Identification of Phenolic Compounds from Peanut Skin using HPLC-MSn By Kyle A. Reed Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Food Science and Technology Committee: Sean O’Keefe (Committee Chair) Rebecca O’Malley (Committee Co-Chair) Susan Duncan Kumar Mallikarjunan Joseph Marcy December 7, 2009 Blacksburg, Virginia Keywords: peanut skin, oligomeric proanthocyanidins, polyphenol, HPLC-MSn Identification of Phenolic Compounds from Peanut Skin using HPLC-MSn By Kyle A. Reed ABSTRACT Consumers view natural antioxidants as a safe means to reduce spoilage in foods. In addition, these compounds have been reported to be responsible for human health benefits. Identification of these compounds in peanut skins may enhance consumer interest, improve sales, and increase the value of peanuts. This study evaluated analytical methods which have not been previously incorporated for the analysis of peanut skins. Toyopearl size-exclusion chromatography (SEC) was used for separating phenolic size-classes in raw methanolic extract from skins of Gregory peanuts. This allowed for an enhanced analysis of phenolic content and antioxidant activity based on compound classes, and provided a viable preparatory separation technique for further identification. Toyopearl SEC of raw methanolic peanut skin extract produced nine fractions based on molecular size. Analysis of total phenolics in these fractions indicated Gregory peanut skins contain high concentrations of phenolic compounds. Further studies revealed the fractions contained compounds which exhibited antioxidant activities that were significantly higher than that of butylated hydroxyanisole (BHA), a common synthetic antioxidant used in the food industry. This indicates peanut skin extracts are a viable antioxidant source, and that synthetic antioxidants can be replaced with those naturally-derived from peanut by-products. Structures contained in each fraction were identified using high performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion trap mass spectrometry (MSn). Prior to this study, approximately 20 compounds have been identified in peanut skins. The combination of Toyopearl SEC with ESI-HPLC-MSn allowed for the identification of 314 phenolic-based compounds, most of which are newly discovered compounds in peanut skins. Many compounds identified are known to have powerful antioxidant effects, and also have been reported to exhibit numerous beneficial chemical and biological activities, including the treatment of various human health-related conditions. It is evident that peanut skins may be a potential untapped source for the extraction of natural food antioxidants, nutracueticals, and even pharmaceuticals. Because peanut skins are largely a wasted resource to peanut processors, the novel polyphenols identified in this research could have a significant financial impact on the peanut industry. Dedication I would like to dedicate this dissertation to my grandfather, the late Sanford (Sandy) H. Stiles. Known to his family as “Pop Pop”, Sandy was an inspiration to both his community and family alike. He was an instrumental driving force in my life, and taught me the necessary values required to succeed both academically, and personally. For everyone who had the pleasure to know him, he made us realize that nothing in life comes easy, and that only through hard work and dedication will you succeed. He lived by the mantra to “Show ‘em what fer!”, which always gave me the hope of achieving my goals during these often tenacious years of graduate school. Pop Pop, although you are no longer here, you will always be with us, and in my heart forever. iii Acknowledgements I would like to thank all the faculty and staff in the Food Technology Department at Virginia Tech, who gave me this fantastic opportunity to not only hone my skills for my future career, but also to make this research project a reality. My sincere appreciation goes to my major advisor, Dr. Sean O’Keefe, for his true friendship, guidance, financial support, and vast sea of never-ending knowledge. A heartfelt thanks goes to my co-advisor, Dr. Rebecca O’Malley, who graciously took me under her wing for this project. In addition to her friendship, she gave me the necessary knowledge, tools, and support required to achieve my goals. Another sincere appreciation goes to my committee members, Dr. Joseph Marcy, Dr. Susan Duncan, and Dr. Kumar Mallikarjunan. Your friendship, guidance, and support were integral to the success of this research, and also made graduate school at VT an enjoyable and memorable experience. A special thanks also goes to Dr. Jodie Johnson, who devoted much of his time and energy toward helping me with this project. Reaching this point has been a long and arduous journey, full of life’s unexpected twists and turns. Traversing this road has been made simpler by my having people in my life who inspired me to never give