Mass Spectrometrical Analysis of Flavonoids and Glucose Esters of Hydroxycinnamic Acids in Dietary Plants By

Mass Spectrometrical Analysis of Flavonoids and Glucose Esters of Hydroxycinnamic Acids in Dietary Plants by

MARIA ALEXANDRA PÃTRAŞ

A thesis submitted in partial fulfilment of the requirements for the degree of

Doctor of Philosophy in Chemistry

Approved Dissertation Committee:

Prof. Dr. Nikolai Kuhnert, Jacobs University Bremen, Germany

Prof. Dr. Matthias Ullrich, Jacobs University Bremen, Germany

Prof. Dr. Ulrich Engelhardt, Technical University Braunschweig, Germany

Date of Defense: 12th April 2018 Department of Life Sciences and Chemistry

Statutory Declaration

Family Name, Given/First Name Patras, Maria Alexandra

Matriculation number 10000571

What kind of thesis are you submitting: Cumulative PhD Thesis Bachelor-, Master- or PhD-Thesis

English: Declaration of Authorship

I hereby declare that the thesis submitted was created and written solely by myself without any external support. Any sources, direct or indirect, are marked as such. I am aware of the fact that the contents of the thesis in digital form may be revised with regard to usage of unauthorized aid as well as whether the whole or parts of it may be identified as plagiarism. I do agree my work to be entered into a database for it to be compared with existing sources, where it will remain in order to enable further comparisons with future theses. This does not grant any rights of reproduction and usage, however.

The Thesis has been written independently and has not been submitted at any other university for the conferral of a PhD degree; neither has the thesis been previously published in full.

German: Erklärung der Autorenschaft (Urheberschaft) Ich erkläre hiermit, dass die vorliegende Arbeit ohne fremde Hilfe ausschließlich von mir erstellt und geschrieben worden ist. Jedwede verwendeten Quellen, direkter oder indirekter Art, sind als solche kenntlich gemacht worden. Mir ist die Tatsache bewusst, dass der Inhalt der Thesis in digitaler Form geprüft werden kann im Hinblick darauf, ob es sich ganz oder in Teilen um ein Plagiat handelt. Ich bin damit einverstanden, dass meine Arbeit in einer Datenbank eingegeben werden kann, um mit bereits bestehenden Quellen verglichen zu werden und dort auch verbleibt, um mit zukünftigen Arbeiten verglichen werden zu können. Dies berechtigt jedoch nicht zur Verwendung oder Vervielfältigung.

Diese Arbeit wurde in der vorliegenden Form weder einer anderen Prüfungsbehörde vorgelegt noch wurde das Gesamtdokument bisher veröfentlicht.

Date, Signature

“If I have seen further, it is by standing on the shoulders of giants„ Sir Isaac Newton

To my husband Marius and our daughters Ilinca and Ioana, without whom I would have finished this work two years earlier but would not have had whom to dedicate it to.

ACKNOWLEDGEMENTS

I would like to begin by expressing my gratitude to my supervisor Prof. Nikolai Kuhnert for entrusting me with this work and for constantly encouraging me with his never-ending patience and understanding, which allowed me to grow as a researcher and as a mother at the same time.

I would also like to acknowledge Prof. Matthias Ullrich and Prof. Ulrich Engelhardt for taking the time to review my thesis.

I would also like to acknowledge all the collaborators to my projects:

Borislav Milev, Dr. Gino Vrancken and Dr. Thorsten Dittmar for the collaborative work on the cocoa investigation.

Dr. Rakesh Jaiswal, Dr. Marius Febi Matei and Viktorija Glembockyte for the collaborative efforts towards synthesis and characterization of caffeoylglucoses.

Dr. Gordon McDougall for providing strawberry samples of different cultivars to be profiled for their hydroxycinnamoylglucose contents.

Dr. Javier Gonzales and Prof. Juan Davalos for performing the IRMPD measurements of isotopically labeled glucoses.

I would like to thank all the current as well as former members of the Kuhnert group for being great colleagues and friends.

I acknowledge financial support from Barry Callebaut in the early stage of my PhD work.

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Contents Thesis outline ...... v Abstract ...... vii List of Figures ...... iix List of Tables ...... xi Chapter 1. Introduction ...... 1 1.1 Polyphenols Biosynthesis and Function ...... 1 1.2 Chemical classification of polyphenols ...... 6 1.3 Methods for polyphenol analysis ...... 19 1.4 Motivation and aim of the study ...... 22 1.4.1 Cocoa Polyphenol analysis ...... 22 1.4.2 Profiling and quantification of hydroxycinnamoyl glucoses in dietary plants ...... 24 1.4.3 Understanding the fragmentation of glucose in mass spectrometry ...... 26 Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin ...... 32 Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn ...... 42 Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose ...... 57 Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits ... 100 Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables ...... 119 Chapter 7. Undestanding the fragmentation of glucose in mass spectrometry ...... 139 Chapter 8. Conclusions ...... 165 Apendix 1. Supporting information for Chapter 4 ...... 170 Appendix 2: Supporting Information for Chapter 5 ...... 173 Appendix 3: Supporting Information for Chapter 6 ...... 224 Appendix 4: Supporting Information for Chapter 7 ...... 241

Thesis outline The thesis is divided into eight chapters, as follows:

Chapter 1 provides a general introduction to polyphenols and comprises of a brief overview of their biosynthetic pathways, a description of their classification into different classes on the basis of their chemical structure, an inquiry into the methodology of their analysis with emphasis on the HPLC-MSn analysis tools available for the complete structural elucidation of isomeric glycosylated compounds, in particular flavonoids and hydroxycinnamic acids and the motivation and aims behind the work which has been carried out throughout the project.

Part I. Cocoa Polyphenol analysis (Chapters 2-3) Chapter 2 comprises of a scientific article published in the Food Research International journal in 2014 and presents the FTICR analysis of raw fermented cocoa beans of diffent origins. Chapter 3 comprises of a scientific article published in the Food Research International journal in 2014 and presents the HPLC-MSn analysis of raw fermented cocoa beans polyphenolic extracts leading to the identification of novel glycosylated and sulphonated compounds not previously mentioned in literature.

Part II. Profiling and quantification of hydroxycinnamoyl glucoses in dietary plants (Chapters 4-6) Chapter 4 comprises of a scientific article published in the Journal of Agricultural and Food Chemistry journal in 2014 and presents the development of an LC-MSn hierarchical scheme for the identification of individual regioisomers of cafeoylglucose using pure synthetic standards. Chapter 5 comprises of a scientific article published in the Journal of Agricultural and Food Chemistry journal in 2017 and presents the qualitative and quantitative analysis of hydroxycinnamoyl glucose esters and hydroxycinnamic acid-O-glycosides present in in a wide variety of edible berries. Chapter 6 comprises of a scientific article published in the Food Chemistry journal in 2017 and presents the qualitative and quantitative analysis of hydroxycinnamoyl glucose esters and hydroxycinnamic acid-O-glycosides present in dietary relevant vegetables of the Solanaceae family.

Part III. Understanding the fragmentation of glucose in mass spectrometry (Chapter 7) Chapter 7 comprises of a manuscript in preparation and presents the detailed analysis of the fragmentation of D-glucose in mass spectrometry, using seven 13C and six 2H isotopomers of glucose. Chapter 8 presents the general conclusions of the entire work and points out the importance of the scientific findings for future research.

Abstract As ubiqutous constituents of most edible and medicinal plants, polyphenols have been the subject of endless studies describing a variety of health effects like antioxidant, cardiovascular protector and antitumoral effects. The present contribution describes the development and use of various mass spectrometry-based analytical tools for the investigation of particular members of the two most prominent classes of polyphenols, namely flavonoids and hydroxycinnamic acids in dietary plant of great economical importance.

The first part of the present work was dedicated to the investigation of the polyphenolic fraction of cocoa, previously reported to contain flavonoids, particularly flavan-3-ols, as major constituents. Cocoa is an agricultural commodity produced annually in quantities over 4 million tons worldwide and converted into cocoa powder and chocolate products with an estimated annual trade value of over 100 billion Euro (FAO 2016 statistics). Both top down and a bottom up approaches were used in the investigation. In the top down approach, methanolic extracts of raw fermented cocoa beans from different origins were analyzed for the first time in literature by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), an ultra-high resolution mass spectrometry technique used mainly for the analysis of unresolved complex mixtures comprising tens of thousands of analytes. With approximately 11 000 peaks, cocoa was shown to be one of the most complex organic mixtures ever analyzed. Class plot diagrams and 3D van Krevelen plots were shown to be valuable data representation tools for the rapid investigation and comparison of the overall chemical profiles of these complex mixtures. In the bottom up approach, the methanolic extracts were analyzed by HPLC-MSn. The optimized method revealed a greater extract complexity than previously reported and allowed the identification of novel compounds, particularly glycosylated and sulphonated flavonoids, present in the mixture in multiple isomeric forms. Given their high dietary burden and reported bioavailability, hydroxycinnamic acids are one of the most important class of plant secondary metabolites present in the human diet. They most oftenly occur in plants bound to polyols (like hydroxycinnamoyl quinates and shikimates, which are the most abundant derivatives) or sugars. Sugar derivatives of hydroxycinnamic acids have been studied on rare occasions and very often aspects of regio- and stereochemistry have been either left open or completely disregarded. Therefore, the second part of the present contribution was dedicated to the investigation of glycosylated derivatives of hydroxycinnamic acids, with particular emphasis on hydroxycinnamoylglucose esters.

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Authentic reference standards of hydroxycinnamoyl glucoses were selectively synthesized and subsequently used to develop a hierarchical HPLC-MSn scheme for the identification of all ten regioisomeric esters. The scheme allows for unambiguous assignment of all isomers in any plant source without compound isolation on the basis of their relative elution from a reversed phase chromaphic column and distinct fragmentation patterns. Over twenty different edible plant sources of great economical relevance, including berries and Solanaceae vegetables, were subsequently profiled for their hydroxycinnamoyl glucose contents. Furthermore, a quantitative HPLC-MS method was developed and validated using authentic reference standards and applied to over fourty different samples, thus providing the first set of quantitative data ever reported for this class of compounds. Elucidation of the fragmentation mechanisms of individual hydroxycinnamoylglucose esters revealed that for nine out of the ten isomers, the most abundant fragment ions are formed by the breakage of the bonds within the sugar moiety, greatly dependent on the regio-and stereochemistry of the O-acyl bond. Moreover different isomeric C- and O-glycosides of flavan-3-ols present in the cocoa extracts could easily be distinguished on the basis of their characteristic fragmentation patterns. Glycosylated derivatives of all classes of natural products, omnipresent in any living tissue, are characterized since over two decades using mass spectrometry. However, the fragmentation mechanism of the simple glucose molecule has never been studied in detail. Therefore, the third part of the present work was dedicated to the in-depth study of the fragmentation mechanism of D-glucose, employing all 13C and all 2H labeled isotopomers. Additionally, a HPLC-tandem MS method able to separate the anomers of D-glucose using a HILIC column was developed and their fragmentation patterns were investigated individually for the first time in literature. The results form the basis of a comprehensive formalism allowing carbohydrate structure elucidation by mass spectrometry.

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List of Figures

1.1. Schematic overview of the shikimate biosynthetic pathway……………………… 4 1.2. Schematic overview of the biosynthesis of different classes of phenylpropanoids………………………………………………………………………. 5 1.3. Structure of Flavan (2-phenyl-1,4-benzopyrane)………………………………….. 6 1.4. Flavan-3-ol backbone and representative structures of the flavan-3-ols class……………………………………………………………………………...... 8 1.5. Proanthocyanidin (condensed tannins) representative structures………………….. 9 1.6. Hydroxyflavone backbone and representative structures of flavonols…………….. 10 1.7. Flavanone backbone and representative structures of flavanones…………………. 10 1.8. Flavone backbone and representative structures of flavones……………………… 11 1.9. 2-phenylbenzopyrillium backbone and the structures of the most common naturally occurring anthocyanidins……………………………………………...... 12 1.10. Structures of cyanidin in aqueous solution under varying pH……………………. 13 1.11. Chemical structures of simple phenolic acids, quinic and shikimic acids, representative hydrolysable tannins and selected examples of hydroxycinnamic acid- sugar derivatives…………………………………………………………...... 16 1.12. Structures of the most representative phenylpropenes…………………………… 17 1.13. Chemical structures of representative coumarins………………………………… 18 1.14: Chemical structures of representative stilbenes…………………………………………... 18 2.1. Class plot diagram of the population (a) and abundance (b) of mass spectrometric signals; …………………………………………………………………………………. 38 2.2. Three-dimensional van Krevelen diagram representing different formula classes in the spectrum of Ivorian bean extract………………………………………………… 39 3.1. Cameroon beans negative ion mode base peak chromatogram……………………. 46 3.2. Ion trap extracted ion chromatogram at m/z 451 (negative ion mode) of Cameroon beans…………………………………………………………………………………….. 47 3.3: Ion trap extracted ion chromatogram at m/z 369 (negative ion mode) of Cameroon beans…………………………………………………………………………………….. 48 3.4: Ion trap extracted ion chromatogram at m/z 605 (negative ion mode) of Cameroon beans………………………………………………………………………… 49 3.5. Ion trap extracted ion chromatograms at m/z: a.) 575, b.) 863 and c.) 861 of Cameroon beans……………………………………………………………...... 50 3.6. Proposed structure for the species identified in the Cameroon beans phenolic extract…………………………………………………………………………………… 54 4.1. Selected structures of typical hydroxycinnamic acids present in the human diet and hydroxycinnamic acid carbohydrate conjugates…………………………………… 80 4.2. Synthesis pathway of 3-caffeoylglucoses (5 and 6) and 6-caffeoylglucoses (9 and 10)……………………………………………………………………………….. 81

4.3. Synthesis pathway of 1-caffeoylglucoses, 2-caffeoylglucoses and 3- caffeoylglucoses (1-6)………………………………………………………………….. 82 4.4. Synthesis pathway of regio- and stereoisomers of caffeoylglucose (1-10)...... 82 4.5. Extracted ion chromatograms (EICs) of regio- and stereoisomers of caffeoylglucose (1-10) at m/z 341 obtained by two different chromatographic methods…………………………………………………………………………………. 83 4.6. MS2 spectra of caffeoylglucoses 1-10 at m/z 341 in negative ion mode…...... 84 4.7. Fragmentation mechanism and pathway of 6-caffeoylglucoses (9 and 10)……….. 85 4.8. Fragmentation mechanism and pathway of 2-caffeoylglucoses (3 and 4)………… 85 4.9. Fragmentation mechanism and pathway of 3-caffeoylglucoses (5 and 6)………… 86 4.10. Fragmentation mechanism and pathway of 4-caffeoylglucoses (7 and 8)……….. 87 4.11. Fragmentation mechanism and pathway of 1-caffeoylglucoses (1 and 2)……….. 88 4.12. MS2 spectra of 1-caffeoylglucoses (1 and 2) at m/z 365 in positive ion mode……………………………………………………………………………………. 88 4.13: MS2 spectra of caffeic acid-3-O-β-glucose 11 and caffeic acid-4-O-β-glucose 12 at m/z 341 in negative ion mode………………………………………………………... 89 5.1: The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass spectrometer and MS2 spectra of labeled peaks from gooseberry extract…………………………………………………………………………...... 111 5.2: a. The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass spectrometer and MS2-4 spectra of 6-β-CG from strawberry; b. The extracted ion chromatogram at m/z 325 (negative ion mode) from an ion trap mass spectrometer and MS2-4 spectra of the tentatively assigned 6-β-p-coumaroylglucose from black currant; c. MS2 fragmentation pathways of the two analogous compounds……………………………………………………...... 112 5.3. Individual structures of the compounds 1-20 found in the investigated berry extracts………………………………………………………………………………….. 113 6.1. Individual structures of all ten isomers of caffeoyl glucose (CG) (1–10) and representative structures of C-glycosides 11, O-Glycosides 12 and 5-Caffeoylquinic acid 13……………………………………………………………….. 122 6.2. Extracted ion chromatograms (EIC) at m/z 341 for: a) Fresh tomato, b) Home- made tomato juice, c) Tomato puree, d) Tomato ketchup…………………………….. 127 6.3. Extracted ion chromatograms (EIC) at m/z 341 for: a) Organic bell pepper, b) Charlie cultivar pepper, c) Turkish green Chilli, d) Dutch red Chilli, e) Indian red chilli…………………………………………………………………………………….. 128 6.4. MS2 spectra of caffeoylglucoses 3–6 and 9–10 from tomato puree (negative ion mode)…………………………………………………………………………………… 129 7.1. Structure of the thirteen glucose isotopomers employed in the study……………... 142 - 7.2. Structures of all the possible [M-H-H2O] ions and the most abundant - [M-H-2*H2O] ions resulting from each of them……………………………………... 152 7.3. Mechanistic pathways for the loss of 30 Da and 60 Da from the [M-H]- ion………………………………………………………………………………………. 153 7.4. Mechanistic pathways for the loss of 90 Da from the [M-H]- ………………… 154 7.5. Total ion chromatograms and MS2 spectra of individual anomers of …...... 2H-C-1ion and………… 2H-C2…………….……………………………………………………………… 160

List of Tables

Table 3.1. Negative ion mode analysis results of the cocoa beans extract from Cameroon……………………………………………………………………………….. 52

Table 4.1. MS3 Data of Caffeoylglucoses (CGs) in Negative Ion Mode……………….. 77 Table 4.2. High resolution MS2 Data of Synthetic Caffeoylglucoses (CGs) in Negative Ion Mode………………………………………………………………………………... 79

Table 5.1: Summary of the profiling results for all the investigated berry extracts…………………………………………………………………………...... 109

Table 5.2: Summary of the quantification results of individual isomers of CG in selected samples………………………………………………………………………... 110

Table 6.1. Caffeoyl glucose individual isomer content of investigated fresh tomato, tomato based products and various pepper samples in mg/100g dried material…………………………………………………………………………………. 134

Table 7.1. Theoretical Ionization energies of the five chemically distinct hydroxyl groups of the glucose molecule………………………………………………………… 146

Chapter 1. Introduction

1.1 Polyphenols Biosynthesis and Function Polyphenols are secondary plant metabolites synthesized through the phenyl propanoid biosynthetic pathway from main products of the shikimate pathway (Figure 1) main products, the amino acids tyrosine and phenylalanine. They are essential to terrestrial plants as they account for: - the mechanical and structural stability of higher vascular plants (for example lignocellulose);1 - regulation of hormonal activity; e.g.depending on their structure they can stimulate or inhibit plant growth by interaction with IAA-oxidase, the enzyme responsible with the degradation of the the growth hormone auxin. o-Diphenols like quercetin (Figure 6.1) , caffeic acid (Figure 2.1) and their glycosylated derivatives are IAA inhibitors whereas monophenols like p- coumaric acid (Figure 2.1), hydroxybenzoic acid and vanilic acid (Figure 10.1) are IAA activators.2 Flavones and flavonols are involved in the transport of auxin to the nodulation sites of certain legumes;3 -providing biochemical resources for successful reproduction; e.g. by selectively attracting pollinators in flowers with their bright colors (flavonoids accounting for bright yellow color of polen4) and by attracting seed dispersers in fruits. Moreover, studies on the effect of phenol rich flower nectar on different species of insects have very interestingly shown appealing effect towards members of the pollinating species (legitimate pollinators) and deterring effect towards occasional visitor species;5 -act as chemical defense substances against pathogenic bacteria, fungi and certain viruses (antibiotic action); parasitic plants and insects and herbivore animals (antifeeding action).6 They have been as well hypothesized to act as protective chemical screens against harmful UV radiation, particularly the class of flavonols (Figure 6.1), due to their UV-light absorbing capacitiy.2,7,8 There is a general scientific agreement on some functions of polyphenols in plants; however, the reason why plants synthesize such a great variety of polyphenolic structures is still subject to scientific debate. The phenyl propanoid pathway generates an enormous array of secondary metabolites based on the few intermediates of the shikimate pathway as the core unit, namely dihydroshikimic acid, shikimic acid, isochorismic acid, phenylalanine, p-coumaric acid9 (Figure1.1).

Chapter 1 Introduction

The resulting hydroxycinnamic acids and esters are amplified in several cascades by combinations of enzymes like oxido-reductases, oxygenases and transferases to result in organ and developmentally specific patterns of metabolites, characteristic for each plant species.9 Some authors hypothesize that throughout their complex evolutionary development process, plants have been creating libraries of secondary metabolites (implicitly polyphenolic compounds) to be screened upon certain stress conditions for specific biological functions.10,11 Therefore, scientists imply that the complexity of an organism’s secondary metabolite profile is a measure of that organism’s ability to survive under hardship conditions.10-12 A summary of the phenylpropanoid metabolic pathway leading to the various classes of polyphenols is schematically represented in Figure 2. Not only are polyphenols of great biological relevance to plants themselves, they have also been proven to be of great importance to humans as well-be it economical or health related. The oldest and most famous economical application of plant polyphenolic extracts is the tanning of animal skin to produce leather (due to precipitation of skin proteins by formation of cross- linkages between selected functional groups of collagen and polyphenolic structures), an application that gave polyphenols their other famous, also commonly used name, namely the one of ‘tannins’. The name comes from the French word ‘tan’, (powdered oak bark extract traditionally used in making of leather from animal skin), which is itself etymologically derived from the ancient Celtic lexical root ‘tann-‘ meaning oak. Certain classes of polyphenols are still commonly referred to as tannins.6,8

As important constituents of edible plants, polyphenols have caught scientific attention for over eight decades, as they have been found to be responsible for the numerous health benefits associated to fruit and vegetable rich diets. In the late 1930s, the Hungarian Nobel Prize winner Albert Szent-Györgyi (who obtained the Nobel prize in 1937 for descovering Vitamin C and elucidating the reactions and components of the citric acid cycle) proposed the classification of the flavanone hesperidin (Figure 7.1) isolated from citrus fruits into the class of vitamins and proposed the name ‘Vitamin P’ for it.8,13 Although the classification was not accepted, many studies conducted until 1950s reported on hesperidin’s protective effects on the vascular system.13 Polyphenols are as well the major constituents of most plant extracts used in traditional folk medicine for thousands of years.14 Countless scientific studies report on polyphenols’ preventative effects on chronic degenerative and cardiovascular diseases, cancer, diabetes, inflammatory diseases and ageing as well as antibacterial,

Chapter 1. Introduction

antifungal and antiviral activities and new research is constantly accumulating.15-24 In the 1990s, polyphenols started being classified as general antioxidants13 and most of their health effects have been exclusively attributed to their antioxidant and radical scavenging properties; however, this simplistic view on such a complex array of effects has recently started to be debated.25 Although very important, the antioxidant properties of polyphenols have been overemphasized, as they cannot account for all their reported health benefits and antimicrobial activity. It is rather suggested that polyphenols act through more complex mechanisms like gene expression modulation and enzyme induction.15,18,25-34

Chapter 1. Introduction

Figure 1.1: Schematic overview of the shikimate biosynthetic pathway9

Chapter 1. Introduction

HO OH HO OMe HO OMe COOH COOH HCAs

O NH2 Phenylalanine Cinnamate Ammonia Lyase Phenylalanine Cinnamate Aromatic ring 4-hydroxylase hydroxylation, O-methylation O2 , NADPH COOH COOH COOH Caffeic acid Ferulic acid Sinapic acid COOH COOH Phenylalanine COOH reduction NH HO 2 Ammonia Lyase HO Tyrosine p-Coumarate Monolignols

p-Coumarate HO OH HO OMe HO OMe CoA ligase CoASH MeO ATP

OH OH OH p-Coumaryl Coniferol Sinapyl alcohol alcohol O HO O O Chalcone Synthase SCoA 3-Malonyl-CoA MeO Lignin Phenylpropenes HO p-Coumaroyl-CoA Coumarins HO OH HO CoAS

O O O O OH OH OH = OH O O OH O

-CO SCoA O -CoASH 2 Enolization = SCoA -H2O COOH O O O O Chalcone Synthase O O O O HO OH Claisen Condensation Stilbenes

OH OH OH HO OH O O HO O

Enolisation OH O Aurones O O O Chalcone OH

OH HO O Intramolecular HO O Michael Addition OH O OH O Flavonoids OH Isoflavonoids

Figure 1.2: Schematic overview of the biosynthesis of different classes of phenylpropanoids8

Chapter 1. Introduction

1.2 Chemical classification of polyphenols Chemically, polyphenols are divided into several structural classes according to the number of phenol rings in their structure and the structural elements linking them to one another.

1.2.1 Flavonoids Flavonoids (from the Latin word ‘flavus’ meaning yellow) are the polyphenols subclass built on the molecular framework of Flavan (2-phenyl-1,4-benzopyrane, C6-C3-C6). They are metabolic hybrids as they are derived from a combination of the shikimate derived phenylpropanoid and the polyketide pathways6 (Figure 1.2).

Figure 1.3: Structure of Flavan (2-phenyl-1,4-benzopyrane)

The most notable functions performedd by plant flavonoids are those of UV protection, antioxidant or free radical protection, modulation of enzymatic activity, alleopathy, insect attraction or repulsion, nectar guides, probing stimulants, defense against pathogen infection, nodulation in leguminous plants, pollen germination,35 etc., and it is likely that many others are still to be elucidated.5 Over the past decade, scientists have become increasingly interested in the potential for various dietary flavonoids to explain some of the health benefits associated with fruit- and vegetable-rich diets. Therefore, up to date, the structure of over 8000 naturally occuringcompounds have been elucidated and classified as members of the flavonoid group.6,8 Flavonoids can be further divided into several subclasses, based on the degree of oxidation of the pyrane ring:

1.2.1.1 Flavanols (Flavan-3-ols) Built on the skeleton of flavan-3-ol (IUPAC: 3-hydroxy-2-phenyl-1,4-benzopyrane), the structure of flavan-3-ols presents two stereogenic centers and therefore each structure can exist as two pairs of enantiomers (four stereoisomers). Plants preferentially produce one over the other stereoisomer. While epimers can be separated with common reversed phase chromatography columns, enantiomers can only be resolved by chiral chromatography, which is a more demanding technique and therefore less commonly used in analytical research.

Chapter 1. Introduction

Therefore, the majority of plant phytochemical profiling studies report the presence of either cis (epi-flavan-3-ol) or trans-flavan-3-ol isomers, leaving aside the absolute stereochemical configuration of the chiral centers. The most important representatives of the class are (+)- catechin (2R,3S) and (-)-epicatechin (2R,3R), reported in high amounts in cocoa beans and cocoa powder,36 (+)-gallocatechin (2R,3S) which is very abundant in green tea and pomegranates21, (-)-epigallocatechin (2R,3R), which is notably found in Hypericum perforatum37 (popularly known as St. John’s wart, a medicinal plant with antidepressant activity) and afzelechin (2R,3S) which is present in some ayurvedic medicinal plants.38 The enantiomeric flavan-3-ols with 2S configuration like ent-catechin and ent-epicatechin occur much more rarely that their 2R epimers.8 Representative structures are given in Figure 4.

1.2.1.1.1 Condensed Flavanols: Proanthocyanidins (Condensed tannins) Flavan-3-ols are the monomeric building units of another important class of polyphenols, the proanthocyanidins, also known as condensed tannins (first isolated from plant tanning extracts). They are chemically stable compounds and do not break down to release their monomeric units upon acid or alkali hydrolysis. Due to the great complexity of structures, which can arise with the increase of the number of monomeric units, most of the literature dealing with the structure elucidation (detailed unambigous description of all aspects of regio and stereochemistry) of members this class of compounds refers only to oligomers with up to 4 monomeric units. However, the number of monomeric units, which can be present in one structure can be as high as 506. Monomers are bound together by oxidative condensation between C4 of the heterocycle of the upper monomeric unit and C8 or C6 of the lower monomeric unit. Double ligation between adjacent units is also possible, like for example, in the case of A-type procyanidin dimer (Figure 1.5).

