr. Antonio, thanks for everything, fatherly correcting me, advising, and also your hospitality in your house in summer vacation time with all CIDAF family members, is also unforgettable. Thank you for everything. I am very grateful to the members of my dissertation committee. Their academic support and input, correcting and advising are greatly appreciated. Thank you. The friendly and supportive atmosphere inherent to the whole FQM 297 Group contributed essentially to the final outcome of my studies. In this context I would like to alphabetically thank: Arantzazu, Ana, Ana Cobo, Cecilia, Celia, Cristina, Hakim, Ihsan, Isa, Javier, Jesús, Maria del Mar, Maria Luz, Nassima, Patricia, Rosa, and Salva, for their friendship, collaboration and the enjoyable and nice times we have passed together. Without their support the completion of this dissertation would not have been possible. And particularly, I’d thank Ihsan, partnership and friendship. Isa, unforgettable moments with Dr. Leopoldo, registration, master classes, I won’t forget your help in everything, many thanks. Jesús, you have correctly got my style of colors, thanks for your compliments. Maria Luz thanks for your humor giving to everybody. Maria del Mar, thanks for everything, collaboration, discussions, and for sweets offering. Salva, thanks for the collaboration, guiding us to the Paris’s congress. Javier; the manager, thank you. Patricia, I’d thank you for everything, cooperation, helping in samples’ preparations, experiments. Also form other members of FQM 297: I’d thank Alegria, Angel, Antonio-Luis Elena, Paco, Paulina, Maria, Marta, and Santi for their encouragement throughout. Santi, Patchi and Marta thank you a lot for your technical help in installing the computer programs and the softwares, intersting discussions. Alegria, you look like your name, I hope you every success. I’d thank in fact collectively, all people and (friends, teachers, relatives, professors) that have helped, encouraged, supported me during all my study periods. Before and after all the thankfulness should be raised to Allah creator of everything the most merciful and Grateful, then to my parents, the factor of my success in this life, thanks for your continues support morally was or financially along all my life stations. Saeeda, thanks for your infinite support throughout everything, in fact, I cannot thank you sufficiently. Kawthar and Mohammad; my lovely kids, your birth were paralleled with my academic progress and advancement. Last but not least, I’d like to thank my family, relatives and friends who always ask, help, encourage also for their support to pursue this study. Finally, I’d like to thank everybody who was part of my success during my entire study career at all levels, apologizing for not being able to mention all since they are lots. Table of Contents TABLE OF CONTENTS SUMMARY --------------------------------------------------------------------------------- 21 RESUMEN ---------------------------------------------------------------------------------- 27 OBJECTIVE -------------------------------------------------------------------------------- 33 OBJETIVO --------------------------------------------------------------------------------- 37 INTRODUCTION -------------------------------------------------------------------------- 43 1. The Mediterranean Diet: An Overview -------------------------------------------- 45 2. Fruits and Vegetables as a Vital Part of The Mediterranean Diet ------------ 50 2.1. Green Beans (Phaseolus vulgaris L.) --------------------------------------- 51 2.2. Cucumber (Cucumis sativus L.) --------------------------------------------- 52 2.3. Watermelon (Citrullus lanatus) --------------------------------------------- 53 2.4. Artichoke (Cynara scolymus) ------------------------------------------------ 54 2.5. Lettuce (Lactuca sativa) ----------------------------------------------------- 56 3. Fruits And Vegetables as Natural Sources of Bioactive Compounds----------- 57 4. Phenolic Compounds: Structure and Classification ----------------------------- 59 5. Phenolic Compound Bioactivity----------------------------------------------------- 67 6. Polyphenols in Highly Consumed Fruits and Vegetables in Relation to 68 Health------------------------------------------------------------------------------------- 7. Analytical Techniques Used For Separating Phenolic Compounds------------- 73 7.1. Liquid Chromatography (LC) ------------------------------------------------ 74 7.2. Detectors Coupled to LC------------------------------------------------------ 77 7.2.1. UV-Visible Absorbance------------------------------------------------- 78 7.2.2. Mass Spectrometry (MS) ---------------------------------------------- 80 7.2.2.1. Ion Source------------------------------------------------------ 82 7.2.2.2. Mass Analyzer------------------------------------------------- 85 Time-Of-Flight (TOF) ---------------------------------------------- 86 Quadrupole-Time-Of-Flight (QTOF) ------------------------------ 88 7.2.2.3. MS Detector --------------------------------------------------- 89 Experimental Section ------------------------------------------------------------------ 91 Chapter 1 Phytochemical Characterization of Green Beans (Phaseolus 93 vulgaris L.) By Using High-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry ------------------ Chapter 2 HPLC-ESI-Q-TOF-MS for a Comprehensive Characterization Of 123 Bioactive Phenolic Compounds in Cucumber Whole Fruit Extract Chapter 3 Profiling Of Phenolic And Other Polar Constituents From Hydro- 155 Methanolic Extract Of Watermelon (Citrullus lanatus) By Means of Accurate-Mass Spectrometry (HPLC–ESI–QTOF–MS) -------------- Chapter 4 Extensive Characterization of Bioactive Phenolic Constituents 181 From Globe Artichoke (Cynara scolymus L.) By HPLC-DAD-ESI- QTOF-MS ------------------------------------------------------------------ Chapter 5 Reversed-Phase Ultra-Performance Liquid Chromatography 205 Coupled To Electrospray Ionization–Quadrupole–Time-Of-Flight Mass Spectrometry as a Powerful Tool for Metabolic Profiling Of Vegetables: Lactuca Sativa as an Example of its Application----- CONCLUSIONS ---------------------------------------------------------------------------- 249 CONCLUSIONS ----------------------------------------------------------------------------