up. I’d like to thank all of them who believed in me; I am grateful to all my friends for their support and encouragement. Also, a special thanks to Denise Gardner, not only for her help and support, but also for being such a huge inspiration in both my academic and my personal life. Most of all, I would like to thank my family for being my rock and my foundation. To my mother and father, Laurie and Paul Dietrich, and my brother, Kent Reed, a special thank you for their love and encouragement. I couldn’t have done it without you! iv TABLE OF CONTENTS Abstract Dedication iii Acknowledgements iv List of Tables and Figures x Introduction 1 CHAPTER I: REVIEW OF THE LITERATURE 3 Peanut Industry 3 Peanut Varieties 4 By-Products of the Peanut Industry 4 Peanut Composition and Health 6 Phenolic Compounds in Peanuts and Other Plants 12 Oxidation and Antioxidants 19 Extraction Methods 26 Antioxidant Activity Methods 29 Total Phenols Method 30 Analytical Methods for Separation and Identification 31 Patentability 38 References 42 CHAPTER II: TOTAL PHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF 50 FRACTIONS OBTAINED FROM SIZE-EXCLUSION CHROMATOGRAPHY (SEC) OF RAW METHANOLIC PEANUT SKIN EXTRACT Abstract 50 Introduction 51 Materials and Methods 52 Results and Discussion 56 Conclusion 60 References 65 CHAPTER III: IDENTIFICATION OF PHENOLIC COMPOUNDS IN PEANUT 66 SKIN EXTRACT USING HPLC WITH NEGATIVE MODE ION TRAP MSn AND ELECTROSPRAY IONIZATION Abstract 66 Introduction 67 Materials and Methods 69 Results and Discussion 74 Fraction A – HPLC Method 1 74 Fraction A – HPLC Method 2 84 v Fraction B – HPLC Method 1 87 Fraction B – HPLC Method 2 97 Fraction C – HPLC Method 1 102 Fraction C – HPLC Method 2 126 Fraction D – HPLC Method 1 146 Fraction D – HPLC Method 2 153 Fraction E – HPLC Method 1 155 Fraction E – HPLC Method 2 159 Fraction F – HPLC Method 1 162 Fraction F – HPLC Method 2 167 Fraction G-Red – HPLC Method 1 168 Fraction G-Red – HPLC Method 2 172 Fraction G – HPLC Method 1 174 Fraction G – HPLC Method 2 178 Fraction H – HPLC Method 1 180 Fraction H – HPLC Method 2 185 Conclusion 186 References 305 Summary 314 Appendix 315 A.1: HPLC-UV Chromatogram of Peanut Skin Raw Extract at 280nm ………… 316 A.2: HPLC-UV Chromatogram of Standard Mix at 280nm ……………………... 317 A.3: Standard Mix: HPLC-UV Chromatogram (bottom) versus TIC …………… 318 A.4: Standard Mix: HPLC-UV Chromatogram (bottom versus TIC’s ………….. 319 A.5: Ion Spectra for Epicatechin …………………………………………………. 320 A.6: Fragmentation Scheme for Epicatechin …………………………………….. 321 A.7: HPLC-UV Chromatogram of Peanut Skin Fraction A (Obtained from Toyopearl Size Exclusion Chromatography at 280nm …………………………... 323 A.8: Peanut Skin Fraction A: HPLC-UV Chromatogram (bottom) versus TIC’s... 324 A.9: Peanut Skin Fraction A: HPLC-UV Chromatogram (bottom) versus TIC …. 325 A.10: HPLC-UV Chromatogram of Peanut Skin Fraction B (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….. 326 A.11: Peanut Skin Fraction B: HPLC-UV Chromatogram (bottom) versus TIC’s 327 vi A.12: Peanut Skin Fraction B: HPLC-UV Chromatogram (bottom) versus TIC ... 328 A.13: Peanut Skin Fraction B: HPLC-UV Chromatogram (bottom) versus TIC ... 329 A.14: HPLC-UV Chromatogram of Peanut Skin Fraction C (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….. 330 A.15: Peanut Skin Fraction C: HPLC-UV Chromatogram (bottom) versus TIC’s 331 A.16: Peanut Skin Fraction C: HPLC-UV Chromatogram (bottom) versus TIC ... 332 A.17: Peanut Skin Fraction C: HPLC-UV Chromatogram (bottom) versus TIC ... 333 A.18: HPLC-UV Chromatogram of Peanut Skin Fraction D (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm …………………………. 334 A.19: Peanut Skin Fraction D: HPLC-UV Chromatogram (bottom) versus TIC’s 335 A.20: Peanut Skin Fraction D: HPLC-UV Chromatogram (bottom) versus TIC ... 336 A.21: Peanut Skin Fraction D: HPLC-UV Chromatogram (bottom) versus TIC ... 337 A.22: HPLC-UV Chromatogram of Peanut Skin Fraction E (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….. 338 A.23: Peanut Skin Fraction E: HPLC-UV Chromatogram (bottom) versus TIC’s 339 A.24: Peanut Skin Fraction E: HPLC-UV Chromatogram (bottom) versus TIC ... 340 A.25: HPLC-UV Chromatogram of Peanut Skin Fraction F (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….. 341 A.26: Peanut Skin Fraction F: HPLC-UV Chromatogram (bottom) versus TIC’s 342 A.27: Peanut Skin Fraction F: HPLC-UV Chromatogram (bottom) versus TIC ... 343 A.28: HPLC-UV Chromatogram of Peanut Skin Fraction G-Red (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….. 344 A.29: Peanut Skin Fraction G-Red: HPLC-UV Chromatogram (bottom) versus TIC’s ……………….…………………………………………………………….. 345 A.30: Peanut Skin Fraction G-Red: HPLC-UV Chromatogram (bottom) versus TIC ……………………………………………………………………………….. 346 vii A.31: HPLC-UV Chromatogram of Peanut Skin Fraction G (Obtained from Toyopearl Size Exclusion Chromatography) at 280nm ………………………….