Chapter 1. Introduction

Figure 1.4: Flavan-3-ol backbone and representative structures of the flavan-3-ols class

1.2.1.2 Flavonols Flavonols are built on the 3-hydroxyflavone (IUPAC: 3-hydroxy-2-phenylchromen-4-one) backbone, and the representatives of the class present different degrees of aromatic

Chapter 1. Introduction

hydroxylation and even methoxylation. The parent structure does not present any stereogenic center and therefore, no stereoisomers exist. Flavonols are present in a wide variety of fruits and vegetables in their free or glycosylated form.39 Most prominent representatives such as quercetin and kaempferol are presented in Figure 1.6.

Figure 1.5: Proanthocyanidin (condensed tannins) representative structures

1.2.1.3 Flavanones: Flavanones are built on the flavanone (IUPAC: 2-phenylchromen-4-one) backbone and can present different degrees of aromatic hydroxylation and methoxylation. They are mainly found in citrus fruits10 in their free or hydroxylated form. Since the parent molecule presents one stereocenter, each structure should exist as a pair of enantiomers. Naturally occurring flavanones present an S absolute configuration of the stereochemical center. Representative structures are given in Figure 1.7.

1.2.1.4 Flavones: Built on the flavone (IUPAC: 2,3-dehydro-2-phenylchromen-4-one) backbone, flavones can present various degrees of hydroxylation and methoxylation. They are mainly found in cereals and herbs (for example parsley and thyme21,40) in their free or hydroxylated form. Representative structures are presented in Figure 1.8.

Chapter 1. Introduction

Figure 1.6: Hydroxyflavone backbone and representative structures of flavonols

Figure 1.7: Flavanone backbone and representative structures of flavanones

Chapter 1. Introduction

Figure 1.8: Flavone backbone and representative structures of flavones

1.2.1.5 Anthocyanidins: Built on the 2-phenylbenzopyrillium backbone, anthocyanidins are widely distributed throughout the plant kingdom, and they most of the time account for the bright colors of certain flowers, fruits (particularly red/purple fruits, e.g berries) and vegetables(e.g. purple potatoes and purple carrots, red cabbage, red beat, aubergine peel). The structures of the most common anthocyanidins in nature are presented in Figure 1.9. Their names originate most of the time from the plant source from which they were first isolated and characterized: rosinidin from roses, petunidin from petunias, peonidin from peonies, delphinidin from delphinium flowers. Cyanidin takes its name from the greek word ‘κύανος’-kyanos, which means blue, and it accounts, among others for the blue color in blueberries and cornflower. Anthocyanins are glycosylated anthocyanidins.

Chapter 1. Introduction

Figure 1.9: 2-phenylbenzopyrillium backbone and the structures of the most common naturally occurring anthocyanidins

Anthocyanidins and anthocyanins are of great economic importance as they can be used as natural food coloring agents. The extended conjugation present in the molecules of anthocyanidins due to the positive charge they are bearing is sensitive to pH conditions. Since anthocyanidins owe their color to this conjugated chromofore, any factor that interferes with the conjugation will have a direct impact on the color of the molecules. The classical example of how anthocyanidins can be differently colored is given by pansy flowers, which show a great range of colors, all due to the same anthocyanidins, in different pHs. The chemical transformations underlying these color changes are presented in Figure 1.10. Although they possess a relatively labile chemical structure, anthocyanins present quite high stability within the plant tissues, due to various stabilizing interactions with other classes of polyphenols.41,42 It has been shown that non covalent (π- π stacking) interactions between colorless flavonoids and anthocyanidins enhance absorbance and sometimes shift absorbtion maximum of the later to higher wavelengths (batochromic shift) and thus enhance their color intensity.43 The phenomenon is called copigmentation and it of great importance to the formation of the red

Chapter 1. Introduction

color in anthocyanin containing plants. The impact that the nature of possible cofactors has on the magnitude of copigmentation has been studied extensively in wines and it has been found out that copigmentation can account for between 30 and 50% of the color in young wines and that it is primarily influenced by the levels of several specific, noncolored phenolic components or cofactors.43-45

Figure 1.10: Structures of cyanidin in aqueous solution under varying pH

1.2.2 Small phenolic acids: Small phenolic acids present a single aromatic ring in their structure bearing one or more phenolic hydroxy or methoxy groups. The most commonly encountered are the shikimate pathway products gallic acid and elagic acid and the hydroxycinnamic acids, namely p- coumaric acid, caffeic acid, ferulic acid and sinapic acid (Structures in Figure 1.1, 1.2), which are products of the phenylpropanoid pathway. They are seldomly present in plant materials in their free form but most often bound through ester- or glyosidic bonds to sugar molecules (most commonly glucose) or cyclitols like shikimic or quinic acid. Substances of the latter group are known as chlorogenic acids and they are by far the most well investigated group of hydroxycinnamic acid derivatives in terms of structural elucidation of the naturally occurring regio- and stereoisomers46-48 as well as in terms of their potential health benefits.49 Coffee, tea, artichokes, eggplants and pomegranates are among the richest sources of chlorogenic

Chapter 1. Introduction

acids.6,20,21 Given their high abundance in most edible plants and their increased bioavailability, hydroxycinnamates can be considered as the most relevant class of polyphenols for the human diet.50 Ferulic and p-coumaric acids in particular are found in their sugar-conjugated form in the structure of plant cell walls (ferulic acid in its dehydrodimeric forms), where they contribute to increasing rigidity by cross-linking to cellulose or lignin.51-53 Ferulic acid in particular presents increased relevance for the food industry as it can serve as a bio based raw material for the sustainable production of vanillin.54

1.2.2.1 Hydrolizable tannins: Hydrolizable tannins are esthers of cyclitols, most commonly shikimic and quinic acid, or sugars with gallic acid (gallotannins) or elagic acid (ellagitannins). They can be easily hydrolyzed in alkali, acids or even hot water: Representative structures are given in Figure 1.11.

1.2.3.2 Conjugates of hydroxycinnamic acids with sugars Although not as abundant as chlorogenic acids, the sugar derivatives of hydroxycinnamic acids are also very frequently encountered in a wide variety of plant extracts. Research dealing with their structure elucidation dates back to the 1920s, with melilotoside (Figure 1.11) being the first ever reported sugar conjugate of a hydroxycinnamic acid in 1925.55 Reports from the early 1960s mention the presence of simple hydroxycinnamoyl glucose esters in over one hundred different plant sources. Before the 1980s, structures of natural products were identified after purification by chemical degradation (acid/base or enzymatic hydrolysis) followed by UV or IR spectroscopy and various chemical assays of the resulting products and comparison to available standards.55 Purification from the crude extracts was achieved by preparative paper and thin layer chromatography wih low-resolution capacity. Therefore, coeluting isomeric species were regarded as one compound and aspects of regiochemistry were very often disregarded; e.g: caffeoyl gucose and/or feruloyl or synapoyl glucose were reported as one compound,56 although each sugar hydroxycinnamic acid-hexose combination presents five different regioisomers each of which can exist as a pair of alpha and beta anomers. A large number of sugar conjugates of hydroxycinnamic acids, including rather complex structures including both esters and O-glycosides, have been elucidated after the mid 1980s due the advances made in NMR spectroscopical techniques. A few examples include

Chapter 1. Introduction

phenylethanoid-hydroxycinnamoyl glycosides like verbascoside (Figure 1.11) and echinacoside from medicinal plants,57 the heavenly blue anthocyanin pigment of morning glory flowers (Ipomea purpurea) containing peonidin, caffeic acid and glucose residues,58 sulphonated hydroxycinnamoyl esters specific to various species of ferns,59 and synapoyl gentiobiosydes from Brasicaceae vegetables.60 Hydroxycinnamic acids-particularly ferulic acid and its dihydrodimers- esterified with cellulose in plant cell walls have as well been suggested to facilitate cross linking of lignin to cellulose in the cell walls of cereals like wheat, oat and barley.48

1.2.3 Monolignols: The structural monomers of lignin, which is the second most abundant polymer on Earth (after cellulose), monolignols are further metabolic products of hydroxycinnamic acids, formed by reduction of the carboxylic moiety to an alcohol (Figure 2). Lignin is a structural polymer of primary importance to vascular plants, since together with cellulose that it is cross-linked to in cell walls, it accounts for tissue rigidity and mechanical strength.61 Due to its hydrophobicity, which dramatically reduces water permeability of the tissues, vascular plants are able to retain and circulate water and the nutrients it carries, which is vital for their development and even their mere existence. As an extremely slowly decomposing material, lignin also plays an important ecological role, as it accounts for a large fraction of soil humus which is mainly responsible for the fertility (quality) of soil.1 The three monolignols are p-coumaryl alcool, coniferyl alcohol and sinapyl alcohol (Figure 1.2) and they are incorporated into lignin as p- hydoxyphenyl (denoted as H), guaiacyl (denoted as G) and syryngyl (denoted as S). Different species of plants present different ratios of the monomeric units. Lignin is formed by radical polymerization of monomers, under the action of oxidative enzymes found in cell walls (for example Laccase and Peroxidase). The complete mechanism is not yet fully elucidated.61 Lignin also presents great economical potential, as in can be used as a bio based raw material for various industrial applications. Volatile compounds formed by the combustion of lignin, namely guaiacol and its methylated derivatives account for the taste and aroma of grilled or smoked food products.

Chapter 1. Introduction

Figure 1.11: Chemical structures of simple phenolic acids, quinic and shikimic acids, representative hydrolysable tannins and selected examples of hydroxycinnamic acid-sugar derivatives

Chapter 1. Introduction

1.2.4 Phenylpropenes: Further metabolites of monolignols, phenylpropenes are volatile compounds present in high amounts in the essential oils of various aromatic plants. Due to their pleasant smell, they are of importance to the cosmetic industry as perfume ingredients. Some phenylpropene rich essential oils are used as anesthetics in traditional medicine (for example eugenol rich clove oil used against dental pain). Some representatives present psychoactive effects or toxicity. Most prominent phenylpropene representatives are shown in Figure 1.12.

Figure 1.12: Structures of the most representative phenylpropenes

1.2.5 Coumarins Coumarins are another subclass of shikimate pathway metabolites present in many plants, including dietary relevant plants like strawberries, apricots, cherries, carrots, celery, parsnip, etc. Their generic name of the entire class comes from the tonka bean’s (native to French Guaiana) French name, ‘coumarou’ from which the simplest representative coumarin was first isolated in in high amounts in 1820. Due to their pleasant sweet smell, they have as well been used in the cosmetic and food industry. However, they have recently been found to present

Chapter 1. Introduction

moderate hepatotoxicity in rodents, fact that has raised concerns regarding their use as flavoring agents in foods (flavoring of tobacco or certain alcoholic beverages) or as odorant agents in cosmetics. Some substituted derivatives (for example warfarin) which act as anticoagulants by inhibiting vitamin K epoxide reductase can be used as drugs against thrombosis. Structures of the most notable representatives of the class are presented in Figure 1.13.

Figure 1.13: Chemical structures of representative coumarins

1.2.6 Stilbenes (Stilbenoids) Stilbenes are a small group of phenylpropanoids possessing a 1,2-dibenzylethene packbone and various degrees of hydroxylation and methoxylation. They are termed as phytoalexins- chemical defense metabolites synthesized by the plants as a response to pathogenic infections. The most prominent representative of this class is resveratrol from grapes, which accounts for most of the beneficial health effects associated with the consumption of red wine.

Figure 1.14: Chemical structures of representative stillbenes

Chapter 1. Introduction

1.3 Methods for polyphenol analysis

The vast majority of studies carried out within the last four decades which deal with the investigation of plant polyphenolic extracts involve separation of the components by high- performance liquid chromatography (HPLC) followed by their individual detection by UV-VIS and MS. The stationary phases mainly employed are reversed phase packing columns (most commonly C-18 or C-18 variants like diphenyl C-18 or amide C-18 packings, less commonly C-8 and C-12 packings). Columns with particle sizes of 3μm or 5 μm and 3-5mm inner diameter are suitable or classical HPLC instruments62, which can optimally operate under pressures of up to 300bar.

Ultra-high pressure liquid chromatography systems developed in the recent years, which can accommodate pressures of up to 1000 bar are optimally coupled with columns with smaller particle size (1.7µm) and smaller inner diameter (1.7-1.9mm),35 with which greatly enhanced resolution (up to 10 fold higher than with conventional HPLC), higher peak efficiency and sensitivity are achieved. This translates into lower limits of detection and lower amounts of solvents used for analysis higher sample throughput.

Gradient elution of the mobile phase-which most commonly consists of a binary mixture of water and an organic solvent (predominantly acetonitrile or methanol)-generally achieves the best separation of polyphenolic mixtures. Small quantities of weak acids are commonly added to the mobile phase for improving peak shape (sharper, narrower peaks).63

Although not as commonly used as reversed-phase column chromatography, hydrophilic interaction liquid chromatography (HILIC) is gaining popularity for the separation of polar and semi-polar samples (e.g: glycosylated derivatives) on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN) using an isocratic elution system.64

Most LC systems have integrated UV-VIS and photodiode array detectors, which record the signals of individual components eluting from the chromatographic column. Polyphenols have characteristic UV absorbance at wavelengths between 190 and 380nm.17,39,62 Although it is not sufficient for unambiguous structural characterization, DAD is an important tool for the tentative assignment of the main phenolic classes present in plants because they show characteristic UV–VIS absorbance. DAD is the method of choice for polyphenol quantification.

Chapter 1. Introduction

The pioneering work performed in the field of structure elucidation of the vast majority of polyphenolic compounds known today was based on preparative chromatographic isolation of most abundant compounds present in plant extracts followed by NMR spectroscopy analysis. Although NMR is the method of choice when it comes to unambiguous structure elucidation of any chemical compound, it allows quite low sample throughput, as the analysis requires pure compounds in relatively high amounts (at least 5mg). Given the great complexity of plant polyphenolic extracts, severeal steps of extensive preparative chromatography are commonly required for achieving pure compounds. Hence, this method of investigation is only suitable for major compounds. Since the development of electrospray ionization, a soft ionization technique which allows for the mass spectrometrical analysis of high and medium polarity compounds, great advances have been achieved towards elucidation of individual components of complex polyphenolic mixtures, even those present in trace amount.

The analytes are delivered in solution and ionized by applying high electrical potential. Deprotonated molecular ions [M-H]- are generated in negative ion mode and protonated molecular ions [M+H]+ or metallic cation adducts like [M+Na]+ or [M+K]+ in positive ion mode. Subsequent fragmentation of the ions present in the MS spectrum occurs by collision with a neutral gas of high energy (collision induced dissociation).

The vast majority of polyphenol studies employ negative ion mode for the analysis, since [M-H]- ions are easily formed due to the increased acidity of the phenolic hydroxyl moieties and their subsequent fragmentation spectra are easier to analyse than in positive ion mode where spectra are more complicated due to the presence of various adducts. Modern mass spectrometers make use of a variety of mass analyzers: single quadrupoles, triple quadrupoles, ion traps, time of flight analyzers, hexapoles, octapoles and hybrids thereof like quadrupole- time of flight (QTof), linear trap-orbitrap and quadrupole-linear ion trap.35

High-resolution mass spectrometry provides information regarding the elemental composition of the analyte, whereas tandem mass spectrometry (MS/MS) provides information about the structure of the investigated compounds. Instruments with hybrid mass analyzers have the advantage of providing high resolution of both the molecular ions [M-H]- and the fragments generated by collision induced dissociation.

Chapter 1. Introduction

Multistage fragmentation (MSn) of ions of interest gives detailed information about the connectivity of the individual chemical moyeties (e.g: flavonoid O-glycosydes can easily be distinguished from C-glycosydes on the basis of their fragmentation pattern;48 regioisomers of hydroxycinnamoyl-quinates and hydroxycinnamoyl-shikimates can easily be assigned on the basis of their distinct fragmentation patterns,48 etc..). Unambigous structure elucidation of compounds still requires authentic reference standards. Experimental tandem MS spectra can be compared to those of available reference standards or literature data. On some occasions (like for example in the case of chlorogenic acids) rules of fragmentation can be developped allowing further reliable structure elucidation of compounds.

Chapter 1. Introduction

1.4 Motivation and aim of the study 1.4.1 Cocoa Polyphenol analysis Cocoa is an agricultural commodity that is produced annually in quantities over 4.5 million tons worldwide and converted into cocoa powder and chocolate products with an estimated annual trade value of over 100 billion Euro (FAO 2016 statistics). Originating from Mesoamerica, cocoa was valued by the indigene ancient Olmec and Maya civilizations for its refreshing and vitalizing effects to such extent that the plant was considered divine in origin. The first scientific work about cocoa’s health effects, entitled: ‘Du The, Du Caffe, et Du Chocolat Pour La Preservation et pour la Guerifon des Maladies’ (Tea, Coffee and Chocolate for preserving and curing illness) was published by a physician at the Court of Louis XIV of France in the year 1687. For centuries cocoa was classified as a medicinal plant. Endless studies involving cocoa and chocolate have been conducted in the last two decades and a multitude of health beneficial properties have been described. In short, all these properties fall under one of the categories: antioxidant, cardiovascular protector and antitumoral. The health effects associated to cocoa consumption are attributed to the high levels of polyphenols therein, particularly the flavonoid subclass, which account for 15-20% of the dried, fat free mass of fresh cocoa beans.

However, scientific attention is almost fully oriented towards the major flavan-3-ol representatives and there is a limited number of reports dealing with the in depth structural elucidation of cocoa’s lower abundance polyphenols. Since in vivo effects depend not only on the concentration of certain chemicals but as well on their bioavailability and enzymatic affinities, it is only fair to assume that a low abundance compound with higher bioavailability and higher in vivo enzymatic affinity/activity could be just as relevant as a more abundant compound which presents lower bioavailability and lower enzyme activity. In these terms, complete structural elucidation of the polyphenolic fraction becomes relevant. Synergistic and additive effects of minor plant secondary metabolites or processing products might as well be of relevance to the overall health effects.

Apart from the relevance with respect to their potential health effects, phytochemicals in cocoa also present a considerable economic relevance, since global cocoa consumption is a hedonistic- indulgence driven phenomenon rather than a health-improving one. During the extensive technological processing steps (fermentation, roasting, alkalization, hydraulic pressing, conching) that cocoa beans are subjected to on their way towards becoming cocoa powder or

Chapter 1. Introduction

chocolate products, the phytochemicals therein undergo various chemical transformations leading to the development of aroma, flavor and color components of the final products. A thorough understanding of the initial chemical composition of the cocoa beans and of the mechanisms underlying their chemical transformations along the industrial processing chain would allow a fine tuning of these processes towards cocoa powders with certain desired properties. Moreover, understanding the chemical differences between cocoa samples from different origins would also be helpful in sourcing the most suitable cocoa samples for specific applications. The aim of the first part the present contribution is to get an insight into the overall polyphenolic composition of raw fermented cocoa beans and compare the profiles of samples of different origins. In order to achieve this, two different approaches are employed for the analysis:

Firstly, methanolic fermented cocoa bean and roasted bean extracts are investigated by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), a non-targeted organic structural spectrometric method that allows for ultra-high resolution of complex organic mixtures like petroleum samples or oceanic dissolved organic matter. Despite their great chemical complexity, different cocoa samples are characterized and compared based on their molecular composition using suitable graphical representations methods of the data, namely class plot diagrams and three dimensional van Krevelen plots presenting classes different classes of compunds color coded based on their heteroatom contents. In addition to the classical two-dimensional van Krevelen plots which depict the hydrogen index (hydrogen/carbon ratio) against the oxygen index (oxygen/carbon ratio), three dimensional van Krevelen plots take into account the relative intensity of the signals. Therefore, a qualitative and relative/quantitative overview the chemical composition of the samples is achieved, allowing for better comparison of the differences.

Secondly, reversed phase HPLC- High resolution MS and HPLC-Tandem MS analysis of cocoa methanolic extracts is performed providing information about molecular formulae and fragmentation behavior of individual species, which aims at the structural elucidation of compounds previously unreported in cocoa.

Chapter 1. Introduction

1.4.2 Profiling and quantification of hydroxycinnamoyl glucoses in dietary plants Conjugates of hydroxycinnamic acids with carbohydrates have been very frequently reported in a wide variety of vegetables, fruits and medicinal plants ever since the middle of the last century.55 Although a significant number of quite complex derivatives (e.g. structures in Figure 10) have been completely elucidated, reports on the structure elucidation of the simplest monohydroxycinnamoyl hexoses are to our best knowledge missing from literature. No biological activity data have been reported so far for monohydroxycinnamoyl hexoses due to the lack of authentic reference standards and difficulties in compound isolation and characterization; however, based on their structural similarities it can safely be assumed that CG derivatives share many beneficial health properties of their chlorogenic acid relatives. Each hydroxycinnamoyl-hexose pair can present five ester regioisomers (each of them existing in equilibrium as a pair of alpha and beta anomers) and at least two epimeric O-aryl-glycoside isomers (number varying according to the number of phenolic hydroxyl groups presented by each hydroxycinnamic acid). Older phytochemical analysis studies completely disregarded the aspects of regio- and stereochemistry, as isolation was carried out on low resolution preparative paper or thin layer chromatography;56 numerous recent studies employing HPLC-MS techniques report on the presence of multiple hydroxycinnamic acid-hexose isomers in different dietary relevant plants. However, they still leave open regiochemistry, stereochemistry, the nature of the linkage between the hexose and the acid and sometimes even the identity of the hexose. Moreover, quantitative data on the simplest representatives of this class of compounds are completely missing from literature. Therefore, a systematic approach towards the complete structural characterization of individual isomers of frequently occurring monohydroxycinnamoyl hexoses is needed. The recently developed LC-MSn hierarchical schemes for identification of isomeric chlorogenic acids, using pure synthetic standards have proven to be very useful tools for the profiling of various plants for their chlorogenic acid contents. They have served as a basis of numerous profiling studies of a great variety of plants, which successfully identified the individual structures based on their dictinct MSn fragmentation and their relative elution order from a reversed phase column omitting compound isolation or using reference standards, which are very often expensive and sometimes even unavailable commercially. The present contribution firstly aimed at synthesizing reference standards to be used for developing a hierarchical LC-MSn scheme for the individual identification of all ten hydroxycinnamoyl-glucose ester isomers. Once developed, the scheme would serve as a

Chapter 1. Introduction

powerful tool for the unambiguous assignment of the respective compounds in any food source. Therefore, the aim of the second part of the present work was to provide complete structural characterization and quantitative data for individual hydroxycinnamic acid-glucose derivatives present in dietary relevant plants. For this purpose, a quantitative HPLC-MS method was developed and validated. The dietary sources investigated include: strawberries (Fragaria ananassa), raspberries (Rubus idaeus), blueberries (Vaccinium corymbosum), blackberries (Rubus fruticosus), red currants (Ribes rubrum), black currants (Ribes nigrum), lingonberries (Vaccinium vitis-idaea), gooseberries (Ribes uva-crispa), purple chokeberries (Aronia melanocarpa), elderberries (Sambucus melanocarpa), cranberries (Vaccinium oxycoccos), goji berries (Lycium chinense), sea buckthorn (Hippophae rhamnoides), açai berries (Euterpe oleracea), sour cherries (Prunus cerasus), pomegranate (Punica granatum), tomatoes (Solanum lycopersicum), peppers (Capsicum spp), chillies (Capsicum annuum,) and aubergines (Solanum melongena).

Chapter 1. Introduction

1.4.3 Understanding the fragmentation of glucose in mass spectrometry Glucose is one of the most important molecules in nature. It constitutes the primary energy source for all living organisms; it is used as a building block in structural polymers and signaling components. Many classes of secondary metabolites are present in living tissues in their glycosylated form. Most polymeric/oligomeric carbohydrates and glycosylated natural products are characterized since over two decades using mass spectrometry based techniques. The fragments resulting from the dissociation of these glycosylated compounds are usually produced by fission of the bonds within the carbohydrate units themselves. It is surprising to note that despite all advances in the mass spectrometrical analysis of glycosylated natural products, the fragmentation mechanisms of the most simple hexose building blocks, most notably glucose has only been studied on rare occasions and no exhaustive mechanistic study using all isotopomers exists in the literature. A thorough understanding of the fragmentation mechanism would evidentiate the influence of the stereochemistry on the relative intensity of the fragment ions. This would have immediate consequences for the full structural analysis of complex carbohydrate-natural product conjugates and form the basis of a comprehensive formalism allowing carbohydrate structure elucidation by mass spectrometry.

This fact has raised interest towards carrying out an exhaustive study of the mechanism of fragmentation of D-glucose using seven 13C labeled isotopomers and six 2H labeled isotopomers by two different fragmentation techniques, namely collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). An additional aim is to develop HPLC- tandem MS method to separate the two anomers of D-glucose on a HILIC packing column and investigate their fragmentation patterns individually.

Chapter 1. Introduction

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Chapter 1. Introduction

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Part I. Cocoa Polyphenol analysis

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin Maria A. Patras, Borislav P. Milev, Thorsten Dittmar, Gino Vrancken, Nikolai Kuhnert.

A b s t r a c t

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is an ultra- high resolution mass spectrometry technique used mainly in analysis of unresolved complex mixtures comprising tens of thousands of analytes. For the ﬁrst time, it was used to analyze samples of raw fermented cocoa beans originating from Cameroon and Ivory Coast. The direct infusion mass spectra of the raw fermented cocoa bean extracts showed 10 091 and 10 911 peaks, resp., rating cocoa among the most complex organic mixtures ever analyzed. Automated molecular formula calculations could assign 2995 and 2968 of the peaks, resp. to formulae containing only C, H, O, N ≤3 and S ≤1 atoms. The formulae were separated into four groups depending on their heteroatom content and the intensities of the groups were compared in class plots, showing the highest population in the CHON species, but the highest abundance in the CHO species. Elemental ratios obtained from the molecular formulae were plotted in an intensity coded three-dimensional modiﬁcation of the van Krevelen diagram. For the CHO species, the van Krevelen diagram showed that most of the intensity belongs to the lipid, polyphenol and carbohydrate regions of the plot. The biggest difference was observed in the CHON group, assigned as peptide degradation products, where the Ivorian beans showed greater variety and molecular diversity and higher total intensity of the nitrogen containing compounds, in accordance with the fact that the Ivorian beans show generally higher nitrogen content than the Cameroon beans. FTICR-MS proves capable not only for high- throughput comparison of major classes of metabolites from cocoa samples from different origins, but also can give insight into the different molecular formulae comprising these compound classes.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

INTRODUCTION

Globally 4.2 Mt of cocoa beans was harvested in 2012 and converted into cocoa powder and chocolate with an estimated value of 100 billion € (fao.org 2012 statistics). Ivory Coast is the world's leading cocoa manufacturer and exporter with an annual production of about 1.2 Mt (2008/2009). Cameroon is currently at the 5th position with an annual production of 0.2 Mt (2008/2009). The Cameroon and Ivorian cocoa beans are of comparable quality, but the Cameroon samples generally show larger bean size with about 85 beans per 100 g, versus about 105 beans per 100 g in Ivorian bean samples, which is a desirable quality when it comes to cocoa. In the Cocoa Atlas (Rohsius, Elwers, & Lieberei, 2010), both their flavor profiles are described as having a pronounced cocoa note and moderate to high bitterness and astringency. The Cameroon samples are described after sensory panel evaluations as fruity, floral or herbal taste, whereas the Ivorian samples often are described as having a tobacco flavor note. As a non-targeted organic structural spectroscopic method, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) allows for an unprecedented resolution of complex organic mixtures, where thousands of compounds are expected to be present. Until recently, the technique was mainly used for petroleum analysis (Fernandez-Lima et al., 2009) or environmental samples like oceanic dissolved organic matter, where it allows for characterization and comparison of complex samples based on their whole molecular composition (Hertkorn, Harir, Koch, Michalke, & Schmitt-Kopplin, 2013). Processed foods like cocoa, coffee, tea or wine are known to show comparable complexity and FTICR-MS has recently been successfully used for similar characterization of the oxidation cascades in tea fermentation (Kuhnert, Drynan, Obuchowicz, Clifford, & Witt, 2010, Kuhnert, Clifford and Mueller, 2010)), roasting of coffee (Jaiswal, Matei, Golon, Witt, & Kuhnert, 2012) or the origins of wood used in wine cask making (Gougeon et al., 2009). Due to the intense processing of cocoa beans prior to production of cocoa powder or chocolate, involving fermentation, roasting, alkalization and possibly conching, the complexity and molecular diversity of cocoa constituents must be expected to exceed that of most other dietary materials (Kuhnert, Dairpoosh, Yassin, Golon, & Jaiswal, 2013). Here, for the first time, we have used direct infusion high resolution and mass precision FTICR-MS to investigate and describe the chemical complexity of the cocoa bean and have successfully used suitable data representation for finding chemical differences between bean samples of Cameroon and Ivorian origin.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

MATERIALS AND METHODS

The two samples of raw fermented Cameroon and Ivorian cocoa (Theobroma cacao) beans were received from Barry Callebaut. The cocoa beans were first macerated to a powder using a knife mill Grindomix GM200 by Retsch (Haan, Germany) for 20 s at 10 000 rpm. 5 g of the powder was then homogenized in 50 ml n-heptane using ULTRA-TURRAX T25 digital, equipped with an S25N 18G dispersing tool by IKA (Staufen, Germany) for 3 min at 24 000 rpm. The resulting suspension was centrifuged using a Centrifuge 5702 by Eppendorf (Hamburg, Germany) at 4400 rpm for 15 min. The supernatant was removed and the resulting powder was dried on air. The extractions were performed by stirring 1 g of the defatted powder in 20 ml of 70% aq. MeOH overnight. The crude extract was diluted 10 times, using MilliQ water and was filtered through a cellulose filter. The analyses were performed on a Solarix 15 T FTICR mass spectrometer from Bruker Daltonics, Bremen, Germany. The analytes were introduced in an electrospray ionization (ESI) source inlet at a rate of 2 μl/min using a kdScientific syringe pump and the analysis was performed in negative mode.

Internal calibration was performed using the inbuilt quadratic calibration equation of the Bruker DataAnalysis 4.0 software with a list of 15 compounds with masses between 200 Da and 1000 Da known to be present in cocoa (Wollgast & Anklam, 2000). The standard deviation of the calibration points was below 0.1 ppm. The peaks were detected using the inbuilt settings for FTMS data using a signal-to-noise ratio of 5 as a threshold. The automatic molecular formula assignment was performed using the SmartFormula plugin with constraints as follows: mass tolerance = 0.5 ppm, H min = 2, O min = 1, N max = 3, S max = 1, H/C min = 0.33, H/C max = 2.2, O/C max = 1, and relative intensity threshold = 0.01%.

RESULTS AND DISCUSSION

In this contribution we decided to analyze alcoholic extracts of raw fermented cocoa beans from two different geographical origins. Since a previous work showed that a cocoa extract after fermentation and roasting exhibited in excess of 30 000 resolved mass spectrometrical signals (Kuhnert et al., 2013), a number too large to allow for a meaningful data interpretation at the current state, we opted in this contribution to investigate phenol rich extracts of raw fermented cocoa beans. When a particular compound class of interest is to be analyzed, selectivity can be achieved by using suitable sample preparation and sample ionization methods.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

Solid–liquid extraction with aqueous alcoholic extracts combined with a soft ionization technique like electrospray ionization has proven to be useful for selectively studying phenolic fractions of phenolic-rich foods by mass spectrometry (Ignat, Volf, & Popa, 2011; Robards, 2003). Raw fermented cocoa bean extracts from two different types of cocoa beans from Cameroon and Ivory Coast were prepared and measured under electrospray ionization conditions in the negative and positive ion mode as direct aqueous methanolic infusions in an FTICR mass spectrometer at 15 T. The peak detection algorithm found 10 090 and 10 910 peaks in the Cameroon and Ivorian raw fermented beans, respectively, with a signal-to-noise ratio higher than 5 in the negative ion mode spectrum. In the roasted beans, the peaks were 9814 and 9203, respectively. In the positive ion mode, the numbers of peaks with the same signal-to-noise ratio in the Cameroon and Ivorian raw fermented beans were found to be ca. 4500 and 5000. This result conﬁrms the fact that a signiﬁcant part of the complexity of the cocoa metabolome is due to compounds, which are acidic and would be expected to be readily ionized by loss of a proton yielding pseudomolecular [M − H]- ions. In both the Cameroon and the Ivorian beans, an empirical molecular formula could be assigned to about 3000 mass-to-charge signals (excluding the same formulae with a different isotopic composition), or close to 30% of them, using the constraints described above. The total intensity that could be assigned, however, was above 50% of the total measured intensity. It is remarkable that these numbers of peaks show complexity exceeding that of the oceanic organic dissolved matter (Hertkorn et al., 2013), thereby placing cocoa among the most complex organic mixtures experimentally investigated. Furthermore, about 850 of the peaks in each sample were unique, suggesting bigger variety in terms of different compounds in beans with different origins. Conservative molecular formula constraints, used normally in formula assignments of oceanic dissolved organic matter with low heteroatom content, were used to compute molecular formulae with errors below 1 ppm. From the molecular formulae calculated from high resolution mass data elemental ratios were determined and grouped into four classes of compounds based on their heteroatom content called CHO, CHON, CHOS and CHONS (the number of each atom is determined by the formula constraints). The population and the abundance of the four classes of compounds are represented in a class plot diagram (Fig. 1a and b). The majority of the number of peaks assigned (Table 1) contained nitrogen as a heteroatom (roughly 50% of the total), but as a percentage of the sum of intensity over all peaks,

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

the molecules containing only carbon, hydrogen and oxygen were most prominent (between 56% for raw fermented Ivorian beans and 86% for roasted Ivorian beans). It was striking that although the difference between the two bean samples in the numbers of peaks assigned as CHO and CHON compounds was small, the the percentage of the sum of the intensities of the nitrogen containing compounds was much higher in the raw fermented Ivorian beans if compared with the Cameroon beans. Furthermore, it was found that only about 80% of all the peaks detected (about 8200 and 9100 in the Ivorian and the Cameroon beans, resp.) accounted for 98% of the total measured intensity, offering a good possibility of data reduction in cases where less rigorous data analysis can prove to be sufficient. For the sake of a better representation of the four formula classes, a van Krevelen diagram (Kim, Kramer, & Hatcher, 2003) was plotted with the elemental ratios between hydrogen and carbon on the y-axis and between oxygen and carbon on the x-axis and the four compound classes color coded. Traditionally used in petrolomics, this type of graph is most commonly seen with two dimensions only. However, adding an additional dimension of data representation, by representing the intensity of each signal by the size of the corresponding circle (Fig. 2), one can significantly increase the information contained in such a diagram. The usefulness of representing the intensity of the signals becomes obvious when one looks at the percentage of the measured intensity of the signals in each class. For instance, the CHO compounds in the Cameroon raw fermented beans are only 11.2% of the number of all assigned peaks, yet they account for 69.7% of the total sum of intensities of the assigned peaks. Furthermore, the formulae of the different classes are grouped together, according to their elemental composition, with lipids, phenolics and carbohydrates appearing in distinct regions (upper left, lower middle, and upper right, resp.). A further observation that can be made is that the majority of the CHO formulae appear between the main regions of lipids, carbohydrates and phenolics. Another distinct group of CHO formulae lies between H/C 1.5–2.0 and O/C 0.5–0.7 suggesting tentatively the presence of lipid derivatives of phenolics and carbohydrates. They mostly contain between 10 and 20 carbon atoms, so they are most likely unreported condensed carbohydrates. Between the two samples, the most significant differences can be found in the CHON set of formulae, which are significantly higher in the case of the Ivorian beans. The difference in nitrogen content in the fermented beans, ca. 5.0% in the Cameroon beans and ca. 5.5% in the Ivorian beans reported in the Cocoa Atlas (Rohsius et al., 2010), is consistent with this finding.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

From their elemental ratios and nitrogen content of up to 3 atoms per molecule, it can be deduced that they are amino acids, short peptides or adducts of amino acids with other small molecules. Such compounds would be a result from the degradation of proteins, which is occurring during the process of fermentation upon the action of exo- and endo-peptidase enzymes (Kirchhoff, Biehl, Ziegelerberghausen, Hammoor, & Lieberei, 1989). The differences in the extent to which the proteins are degraded can be a result of the different climate and bacterial profiles of the fermentation in the two different countries of origin. Differences in the aforementioned compounds will become even more pronounced during the subsequent steps of chocolate or cocoa powder production, and particularly during the roasting and alkalization steps, leading to development of different flavors crucially depending on peptide degradation products as unique flavor precursors (Kirchhoff, Biehl, & Crone, 1989).

CONCLUSION

The high resolution of the FTICR-MS technique has proven to be a valuable tool in studying the molecular composition of complex organic mixtures. Although not commonly regarded for its complexity, cocoa proved to be of a comparable complexity to that of environmental organic samples that were until now most commonly analyzed by FTICR-MS. Based on the number of signals obtained in the mass spectrometry experiments, it would be safe to conclude that the number of different compounds in cocoa is around or even more than 40 000. Even after the first processing step of fermentation the cocoa bean comprises more detectable and resolvable analytes than any other processed food thus investigated. The automated algorithms for peak detection and formula assignment provided by modern software packages offer a good high- throughput method for handling, comparing and visualizing of mass spectral data. Furthermore, molecular formulae were assigned to about one-third of the signals detected and they could be divided into different compound classes, depending on their elemental composition. The FTICR experiments performed in this work revealed a clear difference in the main classes of compounds of beans of Cameroon and Ivorian origin. Although the total number of signals was comparable between the two origins, the class plots and the van Krevelen data representation revealed a significant difference in the abundance and intensity of the nitrogen containing class of compounds. The analysis of the roasted beans showed a reduction of the number of compounds, likely due to degradation and loss of minor components due to the high temperature

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin during roasting. Furthermore, the lists of exact masses can be very useful as supporting evidence, needed sometimes in other mass spectrometric or chromatographic experiments.

ACKNOWLEDGEMENTS We would like to express our gratitude towards Katrin Klaproth for her excellent support during the FTICR-MS measurements, to Jerome Derrey for providing the samples for our study, and to Herwig Bernaert for his support of the project. We would like to thank Barry Callebaut for providing the financial support for this work.

Figure 2.1: Class plot diagram of the population (a) and abundance (b) of mass spectrometric signals; C = Cameroon, IC = Ivory Coast, Cr = Cameroon (roasted), ICr = Ivory Coast (roasted).

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

Figure 2.2: Three-dimensional van Krevelen diagram representing different formula classes in the spectrum of Ivorian bean extract.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

References for Chapter 2. Fernandez-Lima, F. A., Becker, C., McKenna, A. M., Rodgers, R. P., Marshall, A. G., & Russell, D. H. (2009). Petroleum Crude Oil Characterization by IMS — MS and FTICR MS. Analytical Chemistry, 81(24), 9941–9947.

Gougeon, R. D., Lucio, M., De Boel, A., Frommberger, M., Hertkorn, N., Peyron, D., et al. (2009). Expressing forest origins in the chemical composition of cooperage oak woods and corresponding wines by using FTICR-MS. Chemistry — A European Journal, 15(3), 600–611.

Hertkorn, N., Harir, M., Koch, B. P., Michalke, B., & Schmitt-Kopplin, P. (2013). High-field NMR spectroscopy and FTICR mass spectrometry: Powerful discovery tools for the molecular level characterization of marine dissolved organic matter. Biogeosciences, 10(3), 1583–1624.

Ignat, I., Volf, I., & Popa, V. I. (2011). A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chemistry, 126(4), 1821–1835.

Jaiswal, R., Matei, M. F., Golon, A., Witt, M., & Kuhnert, N. (2012). Understanding the fate of chlorogenic acids in coffee roasting using mass spectrometry based targeted and non-targeted analytical strategies. Food & Function, 3(9), 976–984.

Kim, S., Kramer, R.W., & Hatcher, P. G. (2003). Graphicalmethod for analysis of ultrahighresolution broadband mass spectra of natural organic matter, the van Krevelen diagram. Analytical Chemistry, 75(20), 5336–5344.

Kirchhoff, P.M., Biehl, B., & Crone, G. (1989). Peculiarity of the accumulation of free aminoacids during cocoa fermentation. Food Chemistry, 31(4), 295–311.

Kirchhoff, P.M., Biehl, B., Ziegelerberghausen, H., Hammoor, M., & Lieberei, R. (1989). Kinetics of the formation of free amino-acids in cocoa seeds during fermentation. Food Chemistry, 34(3), 161–179.

Kuhnert, N., Clifford, M. N., & Mueller, A. (2010). Oxidative cascade reactions yielding polyhydroxy-theaflavins and theacitrins in the formation of black tea thearubigins: Evidence by tandem LC–MS. Food & Function, 1(2), 180–199.

Kuhnert, N., Dairpoosh, F., Yassin, G., Golon, A., & Jaiswal, R. (2013). What is under the hump? Mass spectrometry based analysis of complex mixtures in processed food —Lessons from the characterisation of black tea thearubigins, coffee melanoidines and caramel. Food & Function, 4(8), 1130–1147.

Kuhnert, N., Drynan, J.W., Obuchowicz, J., Clifford,M. N., &Witt,M. (2010). Mass spectrometric characterization of black tea thearubigins leading to an oxidative cascade hypothesis for thearubigin formation. Rapid Communications in Mass Spectrometry, 24(23), 3387–3404.

Robards, K. (2003). Strategies for the determination of bioactive phenols in plants, fruit and vegetables. Journal of Chromatography A, 1000(1–2), 657–691.

Chapter 2. Fourier transform ion cyclotron resonance mass spectrometrical analysis of raw fermented cocoa beans of Cameroon and Ivory Coast origin

Rohsius, C., Elwers, S., & Lieberei, R. (2010). The cocoa atlas. Leverkusen, Germany: Dresen Funke GmbH.

Wollgast, J., & Anklam, E. (2000). Review on polyphenols in Theobroma cacao: Changes in composition during themanufacture of chocolate andmethodology for identification and quantification. Food Research International, 33(6), 423–447.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Maria A. Patras, Borislav P. Milev, Gino Vrancken, Nikolai Kuhnert.

ABSTRACT Using a detailed HPLC–ESI-MS analysis including both high resolution and tandem MS analysis, a series of fourteen novel flavonoids were identified from alcoholic extracts of raw fermented cocoa beans from Cameroon. The novel compounds include a series of isomeric hexosides of flavan-3-ols and novel proanthocyanidine dimers and trimers and a sulfated flavan- 3-ol. Additionally a jasmonic acid sulfate derivative was identified in the cocoa bean extracts as one of the major metabolites produced under fermentation stress. Therefore, the chosen HPLC–ESI-MS analysis method revealed a greater complexity of the cocoa bean polyphenolic extract than previously reported and allowed individual assignment of novel compounds.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

INTRODUCTION

Cocoa consumption dates back to 1000 BC in Mesoamerica. It was introduced to Europe by Spanish conquistadores in the 16th century and its cultivation was soon after that spread to the overseas colonies of the European empires (Coe & Coe, 2007). The largest producers of cocoa today are Ivory Coast, Ghana and Indonesia. The African continent supplies 75% of the total annual production, which was estimated by The International Cocoa Organization to be 4.3 million tons in 2011. The average price per ton was also estimated to be 3105 US dollars, which translates into over 12.5 billion dollars trade costs per year for raw cocoa beans. During the manufacturing of cocoa powder, cocoa beans undergo a lengthy process including fermentation responsible for the development of flavor precursors, roasting and alkalization during which flavor and color components are formed, and milling which transforms the roasted and alkalized nibs into cocoa liquor and hydraulic pressing which brings down cocoa fat content to 10–12%(Wollgast & Anklam, 2000). Cocoa powder plays the main role in the chocolate industry, a business worth over 90 billion dollars a year. Chocolate consumption has been associated to a multitude of beneficial health effects since ancient times. The first scientific work about cocoa's health effects was published in 1687 (Coe & Coe, 2007). Many recent studies involving cocoa and chocolates with high cocoa content have been performed and a multitude of health beneficial properties have been described. In short, all these properties fall under one of the categories: antioxidant, cardiovascular protector and antitumoral (Cooper, Donovan, Waterhouse, & Williamson, 2008; Rimbach, Melchin, Moehring, & Wagner, 2009). Most of the studies attribute the beneficial health effects of cocoa consumption to the polyphenols therein, particularly the flavonoid subclass, which accounts for 6–8% of the dried, mass of fresh cocoa beans (Rimbach et al., 2009). The other class of compounds present in cocoa in significant amounts are methylxanthines, namely theobromine and theophylline (Brunetto et al., 2007), so it is only fair to take into consideration the fact that these compounds may possess as well health beneficial effects on their own, or at least be involved in synergistic interactions with polyphenols, interactions which may be responsible for the above mentioned health properties. In the case of methylxantines, the identification is quite simple, whereas polyphenols are present in cocoa (and in general in plant materials) as very complex mixtures which require some more elaborated analytical strategies.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Solid–liquid extraction with aqueous alcoholic extracts combined with a soft ionization technique like electrospray ionization (ESI) has proven to be useful for selectively studying phenolic fractions of phenolic rich foods by mass spectrometry (Ignat, Volf, & Popa, 2011). For the case of cocoa, a rather limited number of phenolics has been reported up to date (Hammerstone, Lazarus, Mitchell, Rucker, & Schmitz, 1999; NATSUME et al., 2000; Wollgast & Anklam, 2000). A recent FTICR-MS study of cocoa beans showed great complexity of the phenolic fraction, with over 10 000 peaks in negative ion mode (Kuhnert, Dairpoosh, Yassin, Golon, & Jaiswal, 2013). Considering elemental ratio boundaries from van Krevelen plots, a fermented cocoa bean shows at least 150 compounds, which must be classiﬁed as polyphenols (Kuhnert et al., 2013), an order of magnitude more than previously reported. This ﬁnding prompted us to investigate in this contribution fermented cocoa bean extracts in more detail using LC–tandem MS experiments to identify novel phenolics not previously reported.

MATERIALS AND METHODS:

Sample preparation

One sample of Cameroon cocoa beans was received from Barry Callebaut, Wieze, Belgium. The solid was first macerated to a powder using a knife mill Grindomix GM200 by Retsch (Haan, Germany) for 20 s at 10 000 rpm. The powder was then homogenized in 10 fold (volume/mass) n-heptane using ULTRA-TURRAX T25 digital, equipped with a S25N 18G dispersing tool by IKA (Staufen, Germany) for 3 min at 24 000 rpm. The resulting suspension was centrifuged using a Centrifuge 5702 by Eppendorf (Hamburg, Germany) at 4400 rpm for 15 min. The supernatant was removed and the resulting powder was dried on air at room temperature. The extractions were performed by stirring 1 g of the defatted powder in 20 ml of 70% aq. MeOH at room temperature, overnight. The supernatant was collected after centrifuging for 15 min at 4400 rpm and was kept in a fridge at 4 °C for further analysis. This crude extract was diluted 10 times, using MilliQ water and was filtered through a cellulose filter. The extracts were further used for both HPLC– Ion trap MS analysis and HPLC–TOF MS analysis.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

HPLC–MSn HPLC separations were performed on an Agilent 1100 Series system (Karlsruhe, Germany) using a C18 column at room temperature. The binary solvent system consisted of A 0.05% formic acid in water and B acetonitrile. The gradient used was: 0–2 min 10% B, 2–29 min 50% B, 29– 35 min 80% B, 35–40min 100% B, and 40–65 min 100% B. The LC system was coupled to a quadrupole ion trap mass spectrometer (Bruker Daltonics, HCT Ultra) using an ESI ionization chamber. Tandem MS spectra were acquired in auto MS2 mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. MS operating conditions (negative ion mode) had been optimized to: a capillary temperature of 365 °C, a dry gas ﬂow rate of 10 l/min, and a nebulizer pressure of 10 psi. Data were acquired and processed using Data Analysis 4.0 software package (Bruker Daltonics).

HPLC–TOF MS High-resolution LC–MS was carried out using the same HPLC instrument coupled to a MicrOTOF Focus mass spectrometer (Bruker Daltonics, Bremen, Germany) ﬁtted with an ESI source, and internal calibration was achieved with a 0.1 M sodium formate solution injected through a six-port valve prior to each chromatographic run. Calibration was carried out using the enhanced quadratic mode.

RESULTS AND DISCUSSION:

HPLC-MSn The base peak chromatogram of the negative ion mode analysis of the Cameroon beans extract showed 32 resolved peaks Figure 1. 11 peaks (namely 7, 9, 10, 12–15, 20–22 and 26) of high intensity were assigned to the well-known, previously reported cocoa ﬂavonoids, which are also the major components of this extract (Wollgast & Anklam, 2000).

Compound 16 was assigned to clovamide, based on its molecular formula (C18H16NO7) and MS2 fragmentation pattern, namely the breakage along the α–β unsaturated bond, with loss of the vinyl- catecholic unit, generating the [M − H − 136]− fragment ion of m/z 222, previously reported (Arlorio et al., 2008).

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Int. 7 9 x10 13 1 1.00

20 0.75 14 24 0.50 2 10 22 26 5 -8 27 0.25 3 4 15 --19 30 31 11 28 29 32 0.00 5 10 15 20 25 Time [min] 30

Figure 3.1: Cameroon beans negative ion mode base peak chromatogram

Compound 28 was assigned to dideoxyclovamide (C18H16NO5), which was previously reported in cocoa beans (Sanbongi et al., 1998). However, no previous MS fragmentation data is available in literature so far. Upon fragmentation, the molecular ion ([M − H]-, m/z 326) loses a CO2 molecule, resulting in the base peak daughter ion of m/z 282. A secondary fragmentation pathway involves the loss of the vinyl-phenolic fragment, generating a daughter ion of m/z 206 [M − H − 120]-. The molecular formulae of the compounds corresponding to the remaining 19 peaks, which (to our best knowledge) are not yet reported in cocoa up to date, were assigned based on high resolution HPLC–ESI- TOF MS, accepting an error below 5 ppm. For 7 of the 19 compounds, chemical structures were individually assigned based on their MS2 fragmentation patterns. Table 1 combines the results of the HPLC–MS2 and HPLC–TOF- MS analyses in terms of assigned molecular formula, retention time, molecular ion m/z value, and identity and relative abundance of the MS2 daughter ions generated by the fragmentation of the molecular ion. Upon MS2 fragmentation of compounds 2, 4, 5, 6, 8, 11, 24 and 31, the base peak is generated by a neutral loss of mass 102 Da, which is reminiscent of a cross ring cleavage of a deoxyhexose

(C4H8O3) and therefore suggests rhamnose or fucose derivatives. Another fragmentation feature that they share is the loss of a CO2 molecule (neutral loss of 44 Da), which is an indication of the fact that they possess a carboxylic acid moiety in their structure. Compound 3, upon MS2 fragmentation, gives a base peak of m/z 289, corresponding to a neutral loss of 162 Da, which is a characteristic of ﬂavonoid O-hexosides (Sánchez-Rabaneda et al., 2003). The base peak in MS2 at m/z 289 as a precursor ion in MS3, upon further MS3 fragmentation gives the speciﬁc peaks of (epi)catechin fragmentation, namely those of m/z 245 and 205, as reported in literature (Callemien & Collin, 2008). Therefore the signal can be assigned to an (epi)catechin-O- hexoside. Due to the lack of standards, regio- and

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

stereochemistry remains open. Although the base peak chromatogram only shows one peak of m/z 451, the extracted ion chromatogram shows in total 6 peaks (Fig. 2). In order to avoid confusion with the numbers of the peaks in the base peak chromatogram, the peaks in the extracted ion chromatograms will be named by their m/z (no. of the peak). Therefore, the compound corresponding to the third peak in the base peak chromatogram is referred to as 451(3) in Figures 3.2 and 3.6. MS2 fragmentation is only available for 451(3), 451(4) and 451(5). 451(3) and 451(4) show the same fragmentation and therefore can be assigned as (epi)catechin-O-hexosides. MS2 fragmentation of 451(5) shows a base peak at m/z 331 corresponding to a neutral loss of 120Da, which is a characteristic of ﬂavan-3-ol-C-glycosides (Sánchez-Rabaneda et al., 2003). Therefore 451(5) can be assigned as (epi)catechin-C-glucoside. Regio- and stereochemistry remains open. The peaks observed in the extracted ion chromatogram at retention times bigger than 15.8 minute heterolytic ring ﬁssion fragmentation products of procyanidin dimers, trimers and tetramers eluting at the respective retention times and therefore were disregarded.

Int. 6 x10 2

1.00

0.75

0.50

0.25 1 0.00 10 20 30 40 50 Time [min] 60

Figure 3.2: Ion trap extracted ion chromatogram at m/z 451 (negative ion mode) of Cameroon beans

Compound 19, for which the molecular formula C15H14O9S was assigned, is a sulfated derivative of either catechin or epicatechin, which, upon fragmentation loses the neutral group SO3, generating in the MS2 chromatogram a base peak of m/z 289 which gives upon further MS3fragmentation, the (epi)catechin speciﬁc fragments of m/z 245 and 205

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

(Callemien & Collin, 2008). Due to lack of standards, regio- and stereochemistry remain open. It is interesting to note that ﬂavan-3-ol- sulfates have so far been reported as phase II metabolites (Donovan et al., 1999). One more isomer of lower intensity is observed in the extracted ion chromatogram of m/z 369, shown in Figure 3.3.

Int. 6 3 x10

1.00

0.75 5

0.50 4 1 0.25 2 6

0.00 2 4 6 8 10 12 14 16 18 Time [min] Figure 3.3: Ion trap extracted ion chromatogram at m/z 369 (negative ion mode) from Cameroon beans

Compound 23, showing an m/z value of 737, was assigned the molecular formula C36H34O17, which would correspond to an A- type procyanidin hexoside, as previously speculated in literature (Hammerstone et al., 1999). However, the MS2 fragmentation of the molecular ion does not follow the typical fragmentation pattern of neither flavonoid-O-hexosides, (namely loss of a glycosil unit of mass 162 Da) nor that of flavonoid-C-hexosides (namely loss of a neutral fragment of mass 120 Da) (Sánchez-Rabaneda et al., 2003), but follows the typical fragmentation pattern of proanthocyanidin dimers, namely heterolytic ring fission, retro Diels Alder (RDA) fragmentation and quinone methide fragmentation. (Callemien & Collin, 2008). The same fragmentation behavior as in the case of compound 23 (m/z 707, molecular formula

C35H32O16) is observed for compound 25, which, based on its assigned molecular formula, would correspond to an A-type proanthocyanidin pentoside, as well previously speculated in literature (Hammerstone et al., 1999). Another sulfated compound present in the spectrum, compound 27 is the one corresponding to the m/z 305, to which the C12H18O7S molecular formula was assigned. Upon fragmentation, it loses an SO3 molecule (neutral loss of 80 Da), generating a base peak of m/z 225 which, upon further MS3 fragmentation, generates fragments of m/z 207, corresponding to the loss of a water molecule (neutral loss of 18 Da); 181—corresponding to the loss of a carbon dioxide molecule

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

(neutral loss of 44 Da), indicating a carboxylic acid moiety in the parent ion. Considering the molecular formula and the fragmentation pattern, the proposed structure for this compound is that of a sulfated oxygenated derivative of jasmonic acid reported in the patent literature, namely 12-hydroxy jasmonic acid sulfate. Jasmonic acid and its various metabolites are regulating plant responses to abiotic and biotic stresses as well as plant growth and development. In cocoa bean fermentation, abiotic stress is induced by ethanol, acetic and lactic acid formation leading to death of the bean. Hence the presence of this derivative is suggested to be related to fermentation induced stress. Compounds 29 and 30 (m/z 605) are two isomers of a dimeric compound of formula

C31H25O13 containing catechin or epicatechin as an upper unit, and a methylated flavanone with the molecular formula C16H14O7 in the lower unit. The fragment of m/z 453 corresponds to a retro Diels–Alder type of fragmentation and the neutral loss of 152 Da confirms that there is no methyl unit or additional oxygen atoms in the C ring of the catechin moiety. Fragmentations of dimeric flavonoids were discussed in detail by Jaiswal, Jayasinghe, and Kuhnert (2012). The extracted ion chromatogram at m/z 605 presents 3 more peaks of lower intensity (Figure 3.4).

Int. 6 x10 4 1.5

1.0

0.5 3

0.0 5 10 15 20 25 30 35 Time [min]

Figure 3.4: Ion trap extracted ion chromatogram at m/z 605 (negative ion mode) of Cameroon beans

Fragmentation pattern of 605(1) is identical to that of 605(2) and 605(4). Due to the low intensity of the signals, no fragmentation is available for 605(3) and 605(5).

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

The base peak chromatogram presents only one peak, 30 corresponding to the m/z 575, which was assigned to an A-type proanthocyanidin (C30H24O12) based on its fragmentation pattern (Jaiswal et al., 2012). However, the extracted ion chromatogram for m/z 575 shows 9 more peaks of low intensity (Figure 3.5), for which no fragmentation data is available. Moreover, the base peak chromatogram shows no peak corresponding to A-type procyanidin trimers, where double ligation between the monomeric units is present. Extracted ion chromatograms at m/z 863 (C45H36O18) and m/z 861 (C45H34O18) show four and six peaks respectively (Figure 3.5).

Int. 6 10 x10 a. 1.5 1.0 5 7 2 6 1 3 4 8 9 5 1 x10 b. 3 4 2 2 3 1 0 4 2 x10 c. 3 5 6 6 4 4 1 2 0 5 10 15 20 25 30 35 Time [min] Figure 3.5: Ion trap extracted ion chromatograms at m/z: a.) 575, b.) 863 and c.) 861 of Cameroon beans

The tentative structures of the corresponding compounds are presented in Figure. 6. However, no MS2 data are available at this point, due to the low intensity of the signals and therefore targeted MSn analysis should be performed in order to conﬁrm these structures. For all other peaks, molecular formulae are provided in Table 1.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

CONCLUSION

The complexity of the fermented cocoa phenol fraction was shown to be greater than the previously reported. A series of novel compounds including sulfated flavanols, A- type dimeric and trimeric proanthocyanidins and flavanol–flavanoneadducts could be identified during the present study. HPLC–MSn proved to be an efficient method for crude structure elucidation of these species providing evidence for classes of compounds present. However, a total characterization of regio- and stereochemistry could not be achieved due to the lack of synthetic standards.

ACKNOWLEDGEMENTS

We would like to express our gratitude towards Anja Müller for her excellent support during the measurements and to Jerome Derrey for providing the samples for our study. Last but not least, we would like to thank Barry Callebaut for providing the ﬁnancial framework under which this study was performed.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Table 3.1 Negative ion mode analysis results of the cocoa beans extract from Cameroon Peak Assigned Ret. m/z MS2 no. Compound/ time value m/z value (% abundance) Molecular formula (min) [M-H]- 1 2.2 179 161 2 Quinic acid 4 191 173(36), 111(100) 3 C14H24O10 12.3 351 307(7), 289(31), 249(100) 4 C21H24O11 13.6 451 331(9), 289(100), 245(5) (Epi)atechin-3-O-glucoside 5 C19H21O10 14.5 409 365(20), 347(14), 307(100), 265(45), 247(23) 6 C19H19O10 14.8 407 363(17), 345(14), 305(100), 263(50), 245(84) 7 C19H21O10 15 409 365(18), 347(11), 307(100), 265(30), 247(18) 8 C15H14O6 (Catechin) 15.2 289 245(93), 205(40) 9 C19H21O10 15.4 409 365(16), 347(14), 307(100), 265(47), 247(29) 10 C45H38O18 15.9 865 847(18), 739(29), 713(39), 695(100), (Procyanidin B trimer) 577(81), 425(41), 407 (83) 11 C30H26O12 16 577 425(83), 407(100) (Procyanidin B dimer) 12 C19H21O10 16.1 409 365(21), 347(14), 307(100), 265(46), 247(27) 13 C30H26O12 16.6 577 545(100), 439(60), 425(62), 393(50) (Procyanidin B dimer) 14 C15H14O6 (Epicatechin) 16.9 289 245(93), 205(40) 15 C45H38O18 17.4 865 847(9), 739(41), 711(79), 695(100), (Procyanidin trimer) 577(77), 425(43), 407 (39) 16 C60H28O24 17.9 1153 983(88), 865(100), 739(58), 577(54), (Procyanidin tetramer) 575(81) 17 C45H38O18 18 865 847(15), 739(20), 713(65), 695(95), (Procyanidin trimer) 577(76), 425(26), 407 (100) 18 C18H16NO7 (Clovamide) 18.2 358 312(14), 246(10), 222(73) 19 C43H28O11 18.4 721 635(68), 577(93), 575(88), 407(73), 289(89) 20 C45H38O18 19 865 847(11), 739(29), 695(58), 577(100), (Procyanidin trimer) 425(28), 407 (58) 21 C11H21O9S 19.1 329 329(100), 241(35) 22 C15H14O9S 19.3 369 289(100), 245(22), 217(67), 205(3) (Catechin sulphonic acid) 23 C45H38O18 19.5 865 739(54), 713(39), 695(100), 577(57), (Procyanidin trimer) 425(29), 407 (95) 24 C30H26O12 19.7 577 559(8.7), 451(25), 425(100), 407(96), Procyanidin B dimer 289(25) 25 C21H20O12 20 463 301(100) Quercetin-3-O-glucoside 26 C36H34O17 20.1 737 611(79), 585(14), 539(11), 449(100) (Procyanidin A hexoside) 27 C17H29O10 20.5 393 349(8), 331(37), 291(100), 249(27) 28 C35H32O16 20.7 707 581(50), 539(11), 449(92), 287(9.7) (Procyanidin A pentoside) 29 C20H18O17 21.1 433 301(100) (Quercetin-3-O-arabinoside) 30 C12H18O7S 21.4 305 225(100) 31 C18H16NO5 (dideoxyclovamide) 22.5 326 282(100), 206(68)

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Table3.1 (continued)

32 C30H26O12 23.8 577 451(25), 425(100),407(96) Procyanidin B dimer 33 C31H25O13 23.9 605 453(13), 315(100), 289(42), 245, 205 Procyanidin B-double methylated in the upper unit 34 C31H25O13 24.4 605 453(9), 315(100), 289(44) Procyanidin B-double methylated in the upper unit 35 C30H24O12 24.7 575 449(77), 431(53), 287(51) Procyanidin A dimer 36 C19H33O10 26.7 421 359(23), 319(100), 277(37)

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Figure 3.6: Proposed structure for the species identified in the Cameroon beans phenolic extract (regio- and stereochemistry remains open), m/z values given; number in bracket refers to the nth peak in the extracted ion chromatogram of the respective m/z value.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

3.6 References for Chapter 3 Arlorio, M., Locatelli, M., Travaglia, F., Coïsson, J., Grosso, E. D., Minassi, A., et al. (2008). Roasting impact on the contents of clovamide (N-caffeoyl-L-DOPA) and the antioxidant activity of cocoa beans (Theobroma cacao L.). Food Chemistry, 106, 967–975.

Brunetto, M. d. R., Gutiérrez, L., Delgado, Y., Gallignani, M., Zambrano, A., Gómez, Á., et al. (2007). Determination of theobromine, theophylline and caffeine in cocoa samples by a high- performance liquid chromatographic method with on-line sample cleanup in a switching- column system. Food Chemistry, 100, 459–467.

Callemien, D., & Collin, S. (2008). Use of RP-HPLC–ESI(−)-MS/MS to differentiate various proanthocyanidin isomers in lager beer extracts. Journal of the American Society of Brewing Chemists, 66, 109–115.

Coe, S. D., & Coe, M.D. (2007). The true history of chocolate (2nd ed.). London: Thames & Hudson Ltd.

Cooper, K. A., Donovan, J. L., Waterhouse, A. L., & Williamson, G. (2008). Cocoa and health: A decade of research. British Journal of Nutrition, 99, 1–11.

Donovan, J. L., Bell, J. R., Kasim-Karakas, S., German, J. B., Walzem, R. L., Hansen, R. J., et al. (1999). Catechin is present as metabolites in human plasma after consumption of red wine. Journal of Nutrition, 129, 1662–1668.

Hammerstone, J. F., Lazarus, S. A., Mitchell, A. E., Rucker, R., & Schmitz, H. H. (1999). Iden- tiﬁcation of procyanidins in cocoa (Theobroma cacao) and chocolate using high- performance liquid chromatography/mass spectrometry. Journal of Agricultural and Food Chemistry, 47, 490–496.

Ignat, I., Volf, I., & Popa, V. I. (2011). A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chemistry, 126, 1821–1835. Jaiswal, R., Jayasinghe, L., & Kuhnert, N. (2012). Identiﬁcation and characterization of proanthocyanidins of 16 members of the rhododendron genus (Ericaceae) by tandem LC– MS. Journal of Mass Spectrometry, 47, 502–515.

Kuhnert, N., Dairpoosh, F., Yassin, G., Golon, A., & Jaiswal, R. (2013). What is under the hump? Mass spectrometry based analysis of complex mixtures in processed food— Lessons from the characterisation of black tea thearubigins, coffee melanoidines and caramel. Food & Function, 4, 1130–1147.

Natsume, M., Osakabe, N., Yamagishi, M., Takizawa, T., Nakamura, T., Miyatake, H., et al. (2000). Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal- phase and reversed-phase HPLC. Bioscience, Biotechnology, and Biochemistry, 64, 2581–2587.

Chapter 3. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Rimbach, G., Melchin, M., Moehring, J., & Wagner, A. (2009). Polyphenols from cocoa and vascular health—A critical review. International Journal of Molecular Sciences, 10, 4290– 4309.

Sanbongi, C., Osakabe, N., Natsume, M., Takizawa, T., Gomi, S., & Osawa, T. (1998). Antiox- idative polyphenols isolated from theobroma cacao. Journal of Agricultural and Food Chemistry, 46, 454–457.

Sánchez-Rabaneda, F., Jáuregui, O., Casals, I., Andrés-Lacueva, C., Izquierdo-Pulido, M., & Lamuela-Raventós, R. M. (2003). Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (theobroma cacao). Journal of Mass Spectrometry, 38, 35–42.

Wollgast, J., & Anklam, E. (2000). Review on polyphenols in theobroma cacao: Changes in composition during the manufacture of chocolate and methodology for identiﬁcation and quantiﬁcation. Food Research International, 33, 423–447.

Part II. Profiling and quantification of hydroxycinnamyol glucoses in dietary plants

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Rakesh Jaiswal, Marius Febi Matei, Viktorija Glembockyte, Maria Alexandra Patras, and Nikolai Kuhnert

ABSTRACT: A chromatographic method was developed to separate all 10 regio- and stereoisomers of caffeoylglucose. Following chromatographic separation on reversed phase, the fragmentation behavior of all 10 regio- and stereoisomers of caffeoylglucose has been investigated using LC−MSn. It is possible to discriminate between each of the isomers based on their characteristic fragment spectra and order of elution, including those for which commercial standards are not available. On the basis of the synthesis of authentic standards for 6-caffeoylglucose and 3- caffeoylglucose and nonselective further synthesis of suitable mixtures of isomers, it was possible to fully assign regiochemistry of all 10 isomeric compounds and stereochemistry of eight isomeric compounds. Their fragmentation pattern was rationalized based on assuming different hydrogen-bonding arrays of gas-phase ions opening distinct fragmentation pathways. An analysis of yerba matéextract showed all 10 regio- and stereoisomers of caffeoylglucose to be present in this dietary material, which could all be assigned to regioisomeric level and eight to stereoisomeric level.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

INTRODUCTION Hydroxycinnamates form a subclass of polyphenolic secondary metabolites, found ubiquitously in plants including most fruits and vegetables relevant to the human diet. Hydroxycinnamic acids can occur in their free form with caffeic, ferulic, sinapic and p-coumaric acid being the most widespread derivatives (Figure 1).1-4 Other minor cinnamic acid derivatives such as dimethoxycinnamic, trimethoxycinnamic and isoferulic acid have also been reported in plants.5- 7 Hydroxycinnamates may be conjugated to many other molecules, but conjugates to (-)-quinic acid (chlorogenic acids) are to our present knowledge the most widespread and well investigated compounds in the human diet.

For chlorogenic acids the average daily human intake figures vary depending on the author between 1 g/human/day and 2.5 g/human/day for a heavy coffee drinker. Quantitative data for all other derivatives of hydroxycinnamic acids must be considered as sketchy. However, with a dietary intake figure of around 2 g per day, combined with bioavailability data suggesting a high level of absorption, hydroxycinnamates must be considered as the most abundant, possibly the most relevant, secondary plant polyphenol metabolites for our diet.

Hydroxycinnamic acid conjugates of carbohydrates were frequently reported as secondary plant metabolites in particular as constituents in the human diet. Such compounds can be classified into three distinct structural types: C-glycosides, O-glycosides and O-esters (Figure 1). All three different types are constitutional isomers of one another and examples of O-glycosides and esters were reported in dietary plant material. According to our literature search no C-glycosides were reported in natural sources so far.

8 Caffeic acid-4-O-β-D-glucose was reported in kiwi (Actinidia deliciosa) fruit, Cyathea phalerata,9 Cyathea dregei10 and Moricandia arvensis.11 A series of non 4-substituted glycosides of various hydroxycinnamic acids were further reported in Dendrobium thyrsiflorum12 and investigated by tandem MS.

For hydroxycinnamic acids esterified to carbohydrates a total of ten regio- and stereoisomers exist (five regioisomers each existing as a pair of α- and β-anomers). Out of the ten caffeoylglucoses only a single structure, namely 1-O-β-caffeoylglucose, 2, was unambiguously characterized using NMR spectroscopy by Winterhalter and coworkers.13

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

groups have reported the presence of multiple isomers of hydroxycinnamoyl hexoses, observed by LC-MS in dietary plants, without any assignment of regio- and stereochemistry. Such reports on derivatives include representative mono caffeoylglucoses from maté leaves (Ilex paraguariensis),14 Balanophora japonica15 and Frunallia and Chrysanthemum,16 dicaffeoylglucoses from Picrorhiza scrophulariiflora,17 feruloyl glucose derivatives from barley18 and common beans19 and feruloyl arabinose derivatives from barley.20 Such cinnamoyl esters were isolated from the hydrolysates of the plant cell walls.21-23 Winter and Herrmann24 identified p-coumaroyl-, caffeoyl- and feruloyl-glucosides esterified to the C-1 hydroxyl group from tomatoes, bell peppers and eggplants. In corn and barley, cell walls were found to contain esterified p-coumaric and ferulic acids and also free 5-hydroxyferulic acid and sinapic acid.25 In cereals, the main phenolic acids are ferulic acid and p-coumaric acid, which are found in different parts of the grains such as the wheat bran, the maize bran and the rice endosperm.26 More established is the presence of hydroxycinnamate esters to carbohydrates in the cross linking of lignin in the cell wall of grasses, including wheat, rye or rice. Seminal work by Bunzel and Steinhart showed that ferulic acid moieties bound to the 5-position of arabinose in arabinoglycans readily dimerised through a photochemical mechanism leading to cyclobutane dimers27, 28 and, more importantly, they dimerized through radical coupling mechanisms29 with dehydrodiferulates as reaction products, displaying a surprisingly rich structural chemistry. Caffeoylglucoses were also proposed as biosynthetic intermediates in the syntheses of other caffeoyl derivatives, including chlorogenic acids. In our own experience, we have observed multiple isomers of caffeoylglucose over the last decade in at least half of all the plant extracts analyzed. Following from many reports on caffeoylglucoses and other hydroxycinnamate conjugates to sugars, addressing and solving the structural assignment problem in this class of compounds constitutes an urgent problem. Only if an unambiguous analytical tool for structure elucidation becomes available with authentic reference materials available, will we be able to fully assess the occurrence, dietary burden and biological effects of this widespread but unexplored class of secondary plant metabolites. Unambiguous structure elucidation of regioisomeric chlorogenic acids has become possible through the application of tandem MSn methods. This method has the advantage that chlorogenic acids do not need to be isolated but can be identified and their structure elucidated directly from analytical LC-tandem-MS runs, even if present as minor

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

components or as chromatographically close eluting compounds. For example, we observed that all four regioisomeric mono caffeoylquinic acids and later all six regioisomeric dicaffeoylquinic acids, showed dramatically different tandem mass spectra in the negative ion mode, using an ion trap mass spectrometer.30, 31 Due to the diagnostic differences in the tandem MS fragment spectra, a consistent and predictive structure diagnostic hierarchical key for chlorogenic acids structure elucidation has been established, which allows reliable determination of chlorogenic acids regiochemistry even for minor component chlorogenic acids from tandem MS data exclusively. The basis of these differences in fragment MS spectra was rationalized in terms of different hydrogen bonding arrays found in gas phase ions of regioisomeric chlorogenic acids. In this study we investigated methods for the chromatographic resolution and mass spectrometric characterization of all ten isomers of caffeoylglucose present in yerba maté leaves (ilex paraguayensis).

MATERIALS AND METHODS

All the chemicals were purchased from Sigma-Aldrich, Carl-Roth and Iris Biotech (Bremen, Germany). Green dried maté leaves (of Argentinian origin) and Kiwi fruit (of Italian origin) were purchased from a supermarket in Bremen, Germany.

Methanolic Extract of Yerba Maté. Yerba maté leaves (5 g) were ground to a fine powder and extracted with 70% aqueous methanol (100 mL) using ultra-sonication for 30 min. This extract was filtered through a Whatman no. 1 filter paper. The methanol and the water were removed in vacuo and the residue was stored at -20 oC until required, thawed at room temperature, dissolved in methanol (50 mg/10 mL), filtered through a membrane filter and then used for LC-MS. LC-MSn. The LC equipment (1100 Series, Agilent, Karlsruhe, Germany) comprised a binary pump, an auto sampler with a 100 μL loop and a DAD detector with a light-pipe flow cell, recording at 320 nm and scanning from 200 to 600 nm. This was interfaced with an ion-trap mass spectrometer fitted with an ESI source (HCT-Ultra, Bruker Daltonics, Bremen, Germany) operating in full scan, auto MSn mode to obtain fragment ion m/z values. As necessary, MS2,

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

MS3 and MS4 fragment-targeted experiments were performed to focus only on compounds producing a pseudomolecular ion at m/z 341. Tandem mass spectra were acquired in the Auto- MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. The MS operating conditions (negative ion mode) had been optimized using 3-caffeoylglucoses (5 and 6) and 6- caffeoylglucoses (9 and 10) with a capillary temperature of 365 oC, a drying gas flow rate of 10 L/min and a nebulizer pressure of 10 psi. High resolution LC-MS was carried out using the same HPLC equipped with a high resolution mass spectrometer (MicrOTOF Focus, Bruker Daltonics, Bremen, Germany) fitted with an ESI source and internal calibration was achieved with 10 mL of a 0.1 M sodium formate solution injected through a six port valve prior to each chromatographic run. Calibration was carried out using the enhanced quadratic mode and the mass error was below 5 ppm. HPLC. Separation was achieved on a 250 x 3 mm i.d. column containing 5 μm C18 amide, with a 5 mm x 3 mm inner-diameter guard column (Varian, Darmstadt, Germany). Solvent A was water/formic acid (1000:0.005 v/v) and solvent B was methanol. Solvents were delivered at a total flow rate of 500 μL/min. In the first HPLC method the gradient profile was from 10% B to 70% B linearly in 60 min followed by 10 min isocratic and a return to 10% B at 90 min and finally 10 min isocratic to re-equilibrate. In the second HPLC method 10% B was used isocratically for 80 min. NMR. 1H and 13C NMR spectra were recorded on a JEOL-ECX 400 spectrometer operating at 1 13 400 MHz for H NMR and 100 MHz for C NMR at room temp in CDCl3, acetone-d6 or methanol-d4 using a 5 mm probe. The chemical shifts (δ) are reported in ppm and were referenced to the residual solvent peak. The coupling constants (J) are quoted in Hz.

Synthesis. All synthetic procedures and spectroscopic data for intermediates, reference compounds, equilibration of anomers and synthesis of mixtures of isomers are given below, employing similar strategies to those published before.32, 33

Synthesis of 3-O-Caffeoylglucoses (5 and 6). To a solution of 1,2:5,6-di-O-isopropylidene-α- D-glucofuranose (1.00 g, 3.84 mmol) and 4-dimethylaminopyridine (200 mg, 1.63 mmol) in

CH2Cl2 (50 mL) were added triethylamine (10 mL, 71.70 mmol) and 3,4-di-O-allylcaffeoyl chloride (1.58 g, 5.70 mmol)32 at room temperature. The reaction mixture was refluxed for

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

24h, cooled to room temperature and acidified (pH ≈ 3) with 2 M HCl. The layers were separated and the aqueous phase was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 30-50%) to give 3-O-(3,4-di-O-allyl)caffeoyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (75%) (Figure 2).

δH (CDCl3): 7.61 (1H, d, J 16.0, CAr–CH), 7.11 (1H, dd, J 8.2, 1.9, CArH), 7.09 (1H, d, J 1.7,

CArH), 6.87 (1H, d, J 8.2, CArH), 6.24 (1H, d, J 15.5, CAr–CH CH), 6.06 (2H, m, CH2 CH), 5.90 (1H, d, J 3.6, H-3), 5.39 (2H, d, J 16.9, CHH CH), 5.37 (1H, d, J 2.3, H-1), 5.31 (2H, d, J 9.6, CHH CH), 4.64 (4H, m, CAr–OCH2), 4.56 (1H, d, J 3.7, H-2), 4.28 (2H, m, H-6a, H-

6b), 4.07 (2H, m, H-4, H-5), 1.53 (3H, s, OCH3), 1.41 (3H, s, OCH3), 1.30 (6H, s,

OCH3); δC (CDCl3): 165.8 (-COOC), 151.1 (CAr–OCH2), 148.6 (CAr–OCH2), 146.0 (CAr–CH),

133.0 (CH2 CH), 132.8 (CH2 CH), 127.2 (CAr–CH), 123.1 (CAr), 118.1 (CH2 CH), 114.7 (CH–

COO), 113.4 (CAr), 112.7 (CAr), 112.3 (H3C-O-C-O-CH3), 109.4 (H3C-O-C-O-CH3), 105.1 (C-

3), 83.5 (C-1), 79.8 (C-6), 75.9 (C-2), 72.7 (C-4), 70.0 (CAr–OCH2), 69.7 (CAr–OCH2), 67.2 (C-

5), 26.9 (OCH3), 26.8 (OCH3), 26.2 (OCH3), 25.3 (OCH3).

To a solution of 3-O-(3,4-di-O-allyl)caffeoyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (1.00 g, 2.05 mmol) and p-toluenesulfonic acid (50 mg) in methanol-water (9:1, 50 mL) was added 10% Pd/C (300 mg) at room temperature. The reaction mixture was refluxed for 48 h, cooled to room temperature, filtered and methanol was removed in vacuo. The aqueous reaction mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 50-95%) to give a mixture of 3-O-caffeoyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 3-O-caffeoyl- 1,2-O-isopropylidene-α-D-glucofuranose and 3-O-caffeoyl-5,6-O-isopropylidene-α-D- glucofuranose (Figure 4.2).

The resulting mixture of the esters (500 mg) was dissolved in a mixture of 20 mL of trifluoroacetic acid and water (8:2) at room temperature and stirred for 1 h. The solvents were removed in vacuo to obtain the resulting esters 3-O-caffeoylglucoses (5 and 6) in quantitative yield (Figure 4.2).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

δH (methanol-d3): 7.51 (1H, d, J 16.0, CAr–CH), 7.03 (1H, s, CAr-H), 6.92 (1H, d, J 8.2, CAr-H),

6.77 (1H, d, J 8.2, CAr-H), 6.32 (1H, J 16.0, CAr–CH=CH), 5.31 (1H, t, J 9.6, α-gluc H-3), 5.15 (1H, J 3.2, α-gluc H-3), 5.02 (1H, t, J 9.6, β-gluc H-3), 4.59 (1H, t, J 7.8, β-gluc H-1), 3.60-

3.90 (2H, m, gluc H-6a, gluc H-6b), 3.24-3.57 (3H, m, gluc H-2, gluc H-4, gluc H-5); δC

(methanol-d3): 168.1 (COOR), 148.1 (CAr), 145.6 (CAr-CH), 145.5 (CAr), 126.5 (CAr), 121.5

(CAr), 115.0 (CH2=CH), 114.1 (CH-COO), 114.0 (CH2=CH), 96.8 (β-gluc C-1), 92.6 (α-gluc C-1), 77.7 (β-gluc C-3), 76.5 (α-gluc C-3), 75.7 (β-gluc C-2), 75.5 (β-gluc C-5), 73.4 (α-gluc C-2), 71.5 (α-gluc C-5), 70.9 (α-gluc C-4), 68.6 (β-gluc C-4), 61.4 (β-gluc C-6), 61.1 (α-gluc C-6).

Synthesis of 6-O-Caffeoylglucoses (9 and 10). To a solution of 1,2-O-isopropylidene-α-D- glucofuranose (1.00 g, 4.54 mmol) and 4-dimethylaminopyridine (200 mg, 1.63 mmol) in

CH2Cl2 (50 mL) were added pyridine (5 mL, 62 mmol) and 3,4-di-O-allylcaffeoyl chloride (1.58 g, 5.70 mmol)32 at room temperature. The reaction mixture was stirred for 24 h at room temperature and acidified (pH ≈ 3) with 2 M HCl. The layers were separated and the aqueous phase was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 30-50%) to give 6-O- (3,4-di-O-allyl)caffeoyl-1,2-O-isopropylidene-α-D-glucofuranose (80%) (Figure 4.2).

δH (acetone-d6): 7.62 (1H, d, J 15.6, CAr–CH), 7.32 (1H, dd, J 1.8, CArH), 7.17 (1H, dd, J 8.7,

2.3 CArH), 6.99 (1H, d, J 8.2, CArH), 6.41 (1H, d, J 16.0, CAr–CH CH), 6.08 (2H, m, CH2 CH),

5.85 (1H, d, J 3.6, H-3), 5.43 (2H, m, CHH CH), 5.23 (2H, m, CHH CH), 4.65 (4H, m, CAr–

OCH2), 4.49 (1H, d, J 3.7, H-2), 4.31 (2H, m, H-6a, H-6b), 4.12 (2H, m, H-4, H-5), 1.39 (3H, s, OCH3), 1.24 (3H, s, OCH3); δC (acetone-d6): 166.6 (-COOC), 150.9 (CAr–OCH2), 148.9 (CAr–

OCH2), 144.5 (CAr–CH), 133.9 (CH2 CH), 133.5 (CH2 CH), 127.7 (CAr–CH), 122.9 (CAr),

116.9 (CH2 CH), 116.5 (CH–COO), 115.7 (CAr), 113.7 (CAr), 112.6 (H3C-O-C-O-CH3), 111.2

(H3C-O-C-O-CH3), 105.0 (C-3), 85.3 (C-6), 80.4 (C-1), 74.4 (C-2), 69.4 (C-4), 69.2 (CAr–

OCH2), 67.1 (CAr–OCH2), 66.8 (C-5), 26.4 (OCH3), 25.6 (OCH3).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

To a solution of 6-O-(3,4-di-O-allyl)caffeoyl-1,2-O-isopropylidene-α-D-glucofuranose (1.00 g, 2.23 mmol) and p-TsOH (50 mg) in methanol-water (9:1, 50 mL) was added 10% Pd/C (350 mg) at room temperature. The reaction mixture was heated at 70 oC for 48 h, cooled to room temperature, filtered and methanol was removed in vacuo. The aqueous reaction mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 50-95%) to give 6-O- caffeoyl-1,2-O-isopropylidene-α-D-glucofuranose (62%). The resulting ester 6-O-caffeoyl-1,2- O-isopropylidene-α-D-glucofuranose (400 mg, 1.10 mmol) was dissolved in a mixture of 20 mL of trifluoroacetic acid and water (8:2) at room temperature and stirred for 1 h. The solvents were removed in vacuo to obtain the resulting esters 6-O-caffeoylglucoses (9 and 10) in quantitative yield (Figure 4.2).

δH (methanol-d3): 7.54 (1H, d, J 16.0, CAr–CH), 7.02 (1H, d, J 2.2, CAr-H), 6.92 (1H, dd, J 8.2,

1.4, CAr-H), 6.75 (1H, d, J 8.2, CAr-H), 6.25 (1H, J 16.5, CAr–CH=CH), 5.10 (1H, d, J 3.7, gluc H-1), 4.45 (1H, m, gluc H-6a), 4.30 (1H, m, gluc H-6b), 4.05 (1H, m, gluc H-4), 3.28-3.37 (2H, m, gluc H-2, gluc H-5); δC (methanol-d3): 167.9 (COOC), 148.3 (CAr), 145.8 (CAr-CH), 145.5

(CAr), 126.4 (CAr), 123.6 (CAr), 121.6 (CAr), 115.2 (CH2=CH), 113.7 (CH-COO), 113.6

(CH2=CH), 96.8 (β-gluc C-1), 92.6 (α-gluc C-1), 76.7 (β-gluc C-3), 74.9 (β-gluc C-2), 74.1 (β- gluc C-5), 73.5 (α-gluc C-3), 72.2 (α-gluc C-2), 71.6 (α-gluc C-5), 70.4 (α-gluc C-4), 69.6 (β- gluc C-4), 63.6 (β-gluc C-6), 63.5 (α-gluc C-6).

Synthesis of Stereo- and Regioisomeric Mixture of Caffeoylglucoses (1-6). To a solution of 4,6-O-benzylidene-D-glucopyranose (268 mg, 1.00 mmol) and 4-dimethylaminopyridine (50 mg, 0.04 mmol) in CH2Cl2 (20 mL) were added pyridine (3 mL) and 3,4-di-O-acetylcaffeoyl chloride (387 mg, 1.38 mmol) at room temperature. The reaction mixture was stirred for 10 h and acidified with 1 M HCl (pH ≈ 3). The layers were separated and the aqueous phase was re- extracted with ethyl acetate (2 × 20 mL). The combined organic layers were dried over

Na2SO4 and filtered and the solvents were dried in vacuo. The resulting esters were dissolved in a mixture of 20 mL of trifluoroacetic acid and water (7:3) at room temperature and stirred for 30 min. The solvents were removed in vacuo and the resulting

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

yellowish product was analyzed by HPLC-MS and shown to contain a mixture of caffeoylglucoses 1-6 (Figure 4.3).

Synthesis of Stereo- and Regioisomeric Mixture of Caffeoylglucoses (1-10). To a solution of D-glucose (180 mg, 1.00 mmol) and 4-dimethylaminopyridine (50 mg, 0.04 mmol) in

CH2Cl2 (20 mL) were added pyridine (3 mL) and 3,4-di-O-acetylcaffeoyl chloride (387 mg, 1.38 mmol) at room temperature. The reaction mixture was stirred for 10 h and acidified with 1 M HCl (pH ≈ 3). The layers were separated and the aqueous phase was re-extracted with ethyl acetate (2 × 20 mL). The combined organic layers were dried over Na2SO4 and filtered and the solvents were dried in vacuo. The resulting esters were dissolved in a mixture of 20 mL of trifluoroacetic acid and water (7:3) at room temperature and stirred for 30 min. The solvents were removed in vacuo and the resulting yellowish product was analyzed by HPLC-MS and shown to contain a mixture of caffeoylglucoses 1-10 (Figure 4.4).

Synthesis of 3-O-(3,4-Dimethoxy)cinnamoylglucoses (13 and 14). To a solution of 1,2:5,6- di-O-isopropylidene-α-D-glucofuranose (1.00 g, 3.84 mmol) and 4-dimethylaminopyridine

(200 mg, 1.63 mmol) in CH2Cl2 (50 mL) were added triethylamine (10 mL, 71.70 mmol) and 3,4-dimethoxycinnamoyl chloride (1.29 g, 5.70 mmol) at room temperature. The reaction mixture was refluxed for 24 h, cooled to room temperature and acidified (pH ≈ 3) with 2 M

HCl. The layers were separated and the aqueous phase was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 30-40%) to give 3-O-(3,4-dimethoxy)cinnamoyl-1,2:5,6-di-O- isopropylidene-α-D-glucofuranose (80%).

δH (CDCl3): 7.63 (1H, d, J 16.0, CAr–CH), 7.09 (1H, dd, J 9.0, 1.8 CArH), 7.02 (1H, d, J 1.8,

CArH), 6.85 (1H, d, J 8.2, CArH), 6.28 (1H, d, J 16.0, CAr–CH CH), 5.90 (1H, d, J 3.6, H-3), 5.37 (1H, d, J 2.7, H-1), 4.55 (1H, d, J 3.7, H-2), 4.28 (2H, m, H-6a, H-6b), 4.07 (2H, m, H-4,

H-5), 3.90 (6H, s, CAr-OCH3), 1.52 (3H, s, OCH3), 1.40 (3H, s, OCH3), 1.29 (6H, s,

OCH3); δC (CDCl3): 165.8 (-COOC), 151.5 (CAr–OCH2), 149.3 (CAr–OCH2), 146.0 (CAr–CH),

127.1 (CAr–CH), 123.1 (CAr), 114.7 (CH–COO), 112.4 (CAr), 111.1 (CAr),

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

109.6 (H3C-O-C-O-CH3), 109.3 (H3C-O-C-O-CH3), 105.1 (C-3), 83.5 (C-1), 79.8 (C-6), 76.0

(C-2), 72.6 (C-4), 67.1 (C-5), 56.1 (CAr-OCH3), 56.0 (CAr-OCH3), 26.9 (OCH3), 26.8 (OCH3),

26.4 (OCH3), 25.6 (OCH3).

The resulting mixture of the esters (500 mg) was dissolved in a mixture of 20 mL of trifluoroacetic acid and water (8:2) at room temperature and stirred for 1 h. The solvents were removed in vacuo to obtain the resulting esters 3-O-(3,4-dimethoxy)cinnamoylglucoses (13 and 14) in quantitative yield.

δH (methanol-d3): 7.65 (1H, d, J 16.0, CAr–CH), 7.19 (1H, s, CAr-H), 7.15 (1H, d, J 8.2, CAr-H),

6.95 (1H, d, J 8.2, CAr-H), 6.45 (1H, J 16.0, CAr–CH=CH), 5.32 (1H, t, J 9.6, α-gluc H-3), 5.15 (1H, J 3.2, α-gluc H-1), 5.02 (1H, t, J 9.6, β-gluc H-3), 4.59 (1H, d, J 7.8, β-gluc H-1), 3.65- 3.90 (2H, m, gluc H-6a, gluc H-6b), 3.24-3.57 (3H, m, gluc H-2, gluc H-4, gluc H-5).

δC (methanol-d3): 167.8, 167.6 (COOR); 151.4, 151.3 (CAr); 145.0 (CAr-CH); 148.1, 144.9

(CAr); 127.6, 127.5 (CAr); 122.6, 122.5 (CAr); 115.6 (CH2=CH); 115.5 (CH-COO); 114.0

(CH2=CH); 111.29, 110.16 (CAr-OCH3); 96.9 (β-gluc C-1); 92.6 (α-gluc C-1); 77.8 (β-gluc C- 3); 76.5 (α-gluc C-3); 75.8 (β-gluc C-2); 75.5 (β-gluc C-5); 73.4 (α-gluc C-2); 71.6 (α-gluc C- 5); 70.9 (α-gluc C-4); 68.6 (β-gluc C-4); 61.3 (β-gluc C-6); 61.1 (α-gluc C-6).

Synthesis of 6-O-(3,4-Dimethoxy)cinnamoylglucoses (15 and 16). To a solution of 1,2-O- isopropylidene-α-D-glucofuranose (1.00 g, 4.54 mmol) and 4-dimethylaminopyridine (200 mg,

1.63 mmol) in CH2Cl2 (50 mL) were added pyridine (5 mL, 62 mmol) and 3,4- dimethoxycinnamoyl chloride (1.29 g, 5.7 mmol) at room temperature. The reaction mixture was stirred for 24 h at room temperature and acidified (pH ≈ 3) with 2 M HCl. The layers were separated and the aqueous phase was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate-petroleum ether, 30-40%) to give 6-O-(3,4-di-O-allyl)caffeoyl-1,2-O-isopropylidene-α-D-glucofuranose (75%).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

δH (acetone-d6): 7.68 (1H, d, J 16.0, CAr–CH), 7.32 (1H, dd, J 8.2, 1.8, CArH), 7.17 (1H, d, J 1.8,

CArH), 6.86 (1H, d, J 8.2, CArH), 6.33 (1H, d, J 16.0, CAr–CH CH), 5.97 (1H, d, J 3.6, H-3), 4.54 (1H, d, J 3.6, H-2), 4.31-4.40 (2H, m, H-6a, H-6b), 4.12 (2H, m, H-4, H-5), 3.90 (6H, s,

CAr-OCH3), 1.48 (3H, s, OCH3), 1.31 (3H, s, OCH3); δC (acetone-d6): 168.2 (-COOC), 151.6

(CAr–OCH2), 149.2 (CAr–OCH2), 146.3 (CAr–CH), 127.2 (CAr–CH), 122.9 (CAr), 114.9 (CH2

CH), 114.8 (CH–COO), 111.9 (CAr), 111.2 (CAr), 111.1 (H3C-O-C-O-CH3), 109.6 (H3C-O-C-

O-CH3), 105.2 (C-3), 85.2 (C-6), 79.6 (C-1), 75.6 (C-2), 69.8 (C-4), 66.5 (C-5), 55.8 (CAr–

OCH3), 55.9 (CAr–OCH3), 26.9 (OCH3), 26.2 (OCH3). The resulting esters 6-O-(3,4-dimethoxy)cinnamoyl-1,2-O-isopropylidene-α-D-glucofuranose (400 mg) was dissolved in a mixture of 20 mL of trifluoroacetic acid and water (8:2) at room temperature and stirred for 1 h. The solvents were removed in vacuo to obtain the resulting esters 6-O-(3,4-dimethoxy)cinnamoylglucoses (15 and 16) in quantitative yield.

δH (methanol-d3): 7.55, 7.54 (1H, d, J 16.0, CAr–CH); 7.33 (1H, s, CAr-H); 7.20 (1H, d, J 8.2,

CAr-H); 6.94 (1H, d, J 8.2, CAr-H); 6.55, 6.54 (1H, J 16.5, CAr–CH=CH); 4.88, 4.28 (1H, d, J 3.7, gluc H-1α, 7.6, gluc H-1β); 4.37, 4.38 (1H, m, gluc H-6a); 4.10, 4.15 (1H, m, gluc H-6b);

3.77 (3H, s, OCH3); 3.75 (3H, s, OCH3); 3.41, 3.35 (1H, m, gluc H-4); 3.1 (2H, m, gluc H-2, H-3), 2.9 (1H, t, J 8.2, gluc H-5).

δC (methanol-d3): 167.9 (COOC), 151.5 (CAr), 149.5 (CAr-CH), 145.5 (CAr), 127.4 (CAr), 123.6

(CAr), 115.9 (CH2=CH), 112.0 (CH-COO), 110.8 (CH2=CH), 97.4 (β-gluc C-1), 92.9 (α-gluc C-1), 76.9 (β-gluc C-3), 75.2 (β-gluc C-2), 74.1 (β-gluc C-5), 73.4 (α-gluc C-3), 72.7 (α-gluc C-2), 71.1 (α-gluc C-5), 70.6 (α-gluc C-4), 69.7 (β-gluc C-4), 64.5 (β-gluc C-6), 64.4 (α-gluc

C-6), 56.2 (OCH3), 56.1 (OCH3).

Synthesis of 2-O-(3,4-Dimethoxy)cinnamoylglucoses (17 and 18). To a solution of 4,6-O- benzylidene-D-glucopyranose (268 mg, 1.00 mmol) and 4-dimethylaminopyridine (50 mg, 0.04 mmol) in CH2Cl2 (20 mL) were added pyridine (3 mL) and 3,4-dimethoxycinnamoyl chloride (312 mg, 1.38 mmol) at room temperature. The reaction mixture was stirred for 24 h and acidified with 1 M HCl (pH ≈ 3). The layers were separated and the aqueous phase was re- extracted with ethyl acetate (2 × 20 mL). The combined organic layers were dried over

Na2SO4 and filtered and the solvents were dried in vacuo. The crude product was purified by

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

column chromatography on silica gel (ethyl acetate-petroleum ether, 30-40%) to give 2-O-(3,4- dimethoxy)cinnamoyl-4,6-O-benzylidene-D-glucopyranose (25%). 2-O-(3,4-dimethoxy)cinnamoyl-4,6-O-benzylidene-D-glucopyranose (100 mg) was dissolved in a mixture of 20 mL of trifluoroacetic acid and water (7:3) at room temperature and stirred for 30 min. The solvents were removed in vacuo and the resulting yellowish product was analyzed by HPLC-MS and shown to contain a mixture of 2-O-(3,4- dimethoxy)cinnamoylglucoses 17 and 18.

δH (methanol-d3): 7.68, 7.64 (1H, d, J 16.0, CAr–CH); 7.19 (1H, s, CAr-H); 7.15 (1H, m, J 8.2,

CAr-H); 6.95 (1H, d, J 8.2, CAr-H); 6.45, 6.42 (1H, J 16.0, CAr–CH=CH); 5.30 (1H, t, J 9.6, gluc

H-2); 4.73 (1H, J 3.2, α-gluc H-1); 4.65 (1H, m, H-3); 3.84 (6H, CAr–OCH3); 3.70-3.80 (2H, m, gluc H-6a, gluc H-6b); 3.56 (1H, m, gluc H-4); 3.30-3.40 (1H, m, gluc H-5).

δC (methanol-d3): 167.2, 166.8 (COOR); 151.6, 151.5 (CAr); 145.0 (CAr-CH); 145.3 (CAr), 145.1

(CAr); 127.6, 127.4 (CAr); 122.7, 122.6 (CAr); 115.3 (CH2=CH); 115.3 (CH-COO); 114.9

(CH2=CH); 111.4, 110.2 (CAr-OCH3); 95.4 (β-gluc C-1); 90.2 (α-gluc C-1); 77.1 (β-gluc C-3); 75.4 (α-gluc C-3); 75.0 (β-gluc C-2); 74.1 (β-gluc C-5); 71.7 (α-gluc C-2); 70.9 (α-gluc C-5); 70.7 (α-gluc C-4); 70.4 (β-gluc C-4); 61.4 (β-gluc C-6); 61.3 (α-gluc C-6).

Synthesis of Stereo- and Regioisomeric Mixture of 3,4-Dimethoxycinnamoylglucoses (13- 20). To a solution of 4,6-O-benzylidene-D-glucopyranose (268 mg, 1.00 mmol) and 4- dimethylaminopyridine (50 mg, 0.04 mmol) in CH2Cl2 (20 mL) were added pyridine (3 mL) and 3,4-dimethoxycinnamoyl chloride (312 mg, 1.38 mmol) at room temperature. The reaction mixture was stirred for 24 h and acidified with 1 M HCl (pH ≈ 3). The layers were separated and the aqueous phase was re-extracted with ethyl acetate (2 × 20 mL). The combined organic layers were dried over Na2SO4 and filtered and the solvents were dried in vacuo. The resulting esters were dissolved in a mixture of 20 mL of trifluoroacetic acid and water (7:3) at room temperature and stirred for 30 min. The solvents were removed in vacuo and the resulting yellowish product was analyzed by HPLC-MS and shown to contain a mixture of 3,4- dimethoxycinnamoylglucoses 13-20.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

RESULTS AND DISCUSSION The aqueous infusion of yerba maté was reported to be particularly rich in caffeoylglucoses6, 14 and therefore served as a model system for our investigations. In a typical extracted ion chromatogram (EIC) of an aqueous methanolic yerba maté extract, using a reversed phase LC-

MS method we observed ten peaks with a m/z value of 341.0867 ± 0.005 (M-H, C15H17O9) and MS2 consistent with a caffeoyl ester of glucose. In order to assign the regio- and stereochemistry of the caffeoylglucoses we first required authentic reference substances, both of individual compounds and of a mixture of possibly all ten isomeric compounds, 1-10, and an LC-method capable of resolving all ten isomers. A mixture comprising of all ten isomeric caffeoylglucoses (we refer here exclusively to compounds with a caffeoyl ester linkage, a word on caffeoylglucose phenolic glycoside will follow at the end of the discussion) was obtained by reacting acetyl protected caffeoyl chloride with D-glucose, followed by trifluoroacetic acid mediated deprotection of the acetyl groups (Figure 4). For the analysis of the mixture we optimized two complementary chromatographic methods. In the first method baseline separation of seven isomers could be achieved. The remaining three isomers co-eluted. In the second method we achieved resolution of all ten isomers, however, for four chromatographic peaks not to a baseline separation level. Therefore, the first method is suitable for the quantitation of seven of the ten isomeric compounds, whereas the second method is suitable for compound identification and structure assignment. Typical extracted ion chromatograms are shown in Figure 4.5.

Following the synthesis of a mixture of all ten isomeric caffeoylglucoses we embarked on a series of selective syntheses of caffeoylglucose regioisomers and dimethoxycinnamoylglucose isomers. In the first case we employed diallylcaffeoyl chloride as the caffeic acid building block, while in the latter case we employed dimethoxycinnamoyl chloride. Using a selection of appropriately protected glucose derivatives we could obtain 6-caffeoylglucoses (9 and 10),32, 33 6-dimethoxycinnamoylglucoses (15 and 16), 3-caffeoylglucoses (5 and 6), 3- dimethoxycinnamoylglucoses (13 and 14) and 2-dimethoxycinnamoylglucoses (17 and 18), each as mixtures of α/β anomers (Figures 2 and 3). All the compounds were characterized by NMR spectroscopy with 2-D NMR experiments clearly confirming the position of the

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

caffeoyl and dimethoxycinnamoyl ester. Α β-stereochemistry was assigned based on the 3 characteristic JHCCH coupling constant between the anomeric hydrogen at the hydrogen at C-2 of glucose, with α-caffeoylglucoses showing values between 3 and 4 Hz and β-caffeoylglucoses 33 values between 7 and 8 Hz. On standing in methanol-d3 the mixtures of both 3- caffeoylglucoses and 6-caffeoylglucoses equilibrated, to yield after one day exclusively the β- anomers. We explain this observation by dipole moment considerations. The β-anomer of glucose is reported to have a larger dipole moment if compared to the α-isomer and is thus favored in polar solvents, overriding the anomeric effect. Despite all attempts we failed to obtain 1-caffeoylglucoses and 4-caffeoylglucoses in purified form. Access to the pure β-anomers of 3-caffeoylglucose and 6-caffeoylglucose allowed us to demonstrate that the α-anomer with smaller dipole moment eluted later from a reversed phase packing than the β-anomer with larger dipole moment. It seems reasonable to expect this effect to apply also to the other regioisomers unless there is a tendency for the β-anomer to form an internal hydrogen bond.

It was similarly possible to synthesise a mixture of all ten dimethoxycinnamoylglucose esters. Fortunately, it was possible to synthesis specifically 2-dimethoxycinnamoylglucose, 3, 4-, and 5-dimethoxycinnamoylglucose, (as pairs of anomers) and this allowed the two 1- dimethoxycinnamoylglucose anomers to be identified by default. The order of elution of 3- caffeoylglucose and 6-caffeoylglucose corresponded to the order of elution of 3- dimethoxycinnamoylglucose and 6-dimethoxycinnamoylglucose and we have tentatively concluded that the elution order of the dimethoxycinnamoylglucose regioisomers can be used to predict the elution order of the caffeoylglucose regioisomers. In order to assign the 1-caffeoylglucoses (1 and 2), 1-dimethoxycinnamoylglucoses (19 and 20), 4-caffeoylglucoses (7 and 8) and 4-dimethoxycinnamoylglucoses (21 and 22) we obtained two further synthetic mixtures from 4,6-benzylidene protected glucose. On reaction with diallylcaffeoyl chloride followed by Pd-mediated deallylation a mixture of six isomers was obtained. We assumed that the mixture exclusively contained the anomeric pairs of 1- caffeoylglucose/1-dimethoxycinnamoylglucose, 2-caffeoylglucose/2- dimethoxycinnamoylglucose and 3-caffeoylglucose/3-dimethoxycinnamoylglucose. Both 2- dimethoxycinnamoylglucoses and 3-dimethoxycinnamoylglucoses were previously obtained

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

by selective synthesis and could thus be readily identified based on their retention times and unique tandem MS data. The remaining pair of isomers obtained in this unselective synthesis must therefore be the two anomers of 1-dimethoxycinnamoylglucose. In the initial chromatogram showing all ten isomeric compounds, eight have now been identified, from which follows automatically that the regiochemistry of the two remaining isomers must be assigned as the two anomers of 4-caffeoylglucose with the β-anomer eluting earlier than the α- anomer (Figure 4.5). Tandem Mass Spectrometry and Compound Assignment. In chlorogenic acids chemistry all four regioisomers of mono-caffeoylquinic acids can be readily identified based on their unique tandem mass spectra.31 The differences in observed fragment ions was rationalized by taking into account different hydrogen bonding motifs in the gas phase ions, activating groups for fragmentation depending on their relative stereochemistry.

In order to rationalize the fragmentation behavior of caffeoylglucoses we propose to transfer the same approach successfully employed in chlorogenic acids chemistry, assuming different hydrogen bonding motifs of gas phase anions, depending on compound stereo- and regiochemistry. In the following discussion we report on the fragmentation patterns of all ten compounds 1-10 grouped according to their similarity in fragmentation behavior (Figure 4.6 and Table 4.1). The discussion on fragmentation mechanisms always assumes that in the gas phase anions of caffeoylglucoses exist in their cyclic pyranose form. This is not self-evident since under basic conditions, such as employed in anion chromatography hexoses with a pKa value between 12-13 are deprotonated in basic media at the anomeric hydroxyl group followed by rapid ring opening to shift the chemical equilibrium towards the open chain aldehyde. The latter, however, would result in destruction of the anomeric stereogenic center and consequently in identical fragment spectra for both α/β anomers, which was not consistently observed here with MS2 spectra of both anomers sometimes showing significant and always showing subtle differences, nor in any other tandem MS studies on carbohydrate derivatives carried out and justifies the assumption made above.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Fragmentation of 6-caffeoylglucoses. The two anomers of 6-caffeoylglucose showed very 2 2 similar fragment spectra in MS . The base peak in MS could be observed at m/z 2 (C13H13O7,

D) corresponding to a neutral loss of C2H4O2 through a ring fission fragmentation (Figures 6, + 7 and Tables 1, 2). Further fragment ions at m/z 251 (M-H -caffeic acid, C12H11O6, B), m/z 221

(C11H9O5, C), both obtained through ring fission fragmentation, m/z 179 (C9H7O4, caffeic acid + anion A) and m/z 323 (M-H -H2O, C15H15O8, E) were observed with suggested structures of fragment ions shown in Figure 4.8 (Tables 4.1 and 4.2). Fragmentation of 2-caffeoylglucoses. The two anomers of 2-caffeoylglucose showed very similar fragment spectra in MS2. Anomers were assigned based on their elution order with the more polar β-2-caffeoylglucose eluting earlier. The MS2 base peak at m/z 203 was assigned as - fragment ion G [C11H7O4] with a quinone methide ketene acetal structure (Figure 4.8 and Tables 4.1, 4.2).

The formation of G could be rationalized by assuming an inversion of the 2-caffeoylglucose derivative to its inverted chair conformation. In this inverted chair conformation the caffeoyl moiety and the OH group at C-3 are in an anti-1,2 diaxial orientation, whereas the 3-OH and 6- OH are in a syn-1,3 diaxial position (Figure 8). Hence the 6-OH is able to activate the 3-OH via hydrogen bonding as a leaving group with the 2-caffeoyl substituent acting as an intramolecular nucleophile in a 1,2 acyl-migration step, reminiscent of the Königs-Knorr reaction. This sequence of events results in bicyclic quinone acetal intermediate F seen as a weak MS2 + - - fragment at m/z 323 [M-H -H2O] or [C15H15O8] (Figure 8). Intermediate F undergoes subsequently a ring fission fragmentation to yield fragment ion G at m/z 203. All intermediates and the proposed electron flow are shown in Figure 4.8.

Fragmentation of 3-caffeoylglucoses. The two anomers of 3-caffeoylglucose showed essentially identical fragment spectra in MS2. The base peak of both anomers was m/z 323 [M- + - + - - H -H2O] E/F accompanied by fragment ion signals at m/z 203 [M-H -C4H8O4] [C11H7O4] G and further minor fragments at m/z 233 H and 179 A (Figures 6, 9 and Tables 1, 2). We assigned the MS2 base peak as a quinone methide ketene acetal structure F.

There were significant differences between the anomers at MS3, with the fragment ion at m/z 219 exceeding 50% intensity in α-3-caffeoylglucose but less than 10% in β-3-caffeoylglucose,

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

indicating that its precursor ion at m/z 323 must have retained the anomeric structure in the gas phase and therefore fragmentation occurs from a cyclic structure, most likely the pyranose form. Fragmentation of 4-caffeoylglucoses. The two anomers of 4-caffeoylglucose showed very similar fragment spectra in MS2, however the difference was sufficiently large to allow discrimination through the intensity of the fragment ion at m/z 233, being over 75% for α-4- caffeoylglucose and below 75% for β-4-caffeoylglucose (Figure 6 and Table 1). The base peak for both anomers could be observed at m/z 203 and was assigned again to the quinone methide ketene acetal structure G, but in contrast to the fragment spectra of 2-caffeoylglucose and 3- caffeoylglucose a much larger number of fragments could be observed, at m/z 233 H, 135 I, 179 A, 251 B, 281 D and 323 E/F (Figure 4.10 and Tables 4.1, 4.2).

We assigned the fragments at m/z 233 H, 179 A and 323 E/F as previously. The fragments at m/z 251 B and 281 D were assigned as fragments resulting from a ring fission. It is interesting to note that 4-caffeoylglucoses showed these types of fragments, which could not be observed for 2-caffeoylglucoses and 3-caffeoylglucoses. It appears that the loss of water by a 1,2 acyl participation mechanism is less favored for the 4-caffeoylglucoses allowing ring fission fragmentation to compete with initial dehydration, in comparison to the 3-caffeoylglucoses and 2-caffeoylglucoses.

Multiple reaction monitoring MS3 experiments demonstrated that for 2-caffeoylglucose, 3- caffeoylglucose and 4-caffeoylglucose the MS2 ion of m/z 323 fragmented identically and this quinone methide ketene acetal F cannot be the precursor of the minor fragments that discriminate between the 3-caffeoylglucose and 4-caffeoylglucose anomers.

Fragmentation of 1-caffeoylglucoses. The two anomers of 1-caffeoylglucose showed significantly different fragment spectra in MS2 that could be rationalized mechanistically as set out in Figure 11. Our data showed that the earlier-eluting anomer gave a base peak at m/z 179 corresponding to a neutral loss of glucose and a fragment A corresponding to caffeic acid anion. The later-eluting anomer gave am MS2 base peak at m/z 203 corresponding to the ketene acetal J (Figure 4.11). Following our previous arguments the formation of ketene acetal fragment ion J is only possible for the β-1-caffeoylglucose, 2, stereochemistry. The loss

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

of the caffeic acid moiety should be facile in the α-1-caffeoylglucose, 1, due to the anomeric effect. The assignment of α- and β-anomers was relatively straightforward for all four cases (except for 1-caffeoylglucoses) where pure compounds were obtained by synthesis; isomerization furnished the more stable β-anomers in aqueous solvent which could also be identified based on their retention times. However, none of these arguments hold for 1-caffeoylglucose. β-1- caffeoylglucose was isolated by the group of Winterhalter13 and its structure established by 1H- NMR spectroscopy; however, no tandem MS data on this compound was reported by this group. Other groups have reported tandem MS data on 1-caffeoylglucoses but assignment appeared to be random and the two isomers obtained in the current study have been assigned previously in a non-consistent way. The unambigious assignment of the two anomers of 1-caffeoylglucose must therefore be left open at this point. Positive Ion Mode Tandem Mass Spectra. In the positive ion mode all caffeoylglucoses can + 2 be observed as their sodiated ion at m/z 347 [M+Na ] (C15H17O9Na). All MS spectra display + + fragment ions at m/z 203 (C6H12O6+Na ) and 185 (C9H6O3+Na ) corresponding to sodiated glucose and the sodiated caffeoyl ions, respectively. As representative examples, the MS2 spectra for 1-caffeoylglucoses are shown in Figure 4.12. All positive mode MS2 spectra of all further caffeoylglucose isomers are shown in the supplementary information. As for the negative ion mode spectra, all five different regiochemistries can be distinguished based on the different MS2 spectra. However, it appears that in the positive ion mode the fragmentation is more difficult for all caffeoylglucoses with the exception of 1-caffeoylglucoses, which yield in the fragment spectra a rather large number of fragment ions with low intensities. For this reason we refrain from a rigorous interpretation of the spectra at the current state.

Tandem Mass Spectra of O-Caffeoyl Glycosides (Caffeic Acid-3-O-β-Glucose 11 and Caffeic Acid-4-O-β-Glucose 12). In order to compare the data of the caffeoylglucose esters with that of isomeric O-caffeoyl-glycosides 11 and 12 we recorded tandem MS spectra from an aqueous methanolic extract of kiwi fruit, which was reported to contain those exclusively.8 The MS2 spectra in negative ion mode showed in contrast to the esters a base peak at m/z 179 [caffeic + + acid-H ] and only one secondary peak at m/z 135 [caffeic acid-CO2-H ]. O-caffeoyl glycosides do not show secondary peaks at m/z 323, 281, 251, 221, 203 and 161 if compared

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

to the esters. Compounds 11 and 12 produced identical MS spectra and it was not possible to distinguish between them. However, in this particular case we assigned the first eluting isomer as caffeic acid-3-O-β-glucose, 11, and the later eluting isomer as caffeic acid-4-O-β-glucose, 12. It is interesting to note that caffeic acid glycosides lose the glycone part in MS2 spectra (negative ion mode), which is similar to other known phenolic glycosides, flavonoid glycosides and chlorogenic acid glycosides. Hierarchical Key for Caffeoylglucose Isomers Assignment. From the data shown, we present a hierarchical key or work flow for the analysis and assignment of all isomers of caffeoylglucoses.

All compounds that show in an LC-MS analysis a parent ion in the negative ion mode at m/z 341.0867 [M-H+] and 365.084 in the positive ion mode [M+Na+] with an additional absorption at 320 nm in the UV spectrum should be considered as caffeoylglucose (CG) derivatives. For assignment of the regiochemistry in the negative mode the following key applies:

1. Parent ion at m/z 341 and base peak at m/z 281 in MS2. 6-CGs 2. Parent ion at m/z 341 and base peak at m/z 323 in MS2. 3-CG 2a MS2 fragment at m/z 232 >75% and MS3 fragment at m/z 219 <10% β-3-CG 2b MS2 fragment at m/z 233 <75% and MS3 fragment at m/z 219 >50% α-3-CG 3. Parent ion at m/z 341 and base peak at m/z 179 in MS2. α-1-CG 4. Parent ion at m/z 341 and base peak at m/z 203 in MS2 accompanied β-1-CG by fragment ion at m/z 161 (1-CG pending final sterochemical assignment). 5. Parent ion at m/z 341 and base peak at m/z 203 in MS2 with little 2-CGs further fragments. 6. Parent ion at m/z 341 and base peak at m/z 202.5 in MS2 accompanied 4-CGs by fragment ion at m/z 232.6. 7. The assignment of α and β stereochemistry is based on relative retention time with β-anomers eluting earlier on reversed phase if compared to α-anomers (exception 1-CGs).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Assignment of Caffeoylglucoses from Yerba Maté. A total of ten peaks were detected in the extracted ion chromatograms which produced a pseudomolecular ion at m/z 341.0867 ± 0.005. These isomeric compounds were assigned as the ten regio- and stereoisomeric caffeoylglucoses (1-10) after comparing their retention times, high resolution and tandem MS data with the authentic caffeoylglucoses standards as outlined above. The two anomers of 6-caffeoylglucose are dominate the extract. Similar to chlorogenic acid chemistry this dietary plant produces a mixture of all possible stereoisomers of a given class of natural products.

ACKNOWLEDGEMENTS

Helpful comments and discussions with Prof. M. Clifford and excellent technical support from Ms Anja Müller are acknowledged.

Chapter 4

Table 4.1. MS3 Data of Caffeoylglucoses (CGs) in Negative Ion Mode

. Hierarchical Key for the LC

No. CG MS1 MS2

Parent ion Base peak Secondary peak

m/z m/z int m/z int m/z int m/z int m/z int m/z int m/z int

1 α-1-CG 340.8 178.5 280.6 5 202.6 8 160.5 31 134.6 16 ------

−

2 β-1-CG 340.8 202.5 268.5 5 220.6 5 178.5 5 160.5 99 ------Caff

Identification All of Ten Regio 3 α-2-CG 340.8 202.5 322.7 6 ------eoylglucose

4 β-2-CG 340.8 202.5 322.7 8 ------

5 α-3-CG 340.8 322.7 - - 232.5 54 202.5 72 188.8 4 178.6 29 134.7 5 - -

6 β-3-CG 340.8 322.7 280.6 6 232.6 87 202.6 72 188.6 9 178.6 40 134.6 6 - -

7 α-4-CG 340.8 202.5 322.7 21 280.7 36 250.6 35 232.6 96 178.5 72 160.6 17 134.7 13

8 β-4-CG 340.7 202.5 322.8 14 280.6 25 250.6 26 232.6 63 178.5 56 160.6 7 134.6 15

and

9 α-6-CG 340.8 280.6 322.7 5 250.6 63 220.6 23 178.6 38 ------

Stereoisomers of

10 β-6-CG 340.8 280.6 322.6 7 250.7 60 220.6 24 178.6 38 ------

Chapter 4

No. CG MS1 MS3

. Parent ion Base peak Secondary peak Hierarchical Key for the LC

m/z m/z int m/z int m/z int m/z int

1 α-1-CG 340.8 134.6 ------

2 β-1-CG 340.8 174.6 ------

3 α-2-CG 340.8 174.6 ------

−

78 4 β-2-CG 340.8 174.6 ------MS

Caff

Identification All of Ten Regio

5 α-3-CG 340.8 232.6 260.6 9 242.6 7 218.6 56 188.6 13 eoylglucose

6 β-3-CG 340.8 232.6 260.6 8 242.6 6 218.6 7 188.6 13

7 α-4-CG 340.8 174.5 ------

8 β-4-CG 340.7 174.5 ------

9 α-6-CG 340.8 178.5 220.5 35 134.6 13 - - - -

10 β-6-CG 340.8 178.5 220.6 32 134.6 7 - - - -

and

Stereoisomers of

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Table 4.2. High resolution MS2 Data of Synthetic Caffeoylglucoses (CGs) in Negative Ion Mode

No. CG MS2 fragment ions

1 α-1-CG 281.0644 (C13H13O7), 203.0344 (C11H7O4), 179.0340 (C9H7O4),

161.0239 (C9H5O3), 135.0450 (C8H7O2)

2 β-1-CG 269.0650 (C12H13O7), 221.0448 (C11H9O5), 203.0345 (C11H7O4),

179.0345 (C9H7O4), 161.0239 (C9H5O3)

3 α-2-CG 323.0756 (C15H15O8), 233.0436 (C12H9O5), 203.0332 (C11H7O4),

179.0337 (C9H7O4), 135.0445 (C8H7O2)

4 β-2-CG 281.0654 (C13H13O7), 233.0436 (C12H9O5), 203.0338 (C11H7O4),

179.0336 (C9H7O4), 135.0444 (C8H7O2)

5 α-3-CG 281.0652 (C13H13O7), 233.0438 (C12H9O5), 203.0337 (C11H7O4),

179.0338 (C9H7O4), 135.0449 (C8H7O2)

6 β-3-CG 323.0759 (C15H15O8), 233.0437 (C12H9O5), 203.0331 (C11H7O4),

179.0339 (C9H7O4), 135.0448 (C8H7O2)

7 α-4-CG 323.0760 (C15H15O8), 281.0599 (C13H13O7), 251.0495 (C12H11O6),

233.0436 (C12H9O5), 203.0335 (C11H7O4), 179.0337 (C9H7O4),

161.0238 (C9H5O3), 135.0445 (C8H7O2)

8 β-4-CG 323.0764 (C15H15O8), 281.0597 (C13H13O7), 251.0496 (C12H11O6),

233.0435 (C12H9O5), 203.0332 (C11H7O4), 179.0335 (C9H7O4),

161.0239 (C9H5O3), 135.0442 (C8H7O2)

9 α-6-CG 323.0765 (C15H15O8), 281.0594 (C13H13O7), 251.0499 (C12H11O6),

221.0402 (C11H9O5), 179.0305 (C9H7O4)

10 β-6-CG 323.0760 (C15H15O8), 281.0590 (C13H13O7), 251.0493 (C12H11O6),

221.0399 (C11H9O5), 179.0303 (C9H7O4)

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

OH OH OH OH OH OH O O OH O O O HO HO OH HO HO O OH OH

OH O OH O OH O OH O OH O OH O OH OH Caffeic acid p-Coumaric Ferulic Sinapic acid 1-C-Glycoside 4-O-Glycoside acid acid

OH OH OH OH O O O HO OH HO HO O HO O HO OH HO OH OH OH OH O O OH O O HO 3-O-Glycoside 1-O-Caffeoyl--glucose 1-O-Caffeoyl--glucose -1-CG -1-CG 1 2

OH OH OH OH OH O O O O O O HO HO HO HO O HO HO OH O O OH HO O O OH OH OH OH O OH O OH O O

HO OH OH OH OH OH HO HO HO HO 2-O-Caffeoyl--glucose 3-O-Caffeoyl--glucose 4-O-Caffeoyl--glucose -2-CG -3-CG -4-CG 3 5 7 2-O-Caffeoyl--glucose 3-O-Caffeoyl--glucose -2-CG -3-CG 4 6

OH HO HO O O O O O HO HO HO OH OH O O O O HO HO HO HO OH OH OH HO OH OH 4-O-Caffeoyl--glucose 6-O-Caffeoyl--glucose 6-O-Caffeoyl--glucose -4-CG -6-CG -6-CG 8 9 10 Figure 4.1: Selected structures of typical hydroxycinnamic acids present in the human diet and hydroxycinnamic acid carbohydrate conjugates.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Figure 4.2: Synthesis pathway of 3-caffeoylglucoses (5 and 6) and 6-caffeoylglucoses (9 and 10).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

OH O OH OH HO O O HO O HO O HO O HO O OH OH O O O 1. DMAP, Pyridine, OH O OH + DCM, rt, 24 h O O O + + O OH HO 2. TFA 70%, 30 min O OH O Cl HO HO HO O H HO 1 HO 3 5

HO OH HO OH OH HO HO O HO OH O O + O + O HO O O H OH O O O

HO HO HO OH HO HO 2 4 6

Figure 4.3: Synthesis pathway of 1-caffeoylglucoses, 2-caffeoylglucoses and 3- caffeoylglucoses (1-6).

O O OH O O 1. DMAP, Pyridine, O DCM, rt, 24 h HO HO OH + 1-10 OH 2. TFA 70%, 30 min

O Cl

Figure 4.4: Synthesis pathway of regio- and stereoisomers of caffeoylglucose (1-10).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Intens. EIC 341.0 -All MS x107 3 1.5 4 5 1.0 2 6 9 0.5 1 7 10 8

0.0 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Time [min]

Intens. EIC 341.0 -All MS x107 4

2 2 3 1 1 6 7 10 5 8 9 0 10 20 30 40 50 60 Time [min]

Figure 4.5: Extracted ion chromatograms (EICs) of regio- and stereoisomers of caffeoylglucose (1-10) at m/z 341 obtained by two different chromatographic methods.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

[%] β-1-CG (2) -MS2 [%] α-1-CG (1) -MS2

125 125

160.5 202.5 178.5 100 100

75 75

50 50 160.5 25 25 134.7 132.6 220.5 268.5 202.6 280.7 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 350 m/z

[%] 2 [%] 2 α-2-CG (3) -MS α-3-CG (5) -MS

125 β-2-CG (4) 125 202.5 322.7 100 100 202.5 75 75 232.5 50 50 178.6 25 25 322.7 134.7 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 350 m/z

[%] [%] β-3-CG (6) -MS2 α-4-CG (7) -MS2

125 125 β-4-CG (8) 322.7 202.5 100 100 232.6 75 202.6 75 232.6 178.5 50 50 178.6 250.6 280.6 25 25 134.6 322.7 134.6 160.5 280.6 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 350 m/z

[%] α-6-CG (9) -MS2

125 β-6-CG (10) 280.6 100

75 250.6 50 178.5 25 220.6 322.7 0 100 150 200 250 300 350 m/z

Figure 4.6: MS2 spectra of caffeoylglucoses 1-10 at m/z 341 in negative ion mode.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Figure 4.7: Fragmentation mechanism and pathway of 6-caffeoylglucoses (9 and 10).

Figure 4.8: Fragmentation mechanism and pathway of 2-caffeoylglucoses (3 and 4).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Figure 4.9: Fragmentation mechanism and pathway of 3-caffeoylglucoses (5 and 6).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

Figure 4.10: Fragmentation mechanism and pathway of 4-caffeoylglucoses (7 and 8).

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

H O O OH OH chair O O O O O inversion -C4H10O5 HO O HO HO OH OH O O OH O O 2 O m/z 203 O G

OH OH OH O O HO O HO MS2 OH HO O O O m/z 179 1 A

Figure 4.11: Fragmentation mechanism and pathway of 1-caffeoylglucoses (1 and 2).

[%] β-1-CG +MS2 [%] α-1-CG +MS2

184.8 184.9 100 100

202.9 202.8 162.8 280.9 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 350 m/z

Figure 4.12. MS2 spectra of 1-caffeoylglucoses (1 and 2) at m/z 365 in positive ion mode.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

[%] 2 11 & 12 -MS 150

178.6 100

134.6

0 100 150 200 250 300 350 m/z

Figure 4.13: MS2 spectra of caffeic acid-3-O-β-glucose 11 and caffeic acid-4-O-β-glucose 12 at m/z 341 in negative ion mode.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

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2. Clifford, M.N.; Wu, W.G.; Kuhnert, N. The chlorogenic acids of Hemerocallis. Food Chem. 2006, 95, 574-578.

3. Maruta, Y.; Kawabata, J.; Niki, R. Antioxidative caffeoylquinic acid-derivatives in the roots of burdock (Arctium lappa L). J. Agric. Food Chem. 1995, 43, 2592-2595.

4. Greenaway, W.; Wollenweber, E.; Scaysbrook, T.; Whatley, F.R. Novel isoferulate esters identified by gas chromatography-mass spectrometry in bud exudate of Populus nigra. J. Chromatogr. A 1988, 448, 284-290.

5. Jaiswal, R.; Kuhnert, N. Hierarchical scheme for liquid chromatography/multi-stage spectrometric identification of 3,4,5-triacyl chlorogenic acids in green Robusta coffee beans. Rapid Commun. Mass Spectrom. 2010, 24, 2283-2294.

6. Jaiswal, R.; Sovdat, T.; Vivan, F.; Kuhnert, N. Profiling and characterization by LC-MSn of the chlorogenic acids and hydroxycinnamoylshikimate esters in maté (Ilex paraguariensis). J. Agric. Food Chem. 2010, 58, 5471-5484.

7. Jaiswal, R.; Patras, M.A.; Eravuchira, P.J.; Kuhnert, N. Profile and characterization of the chlorogenic acids in green Robusta coffee beans by LC-MSn: Identification of seven new classes of compounds. J. Agric. Food Chem. 2010, 58, 8722-8737.

8. Fiorentino, A.; D'Abrosca, B.; Pacifico, S.; Mastellone, C.; Scognamiglio, M.; Monaco, P. Identification and assessment of antioxidant capacity of phytochemicals from kiwi fruits. J. Agric. Food Chem. 2009, 57, 4148-4155.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

9. Pizzolatti, M.G.; Brighente, I.M.C.; Bortoluzzi, A.J.; Schripsema, J.; Verdi, L.G. Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid from Cyathea phalerata. Phytochemistry 2007, 68, 1327-1330.

10. Bringmann, G.; Guenther, C.; Jumbam, D.N. Isolation of 4-O-beta-D- glucopyranosylcaffeic acid and gallic acid from Cyathea dregei Kunze (Cyatheaceae). Pharm. Pharmacol. Lett. 1999, 9, 41-43.

11. Braham, H.; Mighri, Z.; Ben Jannet, H.; Matthew, S.; Abreu, P.M. Antioxidant phenolic glycosides from Moricandia arvensis. J. Nat. Prod. 2005, 68, 517-522.

12. Yang, L.; Nakamura, N.; Hattori, M.; Wang, Z.; Bligh, S.W.A.; Xu, L. High-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry for the simultaneous analysis of cis-, trans- and dihydro-2-glucosyloxycinnamic acid derivatives from Dendrobium medicinal plants. Rapid Commun. Mass Spectrom. 2007, 21, 1833-1840.

13. Du, Q.; Xu, Y.; Li, L.; Zhao, Y.; Jerz, G.; Winterhalter, P. Antioxidant constituents in the fruits of Luffa cylindrica (L.) Roem. J. Agric. Food Chem. 2006, 54, 4186-4190.

14. Bastos, D.H.M.; Saldanha, L.A.; Catharino, R.R.; Sawaya, A.C.H.F.; Cunha, I.B.S.; Carvalho, P.O.; Eberlin, M.N. Phenolic antioxidants identifled by ESI-MS from yerba mate (Ilex paraguariensis) and green tea (Camelia sinensis) extracts. Molecules 2007, 12, 423-432.

15. Jiang, Z.H.; Hirose, Y.; Iwata, H.; Sakamoto, S.; Tanaka, T.; Kouno, I. Caffeoyl, coumaroyl, galloyl, and hexahydroxydiphenoyl glucoses from Balanophora japonica. Chem. Pharm. Bull. 2001, 49, 887-892.

16. Clifford, M. N.; Wu, W. G.; Kirkpatrick, J.; Kuhnert, N., Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn. J. Agric. Food Chem. 2007, 55, 929-936.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

17. Zhu, T.F.; Huang, K.Y.; Deng, X.M.; Zhang, Y.; Xiang, H.; Gao, H.Y.; Wang, D.C. Three new caffeoyl glycosides from the roots of Picrorhiza scrophulariiflora. Molecules 2008, 13, 729-735.

18. Norbaek, R.; Aaboer, D.B.F.; Bleeg, I.S.; Christensen, B.T.; Kondo, T.; Brandt, K. Flavone C-glycoside, phenolic acid, and nitrogen contents in leaves of barley subject to organic fertilization treatments. J. Agric. Food Chem. 2003, 51, 809-813.

19. Romani, A.; Vignolini, P.; Galardi, C.R.; Mulinacci, N.; Benedettelli, S.; Heimler, D. Germplasm characterization of Zolfino landraces (Phaseolus vulgaris L.) by flavonoid content. J. Agric. Food Chem. 2004, 52, 3838-3842.

20. Ahluwalia, B.; Fry, S.C. Barley endosperm cell walls contain a feruloylated arabinoxylan and a non-feruloylated β-glucan. J. Cereal Sci. 1986, 4, 287-295.

21. Chen, J.H.; Ho, C.T. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric. Food Chem. 1997, 45, 2374-2378.

22. Levigne, S.; Ralet, M.C.; Quemener, B.; Thibault, J.F. Isolation of diferulic bridges ester- linked to arabinan in sugar beet cell walls. Carbohydr. Res. 2004, 339, 2315-2319.

23. Renger, A.; Steinhart, H. Ferulic acid dehydrodimers as structural elements in cereal dietary fibre. Eur. Food Res. Technol. 2000, 211, 422-428.

24. Winter, M.; Herrmann, K. Esters and glucosides of hydroxycinnamic acids in vegetables. J. Agric. Food Chem. 1986, 34, 616-620.

25. Ohashi, H.; Yamamoto, E.; Lewis, N.G.; Towers, G.H.N. 5-Hydroxyferulic acid in Zea mays and Hordeum vulgare cell-walls. Phytochemistry 1987, 26, 1915-1916.

26. Mathew, S.; Abraham, T.E. Ferulic acid: An antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications. Crit. Rev. Biotechnol. 2004, 24, 59-83.

Chapter 4. Hierarchical Key for the LC−MSn Identification of All Ten Regio- and Stereoisomers of Caffeoylglucose

27. Allerdings, E.; Ralph, J.; Schatz, P.F.; Gniechwitz, D.; Steinhart, H.; Bunzel, M. Isolation and structural identification of diarabinosyl 8-O-4-dehydrodiferulate from maize bran insoluble fibre. Phytochemistry 2005, 66, 113-124.

28. Hartley, R.D.; Morrison, W.H.; Himmelsbach, D.S.; Borneman, W.S. Cross-linking of cell- wall phenolic arabinoxylans in Gramineous plants. Phytochemistry 1990, 29, 3705-3709.

29. Ralph, J.; Quideau, S.; Grabber, J.H.; Hatfield, R.D. Identification and synthesis of new ferulic acid dehydrodimers present in grass cell-walls. J. Chem. Soc. Perkin Trans. 1 1994, 3485-3498.

30. Clifford, M.N.; Knight, S.; Kuhnert, N. Discriminating between the six isomers of dicaffeoylquinic acid by LC-MSn. J. Agric. Food Chem. 2005, 53, 3821-3832.

31. Clifford, M.N.; Johnston, K.L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MSn identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900-2911.

32. Jaiswal, R.; Dickman, M.H.; Kuhnert, N. First diastereoselective synthesis of methyl caffeoyl- and feruloyl-muco-quinates. Org. Biomol. Chem. 2012, 10, 5266-5277.

33. Alakolanga, A.G.A.W.; Siriwardene, A.M.D.A.; Kumar, N.S.; Jayasinghe, L.; Jaiswal, R.; Kuhnert, N. LC-MSn identification and characterization of the phenolic compounds from the fruits of Flacourtia indica (Burm. F.) Merr. and Flacourtia inermis Roxb. Food Res. Int. 2014, 62, 388-396.

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

Maria A. Patras a, Rakesh Jaiswal a1 , Gordon J. McDougallb and Nikolai Kuhnerta* a Department of Life Sciences & Chemistry, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany 1 Present address: Doehler Group SE, Riedstrasse, 64295, Darmstadt, Germany b Environmental and Biochemical Sciences Group, James Hutton Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK *Correspondence to Nikolai Kuhnert, Department of Life Sciences & Chemistry, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany. Tel. 0049 421 200 3120 Email: [email protected]

Adapted with permission from J. Agric. Food Chem. 66, 5, 1096-1104. Copyright © (2018) American Chemical Society Online published version available at: https://pubs.acs.org/doi/abs/10.1021/acs.jafc.7b02446

Abstract: Based on a recently developed tandem MS based hierarchical scheme for the identification of regioisomeric caffeoyl glucoses, selected berry fruits were profiled for their caffeoyl glucose ester content. Fresh edible berries profiled included strawberries, raspberries, blueberries, blackberries, red and black currants, lingonberries, gooseberries, cranberries, juice of elderberries, goji berries, chokeberries, cranberries, açai berries, sea buckthorn berries, Montmorency sour cherries, and pomegranate were investigated. 1-caffeoyl glucose was found to be the predominant isomer in the majority of samples with further profiling revealing the presence of additional hydroxycinnamoyl glucose esters and O-glycosides with p-coumaroyl, feruloyl and sinapoyl substituents. A quantitative LC-MS based method was developed and validated and all caffeoyl glucose isomers were quantified for the first time in edible berries.

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

INTRODUCTION

The impact of the diet on human health has been highlighted by numerous studies in the past decades. Diets rich in fruits and vegetables have been found to be associated to a multitude of health benefits, a fact which has drawn the consumer attention towards the so-called “functional” foods or dietary supplements rich in nutraceuticals.54 Berries in particular are gaining increasing popularity as rich sources of nutraceuticals as new research investigating their health promoting properties is constantly accumulating.55-61 Berries are only seasonally present in the human diet in their fresh form; however, they are widely available all year round in their processed forms such as frozen or dried fruits, juices, purees, jams, etc.,62 and more frequently in recent years in the form of extracts to be used as dietary supplements.56 The most notable health benefits of berries are preventative effects on degenerative and cardiovascular diseases, cancer and ageing.63-65 These properties are mostly attributed to the high levels of phenolics in berries, which act through complex mechanisms like gene expression modulation and enzyme induction.57,58,66-69

The two major classes of phenolics reported in berries are flavonoids and phenolic acids. Flavonoids appear as three main subclasses; anthocyanins (e.g. cyandin, delphinidin and malvidin derivatives) flavonols (e.g. quercetin, myricetin and kaempferol derivatives) and flavanols (catechin and epicatechin derivatives). Phenolic acids are dominated by hydroxycinnamic acids (HCAs) (e.g. caffeic acid, ferulic acid, p-coumaric acid derivaties) being present either in their free form or connected to various polyols like quinic and shikimic acids and simple or complex carbohydrates.70-72 The most abundant and well investigated derivatives of hydroxycinnamic acids (HCAs) are the hydroxycinnamoyl quinates, referred to as chlorogenic acids (CGAs), which have shown a multitude of beneficial health effects.18,19 Although not as abundant as the CGAs, conjugates of HCAs with carbohydrates have also been frequently reported in various vegetables, fruits and in particular in berries, although compounds have never been identified to regioisomeric levels.73,74 Three structural subclasses of constitutional isomers are to be distinguished for each HCA-hexose combination, namely O- esters, C-glycosides and O-glycosides. Representative structures are shown in Figure 5.3. For each hexose O-ester of a particular HCA, five regioisomers are possible, each existing in equilibrium as a pair of α- and β- anomers. Multiple studies have reported the presence of various isomeric HCA-hexose conjugates, observed by LC−MS in a wide variety of plants.74-87 However, these are mostly reported without any assignment of regio- and stereochemistry and without any quantification, as isolation of individual isomers from complex food matrices

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits is a demanding process, frequently failing to provide pure compounds fit for structure elucidation. Recently, Jaiswal et al. introduced a hierarchical scheme using pure synthetic standards, which allowed for the individual identification of all ten caffeoyl glucose (CGs) isomers by a HPLC- tandem-MS based technique that yielded unique fragment spectra for all five regio-isomeric CGs.88 This approach allows unambiguous assignment of each individual isomer in any food source based on their retention time and distinct MS fragmentation patterns, omitting compound isolation. Although no biological data are as yet available for CGs, due to lack of authentic reference standards and difficulties in compound isolation and characterization, we assume that CG derivatives share many beneficial health properties of their CGA relatives. It can be expected that HCs share similar gut micro floral metabolic pathways being substrates to bacterial esterases and glycosidases producing free hydroxycinnamic acids that undergo further bacterial and liver metabolism producing identical bioactive metabolites if compared to CGAs.20, 21

This in depth contribution investigates the identity of HCA-hexose conjugates with a particular focus on caffeoyl glucoses (CGs) present in a series of berries including strawberries, raspberries, blueberries, blackberries, red currants, black currants, lingonberries and gooseberries, and provides both qualitative profiling data as well as quantitative data on this so far neglected class of dietary constituents. Additionally directly pressed juices of purple chokeberry, elderberry, cranberry, goji berry, sea buckthorn, açai berry, sour cherry and pomegranate were also qualitatively investigated. With their very frequent abundance in dietary plants (anecdotal note: we have observed HCA-hexose derivatives in more than 50% of 800 plant extracts investigated in our research group over the last decade) and potential health benefits in mind HCA-hexose form an important class of dietary secondary metabolites requiring scientific attention.

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

MATERIALS AND METHODS: Chemicals and reagents: LC-MS grade methanol was purchased from Carl-Roth (Karlsruhe, Germany). Formic acid and Hesperetin were purchased from Sigma-Aldrich (Steinheim, Germany).

Sample preparation: Extraction: Fresh plant material of various commercial origins was purchased from local markets in different countries (e.g. Germany, Italy & Romania). Some berries were also obtained from a local garden (Bremen, Germany), as mentioned in Table 5.2. 200g of each fresh plant sample was subjected to homogenization with a food blender, followed by immediate freezing and subsequent freeze-drying. The resulting freeze dried plant material was further used for extraction. Five strawberry samples from different cultivars (Adria, Anoi, Elsanta, Romina and Sveva cultivars) were received in freeze-dried form from the James Hutton Institute (Scotland). 0.5g of each freeze dried plant material was extracted with 10mL of methanol/water 70/30 (v/v) by 15 minutes of initial sonication followed by stirring at room temperature for 12 hours. After extraction, the suspensions were centrifuged for 10 minutes at 4400 rpm (3000 g). The supernatant was filtered through a CHROMAFIL® Xtra PTFE syringe filter with a pore size of 0.45μm (Macherey Nagel, Düren, Germany) and transferred into a glass test tube. The solid residue was washed twice with 10mL of methanol/water (70/30 v/v) and centrifuged after each washing step. The supernatants resulting from each washing step were as well filtered through the PTFE syringe filter and combined with the crude extract into the glass test tube. The solvent in the glass test tube was removed under N2 gas using a TurboVap concentration work station (BIOTAGE, Uppsala, Sweden). In order to ensure complete removal of water, the crude extracts were subsequently freeze dried for 12 hours. The mass of each extract was recorded.

SPE purification:

The crude dry extracts were subjected to SPE pre purification. Chromabond C18ec 15mL/2000 mg cartridges (Macherey Nagel, Düren, Germany) were used as stationary phase. Cartridge conditioning was performed by washing with 20mL of methanol followed by 20 mL of water. The crude extracts were dissolved in water in order to be loaded on the SPE cartridge, the amount of water depending on the mass of each extract, such that the total amount of extract

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits contained in 1mL (volume loaded on each cartridge) of water solution did not exceed 200mg. The washing was performed with 20mL of water followed by 20mL of 20% methanol/water (v %). The 20% methanol fraction was further used for HPLC-MS analysis. The SPE method was optimized using a methanolic extact of Ilex Paraguariensis (Maté) which was reported to contain all 10 isomers of CG. Sample concentration prior to the HPLC-MS analysis was performed by total evaporation of the solvent in the 20% methanol fraction, followed by dissolution of each individual sample into a specific amount 70% MeOH. Optimization of the sample concentration was performed such that the compounds of interest showed an intensity fitting into the linearity range of the calibration curve.

Sample preparation from directly pressed juices: Commercially available direct juices of berries were purchased from local stores in Bremen, Germany. Each sample was filtered through a CHROMAFIL® AQ Polyamide syringe filter with a pore size of 0.45μm (Macherey Nagel, Düren, Germany) and diluted 1:10 with 70% methanol/water (v/v). The resulting solutions were used directly for HPLC-ion trap and HPLC- micrOTOF analysis.

HPLC: Separation was achieved on a 250 × 3 mm i.d. C18 amide packing column with 5 μm particle size, with a 5 mm × 3 mm inner-diameter guard column (Varian, Darmstadt, Germany). Solvent A was water/formic acid (1000:0.005 v/v), and solvent B was methanol. Solvents were delivered at a total flow rate of 500μL/minute. The gradient profile used was: starting with 5% B, increasing to 10% B in 3.7 minutes, isocratic 10% until 10 minutes, increasing to 15% in 13,5 minutes, isocratic 15% B until 15 minutes, increasing to 32,2% B until 19 minutes, increasing to 35% B until 30 minutes, followed by increasing at 80% B until 40 minutes, followed by washing with 80% B until 55 minutes and decreasing to 5% B until 60 minutes followed by column re-equilibration at 5% B until 70 minutes.

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

HPLC −Ion Trap MSn: The LC equipment (1100 Series, Agilent, Karlsruhe, Germany) comprised a binary pump, an auto sampler with a 100 μL loop, and a DAD detector with a light-pipe flow cell, recording at 320 nm and scanning from 200 to 600 nm. For the profiling analysis, the LC system was interfaced with an ion-trap mass spectrometer fitted with an ESI source (HCT-Ultra, Bruker Daltonics, Bremen, Germany) operating in full scan, auto MSn mode for generating fragment ions. Tandem mass spectra were acquired in negative ion mode using the auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. The MS operating conditions (negative ion mode) were: capillary temperature of 365°C, drying gas flow rate of 10 L/minute, and a nebulizer pressure of 50 psi. When needed, targeted fragmentation experiments were performed, focusing only on compounds producing a [M-H]- ion of m/z 341.

LC-microTOF: High resolution MS data was acquired using the same the same HPLC equipment described previously coupled with a high-resolution mass spectrometer (MicroTOF Focus, Bruker Daltonics, Bremen, Germany) fitted with an ESI source, and internal calibration was achieved with a 0.1 M sodium formate solution injected through a six-port valve prior to each chromatographic run. Calibration was carried out using the enhanced quadratic mode, and the mass error for the generated molecular formulae was below 5 ppm.

Caffeoylglucose quantification method validation: An LC-MS method was developed for the quantitative analysis of the caffeoyl glucose isomers using 6-Caffeoylglucose synthetic standard prepared and characterized by Jaiswal et al. 88 (see Section 3.3 for detailed discussion). Nine point calibration curves were obtained from serial dilutions of synthetic standards using extracted ion chromatograms at high mass resolution (m/z 341.0878.+/- 0.002 Da). The lower limit of detection was defined as the concentration for which the signal/noise ratio was 5. The LOD was found to be 0.1µg/mL. The calibration curve was constructed from data points corresponding to concentrations in the range 0.5-40 µg/mL, with a Pearson correlation coefficient of 0.9978. The values for each data point were obtained as the average of 3 measurements, with relative standard deviation

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits values below 7% (Supporting information section). Multiple intra and inter day measurements were performed at the LOD. Relative standard deviations obtained for intra and inter day analytical replicates were below 6% (Supporting information). In order to calculate the reproducibility of the extraction and subsequent quantification procedure, triplicate analysis was performed on one representative of each sample type. The extraction as well as the quantification yielded relative standard deviation values below 10% (Supporting information). Hesperetin was used as internal standard for all the investigated samples and integration values normalized to the internal standard, to account for time dependent variations of the detector response.

RESULTS AND DISCUSSION: Profiling of Caffeoylglucoses and Caffeic acid-O-glycosides Methanolic crude extracts of selected dietary berries were obtained, purified by SPE and subjected to targeted LC-ESI-tandem-MS profiling of hexose conjugates of caffeic acid, among them the ester derivatives 1-10. The representative edible berries examined were strawberries (Fragaria ananassa), raspberries (Rubus idaeus), blueberries (Vaccinium corymbosum), blackberries (Rubus fruticosus), red currants (Ribes rubrum), black currants (Ribes nigrum), lingonberries (Vaccinium vitis-idaea) and gooseberries (Ribes uva-crispa). Additionally directly pressed juices of purple chokeberry (Aronia melanocarpa), elderberry (Sambucus melanocarpa), cranberry (Vaccinium oxycoccos), goji berry (Lycium chinense), sea buckthorn (Hippophae rhamnoides), açai berry (Euterpe oleracea), sour cherry (Prunus cerasus) and pomegranate (Punica granatum) were also investigated. The optimized LC-tandem MS method employed provided base line separation of all ten regioisomeric caffeoyl glucoses 1-10 using MS detection in the negative ion mode. In all the investigated samples, assignment of the individual stereoisomeric caffeoyl glucose (CG) structures was carried out on the basis of their high resolution MS data (m/z 341.0878.+/- 0.002 Da) indicating the molecular formula - - [C15H17O9] for the [M-H] ion and respective fragmentation patterns and relative elution times, previously described.88

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

1-O-Caffeoylglucose Our previous study88 found that the first eluting anomer of 1-CG (available as synthetic mixture of both α- and β- anomers) generated upon MS2 fragmentation, the deprotonated caffeic acid of m/z 179 as the base peak which upon further MS3 fragmentation generates the decarboxylated caffeic acid anion of m/z 135 as a single peak. This was speculated to be the α-anomer on the basis of the fragmentation mechanism arguments developed in the study, as the axial orientation of the anomeric hydroxyl group is in favor of the loss of the caffeoyl moiety, due to the anomeric effect. The later eluting anomer generated an MS2 base peak at m/z 203 and another high intensity peak at m/z 161. Based on the same fragmentation mechanisms, this was speculated to be the β-anomer, as only the equatorial orientation of the anomeric OH is in favor of the hexose chair inversion and subsequent sugar ring fission fragmentation leading to the formation of the ketene acetal of m/z 203. The mechanisms have been discussed in detail by Jaiswal et al.88 However, the unambiguous assignment of each anomer could not be made, as it was made for the other eight isomers, for which pure synthetic standard compounds were available.

In order to verify the speculation that the first eluting isomer was most likely the α-anomer, theoretical dipole moment calculations were performed for the two anomers using the Gaussian software. Theoretical dipole moment values retrieved by the software were 7.4 Debye and 8.1 Debye for 1-β-CG and 1-α-CG respectively. Based on these results, the more polar α-anomer is expected to elute first from a reversed phase chromatographic column, which confirms our previous speculation that the first eluting 1-CG can tentatively be assigned as 1-α-CG (2), which was found to be present in relatively higher amounts than other CG isomers in strawberry, raspberry, blueberry, red currant, black currant, gooseberry, lingonberry, aronia puree, cranberry juice, Montmorency sour cherry juice, goji berry juice, pomegranate juice, and absent from blackberry, acai berry juice, elderberry juice and sea buckthorn juice. The specific fragmentation pattern of the polar 1-β-CG (1) was not found in any of the investigated samples.

2-O-Caffeoylglucose Both anomers of 2-CG (3-4) were found in extracts of gooseberry, raspberry, aronia puree, Montmorency sour cherry and goji berry juice. The 2- α -CG was detected in black currant as a single anomer.

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

3-O-Caffeoylglucose Both anomers of 3-CG (5-6) were found in gooseberry in very low amounts. 3-α-CG was found in low amounts in elderberry juice, but was absent from all other samples.

4-O-Caffeoylglucose Both anomers of 4-O-Caffeoylglucose (7-8) were found in gooseberry in very low amounts but absent from all other samples.

6-O-Caffeoylglucose Both anomers of 6-O-Caffeoylglucose (9-10) were found in gooseberry, strawberry, aronia puree, Montmorency sour cherry juice, elderberry juice, Goji berry juice, and pomegranate juice. The 6-α-CG (10) was detected as single anomer in lingonberry and black currant.

Caffeic acid-3-O-glucose and caffeic acid-4-O-glucose Caffeic acid-4-O-β-D-glucose and caffeic acid-3-O-β-D-glucose were reported in kiwi fruits after isolation by preparative chromatography and NMR identification.81 Therefore, a methanolic extract of kiwi was used as a surrogate standard89 for the identification of these two glycosides in all the berry samples analyzed. The extracted ion chromatogram at m/z 341.0878.+/- 0.002 Da (m/z 341 recorded with the ion trap spectrometer), corresponding to the - - 2 molecular formula of [C15H17O9] for the [M-H] ion showed 5 peaks, all of which upon MS fragmentation generated the deprotonated caffeic acid of m/z 179 as the base peak. Further MS3 fragmentation of this peak generated the decarboxylated caffeic acid anion of m/z 135 as a single peak. In the absence of authentic standards, given that all isomers have identical MS2 fragmentation patterns, caffeic acid-4-O-β-D-glucose and caffeic acid-3-O-β-D-glucose were considered to correspond to the two most intense peaks (since they were reported exclusively after preparative HPLC) and the individual structures were tentatively assigned to each peak on the basis of their relative elution order from a reversed phase column. Dipole moment calculations were performed using the Gaussian software and the values retrieved were 5.45 Debye and 5.20 Debye for caffeic acid-3-O-β-D-glucose and caffeic acid-4-O-β-D-glucose respectively. Therefore, the first eluting peak was tentatively assigned as caffeic acid-3-O-β-D- glucose (11) and the second eluting isomer as caffeic acid-4-O-β-D-glucose (12) (Figure 5.3). The two regioisomers were found in many of the investigated samples, as summarized in Table 5.1.

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

Assignment of other compounds by tandem MS

All the investigated samples showed a higher complexity than previously reported of isomers - - - with [M-H] ions at 341.0878.+/-0.002 ([C15H17O9] ), m/z 325.0929.+/-0.002 ([C15H17O8] ), - - m/z 355.1035.+/-0.002 ([C16H19O9] ) and m/z 385.1140.+/-0.002 ([C17H21O10] ) corresponding to hexose conjugates of caffeic, p-coumaric, ferulic and sinapic acids respectively. The following section presents compound assignment within chosen samples with a subsequent discussion of fragment spectra allowing such assignment. (MSn data from the ion trap spectrometerfor all peaks with precursor ions at m/z 341, m/z 325, m/z 355 and m/z 385 for individual samples is presented in the Supporting information section).

Caffeic acid conjugates with hexoses other than glucose (C15H18O9)

The optimized HPLC method achieved good separation of a large number of isomers corresponding to the molecular formula C15H18O9 confirmed by high resolution m/z values of 341.0878.+/-0.002 for their [M-H]- ions. The largest isomeric complexity was found for gooseberry, with 20 peaks in the extracted ion chromatogram at m/z 341.0878.+/-0.002 (m/z values of 341 +/- 0.5 in ion trap spectra). Peaks with retention times below 10 minutes were not taken into account as they correspond to isomeric disaccharides such as sucrose (with molecular formula of C12H22O10 confirmed by high resolution m/z values of 341.1089.+/-0.002 for their [M-H]- ions and characteristic disaccharide fragmentation patterns.90

Six of the peaks presented a fragmentation pattern characteristic to caffeic acid O-glycosides, 2 - - generating in MS from the [M-H] ion at m/z 341 a single peak of m/z 179 ([C9H7O4] ) through the loss of the hexosyl unit of 162 Da. Further MS3 fragmentation of the peak at m/z 179 generated a single peak at m/z 135, through the loss of a carbon dioxide molecule, which is indicative of the caffeic acid moiety in the parent structure. Therefore, the peaks giving this specific fragmentation can be tentatively identified as O-glycosides of caffeic acid with various hexoses (e.g. mannose, galactose or others), with each combination generating in theory 4 possible isomers. The remaining peaks show a fragmentation pattern resembling that of the caffeoyl glucose esters (fragments of m/z 323, 281, 251, 233, 203 etc.). Therefore, these peaks can be tentatively identified as caffeoyl esters of a hexose different from glucose, with each combination giving in theory ten possible isomers.

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits p-coumaric acid conjugates with gluconic acid (C15H18O9) The gooseberry, red currant and black currant extracts show six isomeric compounds with the same molecular formula as the caffeic acid hexose conjugates, C15H18O9-confirmed by high resolution m/z values of 341.0878.+/-0.002 (m/z 341 recorded with the ion trap spectrometer) for their [M-H]- ions- but with different fragmentation behavior compared to both caffeoyl glucose esters and caffeic acid-O-glycosides. The first eluting isomer generated upon MS2 - - fragmentation of the [M-H] ion of m/z 341 a base peak of m/z 163 ([C9H7O3] ) by a neutral 3 loss of 178 Da (C6H10O6). MS fragmentation of the base peak at m/z 163 generated a single peak of m/z 119, by the loss of carbon dioxide molecule, which is indicative of a p-coumaric acid moiety in the parent structure. The remaining four isomers generated upon MS2 - fragmentation a base peak of m/z 195 ([C6H11O7] , by a neutral loss of 146 Da (p-coumaroyl - moiety C9H6O2) and another high intensity peak of m/z 163 ([C9H7O3] ) by a neutral loss of 178 3 Da (C6H10O6). Further MS fragmentation of the base peak of m/z 195 gave a base peak of m/z - - 159 ([C6H11O7-2H2O] ), another high intensity peak of m/z 129 ([C6H11O7-2H2O-CH2O] and - - smaller intensity peaks of m/z 177 ([C6H11O7-H2O] ), m/z 149 ([C6H11O7-H2O-CO] ), m/z 141 - - 4 ([C6H11O7-3H2O] ), and m/z 111 ([C6H11O7-3H2O- CH2O] ). MS fragmentation of the peak of - - m/z 159 ([C6H7O5] ), gave the base peak of m/z 129 ([C6H7O5-CH2O] ) and a low intensity peak 3 - of m/z 97 ([C6H7O5 -H2O-CO2] ). This fragmentation behavior which is characteristic to sugars,90 together with high resolution MS data which indicates the molecular formula of

C6H12O7, strongly suggests that this moiety is gluconic acid, which has been previously reported in its free form in various fruits including berries.91,92 Therefore, the compounds are tentatively assigned as p-coumaric acid conjugates of gluconic acid. Selected representative structures are presented in Figure 5.3, Structures 13-14. The regiochemistry and nature of the linkages remain unclear.

p-coumaric acid O-glycosides and p-coumaroyl glucoses (C15H18O8)

Isomeric p-coumaric acid-hexose conjugates with molecular formula of C15H18O8 (confirmed by the high resolution m/z values of 325.0929.+/-0.002 for their [M-H]- ions) have been found in most of the investigated samples (the number of isomers present in each sample is given in Table 5.1; high resolution extracted ion chromatograms at m/z 325.0929.+/-0.002 for individual samples are given in Supporting Information).

104

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits p-Coumaric acid-4-O-β-D-glucopyranose (13), synthesized and characterized by Galand et. al.,93 was used as a reference standard. The standard generated a high resolution m/z value of 325.0920. for its [M-H]- ion (theoretical m/z value of 325.0929, which confirms the molecular 2 formula of C15H18O8 (error of 2.7 ppm from the theoretical m/z value of 325.0929). MS - - fragmentation of the [M-H] ion of m/z 325, generated the base peak of m/z 163 ([C9H7O3] ) through the loss of the glycosyl unit of 162 Da, and a small intensity peak of m/z 119. Further MS3 fragmentation of the MS2 base peak (m/z 163) generates a single peak of m/z 119, through the loss of a carbon dioxide molecule, characteristic for p-coumaric acid moiety. This fragmentation behavior is in agreement to that of the caffeic acid-O-glycosides. Some of the investigated isomers presented this O-glycoside characteristic fragmentation pattern and some isomers presented distinct fragmentation patterns, identical to the fragmentation patterns of their caffeoyl glucose analogues. p-Coumaric acid-4-O-β-D-glucopyranose was successfully identified in black currant, strawberry, lingonberry, blueberry, elderberry juice, sea buckthorn berry juice and sour cherry juice, on the basis of high resolution MS data, fragmentation pattern and retention time.

Two C15H18O8 isomers-confirmed by the high resolution m/z values of 325.0929.+/-0.002 for their [M-H]- ions- found in red currant, black currant and strawberry samples, presented identical MS2 fragmentation behavior as the two anomers of 6-CG, namely sugar ring fission fragmentations generating neutral losses of 60Da, 90Da and 120 Da and loss of the glycosyl unit of 162 Da to generate the deprotonated acid. MS2 fragmentation of the [M-H]- ion of m/z - - 325 ([C15H17O8] ) generated the base peak at m/z 265 ([C15H17O8-C2H4O2] ) by a neutral loss of - 60 Da, another high intensity peak at m/z 235 ([C15H18O8-C3H6O3] ) by a neutral loss of 90 Da - and smaller intensity peaks at m/z 205 ([C15H17O8-C4H8O4] ) by a neutral loss of 120 Da and - 3 m/z 163 ([C15H17O8-C6H10O6] ) through the loss of the glycosyl unit of 162 Da. Further MS fragmentation patterns are identical to the MS3 fragmentation patterns (Figure 5.2) of the analogous caffeoyl species. Therefore, the two isomers were tentatively assigned as the two anomers of 6-O-p-coumaroyl-glucose (16-17).

Ferulic acid-O-glycosides (C16H20O9)

Ferulic acid-hexose conjugates with molecular formula C16H20O9 (m/z values of 355.1035.+/-0.002 for their [M-H]- ions) were found to be present in most of the investigated samples. The number of isomers present in each sample is given in Table 5.1. Ferulic acid-4- O-β-D-glucopyranoside (16) synthesized and characterized by Galland et al.,93 was used as a

105

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits reference standard. The standard generated a high resolution m/z value of 355.1028 for its [M-H]- ion (theoretical m/z value of 355.1035, which confirms the molecular formula of 2 C16H20O9 (error of 1.9 ppm from the theoretical m/z value of 355.1035). Ion trap MS fragmentation of the [M-H]- ion of m/z 355 generating a single peak of m/z 193 (deprotonated - 3 ferulic acid [C10H9O4] ) through the loss of the glycosyl unit of 162 Da. Further MS 2 fragmentation of the MS base peak (m/z 193) generated a base peak of m/z 149 ([C10H9O4- - CO2] ), through the loss of a carbon dioxide molecule, and another peak at m/z 177 ([C10H9O4- - 4 CH3] ) through the loss of a methyl group. Further MS fragmentation of the peak at m/z 149 - - ([C10H9O4-CO2] ) gave a single peak at m/z 134 ([C10H9O4-CO2-CH3] ) through the loss of a methyl group. All the MS2-4 spectra confirmed the presence of the ferulic acid moiety in the parent structures.94 Therefore, based on the high resolution MS data as well as fragmentation data and retention time ferulic acid-4-O-β-D-glucopyranoside (18) was successfully identified in Goji Berry.

Sinapic acid hexose conjugates (C17H22O10)

Sinapic acid-hexose conjugates with molecular formula C17H22O10 (m/z values of 385.1140.+/-0.002 for their [M-H]- ions) were found to be present in most of the investigated samples. The number of isomers present in each sample is given in Table 5.1. The black currant sample investigated showed a high intensity peak at m/z 385.1135 (m/z 385 recorded with the ion trap spectrometer). MS2 fragmentation of the [M-H]- ion generated the deprotonated acid of - m/z 223 ([C11H11O5] ), through the loss of the glycosyl unit of 162 Da, which is a fragmentation characteristic to O-glycosides, confirmed during our study by all the O-glycosides investigated. caffeic acid-, p-coumaric acid- and ferulic acid-O-glycosides. In the absence of reference standards, the identity of the hexose remains unclear. MS3 fragmentation of this ion generated - a base peak at m/z 208 ([C11H11O5-CH3] ) by the loss of the first methyl group and lower - intensity peaks at m/z 179 ([C11H11O5-CO2] ) by the loss of the carbon dioxide molecule and - m/z 164 ([C11H11O5-CO2-CH3] ) from the loss of the carbon dioxide and the second methyl 4 group. MS fragmentation of the peak at m/z 208 gave a base peak at m/z 164 ([C11H11O5-CH3- - - CO2] ) and lower intensity peaks at m/z 193 ([C11H11O5-2CH3] ) and - m/z 149 ([C11H11O5-2CH3-CO2] ).

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

Therefore, this isomer is preferentially losing the methyl group over the carboxylate group from - the carboxylate ion ([C11H11O5] ). The fragmentation of deprotonated sinapic acid however, previously reported for 3-sinapoylquinic acid, occurs preferentially through the loss of a carbon dioxide molecule, generating a base peak of m/z 179.95 The same difference in fragmentation behavior has been previously reported for ferulic and isoferulic acid, with ferulic acid preferentially losing a carbon dioxide molecule from the deprotonated ion and isoferulic acid preferentially losing the methyl group.94 Since the

C17H22O10 isomer from black currant shows the fragmentation pattern specific to isoferulic acid, it was tentatively assigned as an O-hexoside of 3,4-dimethoxy-5-hydroxy-cinnamic acid, with two different anomeric structures being possible. Representative structures are presented in Figure 5.3. A summary of the profiling results is given in Table 5.1.

Quantification of CGs

Prior to quantification, the caffeoyl glucose derivatives from the methanolic crude extracts were enriched using a C18 SPE cartridge using a water wash step and elution with 20% MeOH, as described in section 2.2.2. The more sensitive LC-MS technique was chosen for further quantification work, as the extracts showed a satisfactory signal in the UV chromatogram at 320 nm only for the major isomer 1-α-CG. From our previous work, synthetic reference standards were available for 6-CG, 3-CG and 1-CG. 6-CG was found to be by far the most stable derivative showing a small degree of epimerization and no acyl migration chemistry after dissolution in aqueous solution.88 All other derivatives were less stable, at least 10% of acyl migration products after 20 min in aqueous solution. For this reason we related all quantifications to a 6-CG calibration curve, assuming a response factor of unity for all isomers quantified here. This assumption is supported by comparison of the slope of different calibration curves obtained for all derivatives (three available as authentic reference standards) and by the observation that in isomeric mixtures obtained through acyl migration, the sum of all LC-MS peak areas remained constant (+/- 5 %) within acceptable boundaries.

Since all samples were thoroughly desiccated prior to extraction, the results are normalized to 100g of dry weight (DW). Quantitative data for caffeoyl glucoses are provided for the first time for all the berry samples investigated. CGs were quantified in nineteen samples (Table 5.2). For calculating the reproducibility of the quantification procedure, triplicate extraction was performed on one representative of each sample type (namely samples 7, 10, 12, 15, 18 and

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

19-Table 5.2) serving as technical replicates and each extract was analyzed per duplicate of injection, serving as analytical replicates. For all the other samples, single extracts were analyzed per duplicate of injection. Table 5.2 presents the average value resulting from the duplicate injections. Relative standard deviations were always below 5 %. 1-α-CG was the most abundant CG isomer in all the investigated samples. The highest concentration of CGs was found for the lingonberry sample (35 mg/100g DW), followed by gooseberry samples (13 mg/100 g DW). The lowest concentration was found for blueberry samples, with an average content of 1.5 mg/100 g DW. Five different cultivars of strawberry were investigated. No outstanding differences were observed between samples. The highest CG content was found for the cultivar Romina (4.33 mg/ 100g DW) and the lowest content was found for Elsanta (2.65 mg/ 100g DW). The Food and Agriculture Organization of the United Nations (FAO) reports a continuous increase in the world production of strawberry, raspberry and blueberry, over the last decade, with 8114373 tons of strawberry, 612571 tons of raspberry and 525620 tons of blueberry produced worldwide in 2014. Given the average contents of CGs found for these 3 main berry crops, we have estimated an annual world natural production of 25 tons of CGs from only these fruit sources.

Acknowledgements: IT support from Dr. Abhinandan Shrestha and technical support from Ms Anja Müller is greatly acknowledged. Gordon McDougal is grateful for support from the Scottish Government’s Rural and Environment Science and Analytical Services Division. We thank Prof O. Dangles for the provision of authentic reference samples.

Statement: This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Supporting information: The supporting information document contains high resolution MS data as well as fragmentation data for individual samples. It also contains statistical information regarding the quantification method.

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Table 5.1: Summary of the profiling results for all the investigated berry extracts (‘yes’ indicates the presence of the respective isomer, ND-not detected-indicates

Chapterquantification 5. Profiling and of caffeoyl regioisomeric in berry glucoses fruits the absence of the respective isomer)

No. of isomers No. of isomers No. of isomers No. of isomers 1-α-CG 2 - CG 3 -CG 4 -CG 6 -CG Caffeic acid Caffeic acid Sample m/z 341.0878. +/- m/z 325.0929. +/- m/z 355.1035+/- m/z 385.1140. +/- (2) (3-4) (5-6) (7-8) (9-10) 3-O-β-glucose 4-O-β-glucose 0.002 0.002 0.002 0.002

Strawberry yes ND ND ND yes yes yes 10 2 1 1 Raspberry yes yes ND ND ND yes yes 9 0 0 0 Blueberry yes ND ND ND ND yes ND 4 3 4 3 Blackberry ND ND ND ND ND ND ND 5 3 0 4 Red currant yes ND ND ND ND ND ND 11 3 0 2

109 LingonBerry yes yes ND ND yes yes ND 7 7 8 5

Gooseberry yes yes yes yes yes yes yes 20 5 2 3 Black currant yes yes ND ND yes yes ND 13 5 5 1 Aronia juice yes yes ND ND yes yes ND 9 4 2 2 Elderberry juice ND ND yes ND yes yes ND 13 3 4 5 Cranberry juice yes ND ND ND ND yes yes 8 5 4 4 Goji Berry juice yes yes ND ND yes yes yes 9 7 7 4 Açai Berry juice ND ND ND ND ND ND ND 0 1 3 2 Sea buckthorn ND ND ND ND ND ND ND 0 4 3 6 Sourjuice Cherry yes yes ND ND yes yes yes 12 9 3 2 Pomegranatejuice yes ND ND ND yes yes ND 5 5 5 2 juice

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

Table 5.2: Summary of the quantification results of individual isomers of CG in selected samples; results given in mg CG/100g of dry weight (DW). Analyses were performed in replicate. Mean values are stated.

2-β-CG 2-α-CG 1-α-CG 6-β-CG 6-α-CG Total No Sample [mg/100g DW] [mg/100g DW] [mg/100g DW] [mg/100g DW] [mg/100g DW] [mg/100g DW] (%STDEV) (%STDEV) (%STDEV) (%STDEV) (%STDEV) Strawberry 1 ND ND 2.13 0.39 0.42 2.94 +/- 0.14 (c.v. Adria) Strawberry 2 ND ND 2.31 0.40 0.42 3.13 +/-0.16 (c.v. Anoi) Strawberry 3 ND ND 3.40 0.46 0.47 4.33 +/-0.22 (c.v.Romina) Strawberry 4 ND ND 1.56 0.53 0.54 2.63 +/- 0.13 (c.v. Elsanta) Strawberry 5 ND ND 2.42 0.40 0.40 3.22 +/- 0.16 (c.v.Sveva) Strawberry 6 ND ND 2.46 0.72 0.72 3.89 +/-0.19 Italy Strawberry 1.44 0.38 0.39 7 ND ND 2.22 +/- 0.12 Romania (2.39%) (5.58%) (3.04%) Strawberry 8 ND ND 1.58 0.44 0.43 2.45 +/- 0. 22 Romania Strawberry 2.63 0.34 0.34 3.31 +/- 0.17 9 ND ND Germany Blueberry 1.43 10 ND ND ND ND 1.43 +/- 0.08 Germany (2.97%) Blueberry 1.68 11 ND ND ND ND 1.68 +/- 0. 09 Morocco Raspberry 4.50 12 ND ND ND ND 4.50 +/- 0.25 Germany (5.17%) Raspberry 4.29 13 ND ND ND ND 4.29 +/- 0.22 Romania Red currant 3.40 14 ND ND ND ND 3.40 +/- 0.17 Germany Red currant 5.20 15 ND ND ND ND 5.20 +/- 0.26 Germany (2.81%) Black 1.61 16 (garden) ND ND ND ND currant 1.61 +/- 0.08 Gooseberry 0.89 1.04 10.48 17 Germany 0.83 ND 13.23 +/ - 0.62 Germany(garden) Gooseberry 0.38 0.4 8.81 0.76 18 ND 10.34 +/-0.51 Germany (6.4%) (6.09%) (3.48%) (3.79%) Lingonberry 15.8 20.1 19 (garden) ND ND ND 35.9 +/- 0.95 (2.98%) (3.65%)

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Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

Figure 5.1: The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass spectrometer and MS2 spectra of labeled peaks from gooseberry extract. Numbering of peaks refers to structures shown in Figure 3.

111

Chapterquantification 5. Profiling and of caffeoyl regioisomeric in berry glucoses fruits

112

Figure 5.2: a. The extracted ion chromatogram at m/z 341 (negative ion mode) from an ion trap mass spectrometer and MS2-4 spectra of 6-β-CG from strawberry; b. The extracted ion chromatogram at m/z 325 (negative ion mode) from an ion trap mass spectrometer and MS2-4 spectra of the tentatively assigned 6-β-p-

coumaroylglucose from black currant; c. MS2 fragmentation pathways of the two analogous compounds. Numbering of peaks refers to structures shown in Figure 3.

Chapterquantification 5. Profiling and of caffeoyl regioisomeric in berry glucoses fruits

113

Figure 5.3: Individual structures of the compounds 1-20 found in the investigated berry extracts.

Chapter 5. Profiling and quantification of regioisomeric caffeoyl glucoses in berry fruits

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Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables

Maria A. Patras, Rakesh Jaiswal and Nikolai Kuhnerta*

Online published version available at: https://doi.org/10.1016/j.foodchem.2017.05.150

ABSTRACT: Based on the recently developed tandem MS based hierarchical scheme for the identification of regioisomeric caffeoyl glucoses, selected vegetables were profiled with respect to their caffeoyl glucose content. The dietary plants profiled were tomato, pepper, chili and aubergine, all members of the Solanaceae family. 6-O-caffeoyl glucose was found to be the predominant isomer. In processed food such as tomato puree and ketchup a larger number of caffeoylglucose isomers formed through acyl migration reactions were observed. A LC-MS based quantitative method was developed, validated and caffeoyl glucose regioisomers quantified for the first time in dietary plants with quantitative data obtained from representative 30 food samples.

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INTRODUCTION Hydroxycinnamic acids (HCAs) are a major subclass of phenolic plant secondary metabolites. They are biosynthetically derived from the amino acids phenylalanine and tyrosine with p- coumaroyl-Coenzyme A as the key biosynthetic intermediate RW.ERROR - Unable to find reference:29. Among the most abundant and well-studied hydroxycinnamic acids from plant sources are cinnamic acid, ortho-, meta- and para-coumaric acids, caffeic acid, ferulic acid and sinapic acid. HCAs occur in nature either in their free form or bound to various polyols. Major examples of polyols include hydroxy acids like tartaric, malic, quinic and shikimic acids, and carbohydrates or carbohydrate derivatives like glucose or glucuronic acid respectively. The most commonly encountered and well investigated derivatives of HCAs are by far their congujates with quinic acid, referred to as chlorogenic acids 18,19. Although not as abundant as their quinic acid analogues, conjugates of HCAs with carbohydrates have also been frequently reported in various dietary plants. Based on the connectivity between the HCA moiety and the carbohydrate moiety, these derivatives can be divided into three distinct structural subclasses of constitutional isomers, namely O-esters 1-10, C-glycosides 11 and O-glycosides 12. Representative structures are shown in Figure 6.1. O-glycosides of various hydroxycinnamic acids have also been investigated in several LC-MS studies. Caffeic acid-4-O-β-D-glucose and Caffeic acid-3-O-β-D-glucose were isolated from kiwi fruits by preparative HPLC and characterized by NMR by Fiorentino et al. 81. Tandem MS data of these two isomers- reported by Jaiswal et al. 88 are diagnostic to allow differentiation from Caffeoyl-glucose esters (CGs). For each ester combination between a hydroxycinnamic acid and a hexose sugar, a total of ten isomeric arrangements exist (5 regioisomers each existing in equilibrium as a pair of α and β anomers). Many studies have reported the presence of multiple isomers of hydroxycinnamoyl hexoses, observed by LC−MS in a wide variety of plants, without any assignment of regio- and stereochemistry and without any quantifications carried out 75,76,83,84,100,101. The Solanaceae family of plants includes species of major economical and dietary importance, like the tomato (Lycopersicon esculentum) - second most important fruit crop worldwide 77 and a key component of the mediterranean diet which is associated to a variety of health benefits 102 - the pepper (Capsicum anuum), the aubergine (Solanum melongena)- reported to be among the richest sources of chlorogenic acids investigated so far 18,19,103,104-and the potato (Solanum tuberosum).

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In 1986, Winter and Hermann reported caffeoyl glucose in tomato, bell pepper and aubergine samples, by HPLC-UV 82. The standard used for identification was isolated from black currant fruits and characterised by 1H-NMR as 1-O-Caffeyl-β-D-glucopyranose (1-β-CG) by Koeppen and Hermann 74. More recent studies involving HPLC-MS also report the presence of caffeic- ferulic- and p-coumaric acid-O-hexoside isomers in of the above mentioned plant materials. Vallerdu-Queralt et al. reported four isomeric caffeic acid-O-hexosides, as well as one ferulic acid hexoside and one coumaric acid hexoside in fresh tomato samples and tomato based products 77,78,101. No details regarding the type of bonding, regio- or stereochemistry were provided. Similarly quantitative data on this class of compounds are largely absent from the literature. Marin et al. reported the presence of a p-coumaroyl-glucopyranoside and other p- coumaric acid and caffeic acid derivatives in sweet pepper (Capsicum anuum L.) 83. Recently, Jaiswal et al. - using pure synthetic standards-developed a hierarchical scheme, which allows for the individual identification of all ten caffeoyl glucose isomers by HPLC-tandem- MS based on unique fragment spectra of all five regioisomeric caffeoyl glucose esters (CGs) 88. Therefore, it is now possible to assign individual structural identities of each of these isomers in any food source based on their retention time and distinct MS fragmentation patterns, without compound isolation. The present study investigates in depth the identity of HCA-hexose conjugates with a particular focus on caffeoyl glucoses (CGs) present in a series of tomato and tomato based products, bell pepper, chili pepper and aubergine samples by means of HPLC- ESI-tandem-MS, providing both qualitative profiling data as well as quantitative data on this so far neglected class of dietary constituents. It should be noted that due to previous difficulties in compound assignment no biological data are available; however, it can be assumed that CG derivatives might share many beneficial health properties of their chlorogenic acid relatives 13.

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Figure 6.1: Individual structures of all ten isomers of caffeoylglucose (CG) (1–10) and representative structures of C-glycosides 11, O-Glycosides 12 and 5-Caffeoylquinic acid 13.

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MATERIALS AND METHODS:

Chemicals and reagents: LC-MS grade methanol was purchased from Carl-Roth (Karlsruhe, Germany). Formic acid and Hesperetin were purchased from Sigma-Aldrich (Steinheim, Germany).

Sample preparation: Extraction: Fresh plant material of various origins was purchased from local markets in different countries (Germany, Spain, Italy, Romania, and Great Britain). 200g of each fresh plant sample was subjected to homogenization with a food blender, followed by immediate freezing and subsequent freeze-drying. The resulting freeze dried plant material was further used for extraction.

0.5g of each freeze dried plant material was extracted with 10mL of methanol/water 70/30 (v/v) by 15 minutes of initial sonication followed by stirring at room temperature for 12 hours. After extraction, the suspensions were centrifuged for 10 minutes at 3000 g (4400 rpm). The supernatant was filtered through a CHROMAFIL Xtra PTFE syringe filter with a pore size of 0.45μm (Macherey Nagel, Düren, Germany) and transferred into a glass test tube. The solid residue was washed twice with 10mL of methanol/water (70/30 v/v) and centrifuged after each washing step. The supernatants resulting from each washing step were as well filtered through the PTFE syringe filter and combined with the crude extract into the glass test tube. The solvent in the glass test tube was removed under N2 gas using a TurboVap concentration work station (BIOTAGE, Uppsala, Sweden). In order to ensure complete removal of water, the crude extracts were subsequently freeze dried for 12 hours. The mass of each extract was recorded.

SPE purification: The crude dry extracts were subjected to SPE pre purification. Chromabond C18ec 15mL/2000 mg cartridges (Macherey Nagel, Düren, Germany) were used as stationary phase. Cartridge conditioning was performed by washing with 20mL of methanol followed by 20mL of water. The crude extracts were dissolved in water in order to be loaded on the SPE cartridge, the amount of water depending on the mass of each extract, such that the total amount of extract contained in 1mL (volume loaded on each cartridge) of water solution did not exceed 200mg. The washing was performed with 20mL of water followed by 20mL of 20% methanol/water 123

Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables

(v%). The 20% methanol fraction was further used for HPLC-MS analysis. The SPE method was optimized using a methanolic extact of Ilex Paraguariensis (Maté) which was reported to contain all 10 isomers of CG. Sample concentration prior to the HPLC-MS analysis was performed by total evaporation of the solvent in the 20% methanol fraction, followed by dissolution of each individual sample into a specific amount 70%MeOH Optimization of the sample concentration was performed such that the compounds of interest showed an intensity fitting into the linearity range of the calibration curve.

HPLC: Separation was achieved on a 250 × 3 mm i.d. column with 5 μm C18 amide packing, with a 5 mm × 3 mm inner-diameter guard column (Varian, Darmstadt, Germany). Solvent A was water/formic acid (1000:0.005 v/v), and solvent B was methanol. Solvents were delivered at a total flow rate of 500μL/minute. The gradient profile used was: starting with 5% B, increasing to 10% B in 3.7 minutes, isocratic 10% until 10 minutes, increasing to 15% in 13,5 minutes, isocratic 15% B until 15 minutes, increasing to 32,2% B until 19 minutes, increasing to 35% B until 30 minutes, followed by increasing at 80% B until 40 minutes, followed by washing with 80% B until 55 minutes and decreasing to 5% B until 60 minutes followed by column re- equilibration at 5% B until 70 minutes:

LC−MSn: The LC equipment (1100 Series, Agilent, Karlsruhe, Germany) comprised a binary pump, an auto sampler with a 100 μL loop, and a DAD detector with a light-pipe flow cell, recording at 320 nm and scanning from 200 to 600 nm. For the profiling analysis, the LC system was interfaced with an ion-trap mass spectrometer fitted with an ESI source (HCT-Ultra, Bruker Daltonics, Bremen, Germany) operating in full scan, auto MSn mode for generating fragment ions. Tandem mass spectra were acquired in negative ion mode using the auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. The MS operating conditions (negative ion mode) were: capillary temperature of 365°C, drying gas flow rate of 10 L/minute, and a nebulizer pressure of 50 psi. When needed, targeted fragmentation experiments were performed, focusing only on compounds producing a pseudomolecular ion with an m/z value of 341. For the quantitative analysis of the caffeoyl glucose isomers, the same HPLC equipment was coupled with a high-resolution mass spectrometer (MicroTOF Focus, Bruker Daltonics,

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Bremen, Germany) fitted with an ESI source, and internal calibration was achieved with 10 mL of a 0.1 M sodium formate solution injected through a six-port valve prior to each chromatographic run. Calibration was carried out using the enhanced quadratic mode, and the mass error was below 5 ppm. The calibration curve for quantification of caffeoyl glucose isomers in the analyzed plant samples was obtained using 6-Caffeoylglucose synthetic standard, prepared and characterized by Jaiswal et al.88

RESULTS AND DISCUSSION:

Profiling Methanolic crude extract of selected dietary plants of the Solanaceae family were obtained and subjected to targeted LC-ESI-tandem-MS profiling of caffeic acids conjugates of hexoses, among them ester derivatives 1-10. As representative dietary plants Solanaceae providing the highest dietary burden were chosen including tomato (Solanum lycopersicum), pepper (Capsicum spp), chilli (Capsicum annuum,) and aubergine (Solanum melongena). As LC- tandem MS method our previously established method was employed, providing base line separation of all ten regioisomeric caffeoyl glucoses 1-10 using MS detection in the negative - ion mode. In all samples investigated, the extracted ion chromatograms at m/z 341 ([C15H17O9] ) show a relatively large number of peaks, varying between different samples from 8 to 23 peaks. Assignment of the CG isomers in individual samples was achieved on the basis of retention times and characteristic fragmentation patterns described recently (shown in Figure 6.3). (MS2 data for all peaks with a precursor ion at m/z 341 is presented in the Supporting information section). In the present study, peaks of m/z 341 with retention times below 10 minutes were disregarded as they correspond to isobaric disaccharides according to their measured high resolution m/z values of 341.1089 +/- 0.002 Da (corresponding to the - pseudomolecular ion with formula [C11H21O11] ) and fragment spectra characteristic for disaccharides. The following section presents compound assignment within chosen samples with a subsequent discussion of fragment spectra allowing such assignment. Fresh tomato samples: The extracted ion chromatogram at m/z 341 of fresh tomato samples, depicted in Figure 6.2a shows 16 peaks. 2-α-CG 4 and 6-α-CG 10 were successfully identified based on their retention times and characteristic fragmentation behaviors, shown individually in Figure 6.3.

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Homemade tomato juice: The extracted ion chromatogram at m/z 341 of homemade tomato juice, depicted in Figure 6.2b, shows 21 peaks. The same two esters found in fresh tomato samples, namely 2-α-CG 4 and 6- α-CG 10 were also present here. Commercial tomato puree: The extracted ion chromatogram at m/z 341 of commercially available tomato puree depicted in Figure 6.2c shows 18 peaks. Apart from the two CGs found in fresh tomato samples, 2-β- CG 3, 3-β-CG 5, 3-α-CG 6 and 6-β-CG 9 were also identified in tomato puree as minor components. This is an indication that isomerization through acyl migration occurs during the processing of tomato into tomato puree, a reaction which is highly favored by the elevated processing temperature as well as the acidic medium provided by the tomato matrix. Such acyl migration was reported in chlorogenic acid chemistry and its mechanism elucidated in detail 105. Furthermore acyl migration in caffeoyl glucoses was recently reported in the brewing process of maté infusions 106. Tomato ketchup: The extracted ion chromatogram at m/z 341 of tomato ketchup shows 13 peaks, out of which two correspond to the esters also identified in fresh tomato, namely 2-α-CG 4 and 6-α-CG 10 (Figure 6.2d). Tomato ketchup is industrially produced from tomato puree. The absence of isomers 3, 5, 6 and 9 could be explained by degradation of these compounds occurring due to the extended processing time at elevated temperatures. The fragmentation of 6-β-CG 9 gives a base peak ion of m/z 281, followed by lower intensity ions of m/z 251 and m/z 221. This fragmentation has not been encountered for the case of tomato, home-made tomato juice of ketchup samples. However, the ion of m/z 281 is present with high relative intensity in the MS2 chromatogram of the peaks corresponding to retention time of 30.2-30.5 min, present in al the 3 samples. This is an indication of the fact that 6-β-CG is also present there in low amounts, and it co-elutes with an isobaric species whose fragmentation gives a base peak of m/z 179. However, for the case of tomato puree and tomato juice, the co-eluting species is no longer present, probably due to degrading during thermal treatment. (Fragmentation presented in Tables S1-S3, Supporting information section).

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Figure 6.2: Extracted ion chromatograms (EIC) at m/z 341 for: a) Fresh tomato, b) Home-made tomato juice, c) Tomato puree, d) Tomato ketchup. (The low intensity peaks in the retention time area between 12 and 28 min (a–d) and 34–38 min (d) were enlarged for better visualization of individual chromatograms.).

Pepper: Methanolic extracts of bell pepper, banana pepper and chili pepper were all analyzed and their caffeoyl glucose profiles vary slightly depending on their type and origin (Figure 6.3). Organic bell pepper was found to contain both anomers of 2-CG 3 and 4 as well as both anomers of 6- CG (9 and 10), whereas non-organic bell pepper was found to contain only 2-α-CG 4. Banana pepper of Turkish origin (Charlie and Sivri cultivars) was found to contain 2-CG 3 and 4, 3-CG 5 and 6 and 6-CG 9 and 10. The same profile was also found in green chili of Turkish origin, whereas red chili of Dutch origin was found to contain only compounds 3, 4, 9 and 10. No CG was found in red chili of Indian origin. Results are summarized in Table 6.1. Also, the extracted ion chromatograms at m/z 341 vary significantly. MSn fragmentation tables are available in the Supporting information section.

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Figure 6.3: Extracted ion chromatograms (EIC) at m/z 341 for: a) Organic bell pepper, b) Charlie cultivar pepper, c) Turkish green Chilli, d) Dutch red Chilli, e) Indian red chilli. (The low intensity peaks in the retention time area between 12 and 40 min were enlarged for better visualization of individual chromatograms.)

Aubergine The extracted ion chromatogram at m/z 341 of aubergine shows 8 peaks, none of them showing a fragmentation pattern identical to any of the CG isomers.

5.3.1.7 Compound assignment by tandem MS In all the investigated samples, assignment of the individual stereoisomeric structures was carried out on the basis of their respective fragmentation patterns and relative elution times. MS2 fragmentation of the pseudo molecular ions at m/z 341 of 2-CGs 3 and 4 generated a base peak at m/z 203 and low intensity peaks at m/z 323. MS2 fragmentation of the pseudo molecular ions at m/z 341 of 3-CGs 5 and 6 generated a base peak at m/z 323, and other lower intensity peaks at m/z 233, 203 and 179. MS2 fragmentation of the pseudo molecular ions at m/z 341 of 6-CGs 9 and 10 generated a base peak at m/z 281, and other lower intensity peaks at m/z 251, 233, 221 and 179. MS2 spectra of all identified regio- and stereoisomers are depicted in Figure

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6.4. Fragmentation mechanisms of individual regioisomers as well as their relative retention times were explained in detail by Jaiswal et al.88 and are therefore not discussed further here.

Figure 6.4: MS2 spectra of caffeoylglucoses 3–6 and 9–10 from tomato puree (negative ion mode).

In all analyzed samples a large number of peaks of m/z 341, (which in most of the cases corresponded to the highest intensity peaks) generated, upon MS2 fragmentation of the pseudomolecular ion, the molecule loses the glycon part as a neutral loss (162 Da), therefore generating the base peak at m/z 179 and a second low intensity peak at m/z 135, indicative of the caffeoyl moiety. Upon MS3 fragmentation of the base peak ion at m/z 179, a single fragment of m/z 135 is observed, indicative of a carbon dioxide loss from caffeic acid. According to Jaiswal et al., this fragmentation pattern is encountered for the caffeic acid 3-O-glycoside and caffeic acid 4-O-glycoside reported in kiwi fruits 81,88. Therefore, the peaks giving this specific fragmentation can be tentatively identified as O-glycosides of caffeic acid with various hexoses (mannose, galactose or other), with each combination generating in theory 4 possible isomers. The remaining peaks show a fragmentation pattern resembling that of the caffeoylglucose esters (fragments of m/z 323, 281, 251, 233, 203 etc.). Therefore, these peaks can be tentatively identified as caffeoyl esters of a hexose different from glucose, with each combination giving in theory ten possible isomers. The MS2 fragmentation pattern generating the deprotonated caffeic acid of m/z 179 as the base peak was also encountered for the first eluting anomer of 1-CG, speculated to be the α-anomer on the basis of fragmentation mechanism arguments. The 1-β-CG was speculated to generate an MS2 base peak at m/z 203 and another high intensity peak at m/z 161, based on the same

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Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables fragmentation mechanism arguments 88. However, the unambiguous assignment of the two anomers of 1-CG could not be made, as it was made for the other eight isomers, for which pure synthetic standard compounds were available. In order to verify the speculation that the first eluting isomer is most likely the α-anomer, theoretical calculations of the dipole moments were performed using the Gaussian software. Theoretical dipole moment values retrieved by the software were 7.4 Debye and 8.1 Debye for 1-β-CG and 1-α-CG respectively. Therefore, based on these results, the more polar α-anomer is expected to elute first from a reversed phase chromatographic column, which is in agreement with the speculation of Jaiswal et al., made on the basis of the fragmentation mechanism.

β-1-Caffeoyl-O-glucose was reported in different plant sources, including tomato, bell pepper and aubergine 79,82. Its identification was performed by 1H-NMR spectroscopy. However, tandem MS data for this compound is- to our current knowledge- not available in literature. Based on the arguments presented above, 1-β-CG should show an MS2 base peak at m/z 203 and another high intensity peak at m/z 161. However, this fragmentation behavior was not encountered in any of the investigated samples, although 1-β-CG has been reported as a major component in all the presently investigated plant materials. Based on our observation this isomer was miss assigned in several reports in the literature with the major isomer of caffeoyl glucose being 6-O-caffeoyl glucose in all Solanaceae.

Quantification of CGs in Lycopersicon esculentum and derivatives and Capsicum annuum: Prior to quantification the caffeoyl glucose derivatives from the methanolic crude extracts were enriched using a C18 SPE cartridge using a water wash step and elution with 20 % MeOH. Since the resulting extracts showed only for the major isomer 6-CG a satisfactory signal in the UV chromatogram at 320 nm, the more sensitive LC-MS technique was chosen for further quantification work. Quantification was carried out using the previously optimized LC-MS method on a C-18 reverse phase packing with detection in the negative ion mode using a LC- ESI-TOF MS system. For determination of the calibration curve, synthetic 6-CG was employed.

It should be noted that from previous work we had synthetic reference standards available for 6-CG as pure single anomer, 3-CG and 1-CG as 1:11 mixture of anomers. 6-CG was found to be by far the most stable derivative showing a small degree of epimerization and no acyl migration chemistry once dissolved in aqueous solution. All other derivatives displayed a degree of instability yielding after 20 min in aqueous solution at least 10 % of acyl migration products. For this reason we decided to relate all quantifications to a 6-CG calibration curve,

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Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables assuming a response factor of unity for all isomers quantified here. This assumption is supported by comparison of the slope of different calibration curves obtained for all derivatives three available as authentic references and by the observation that in isomeric mixtures obtained through acyl migration, the sum of all LC-MS peak areas remained constant (+/- 5 %) within acceptable boundaries.

Eight point calibration curves were obtained from serial dilutions of synthetic standards using extracted ion chromatograms at high mass resolution (m/z 341.0878.+/- 0.002 Da). The lower limit of detection was defined as the concentration for which the signal/noise ratio was 5. In this case, we found the LOD to be 0.1µg/mL. The calibration curve was constructed from data points corresponding to concentrations in the range 0.5-25 µg/mL, for which the best Pearson correlation coefficient (0.9984) was obtained. The values for each data point were obtained as the average of 3 measurements, with relative standard deviation values below 10% (Supporting information section). Multiple intra and inter day measurements were performed at the concentration of 0.5 µg/mL, the lower limit of quantification. Relative standard deviations obtained for intra and inter day analytical replicates were higher if compared to CGA quantification, however at acceptable levels around 6% (Supporting information). The absence of a matrix effect was confirmed by spiking of tomato extracts, at the concentrations of 0.5μg/mL and 1μg/mL. The apparent recovery was determined to be 98%. In order to calculate the reproducibility of the extraction and subsequent quantification procedure, triplicate analysis was performed on one representative of each sample type (namely sample numbers 1, 8, 11, 15, 21, 24). The extraction procedure yielded relative standard deviation values below 12% (Supporting information). The best extraction reproducibility was obtained for organic bell pepper and the lowest for conventional bell pepper. The quantification of caffeoyl glucoses yielded relative standard deviation (RSD) values between 8-16%.

Since all samples were thoroughly desiccated prior to extraction the results were normalized to 100 g of dry plant material 1µg/mL Hesperetin was used as internal standard for all the investigated samples and integration values were normalized to the internal standard, to account for time dependent variations of the detector response. Results are summarized in Table 6.1.

All investigated tomato samples showed a much higher content of 6-α-CG compared to the content of 2-α-CG (more than 10 fold). The average content of 2-α-CG from all investigated samples was found to be 1.58 mg/100g of dry material and that of 6-α-CG was found to be 21.37mg/100g of dry material. As discussed in Section 3.1.4, 6-β-CG co-elutes with an isobaric

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Chapter 6. Profiling and quantification of regioisomeric caffeoyl glucoses in Solanaceae vegetables compound, and therefore, its quantification was not possible. The highest content of CGs was found in Kumato cultivar tomatoes of Spanish production (See sample 6, Table 6.1). The tomato juice and tomato puree samples investigated all showed more isomers and in considerably larger amounts compared to the fresh tomato samples. The hypothesis of the acyl migration which is favored by high cooking temperatures, leading to the formation of 3-CG 5 and 6 from 2-O-caffeoyl glucose 4 initially present in fresh tomato, cannot account for the larger contents. Responsible for the relatively larger contents of caffeoyl glucoses in tomato based products could be the hydrolysis of larger molecules (e.g dicaffeoyl glucoses, glucose containing oligosaccharides bound to caffeic acid through an ester bond) with release of the specific caffeoyl glucoses. Tomato ketchup samples investigated show lower amounts of CGs compared to fresh tomato and absence of isomers 5 and 6 (See section 3.1.4). Since tomato ketchup is industrially produced from tomato puree, it can be hypothesized that the products formed during the first steps of processing from fresh tomato to tomato puree get degraded during the subsequent steps of extended thermal treatment. Additionally, tomato ketchup also contains relatively large amounts of added sugar, which reduces the final concentration of CGs in the final product.

All the investigated pepper samples showed roughly five fold lower contents of CG, compared to the tomato samples. The highest amount of CG was found for the Chilli samples (Samples 15 and 16), followed by the banana type pepper (Sivri cultivar) of Turkish origin (Sample 24), and the organic bell pepper of Dutch origin (Samples 17 and 18). Conventional bell pepper samples investigated were all found to contain lower amounts of caffeoyl glucoses. All the results are presented in Table 6.1.

The total amount of CGs in tomato was determined to be 23mg/100g of dry plant material and in pepper 3.2mg/100g of dry plant material. Given the average water content of fresh tomato- about 95%-and that of pepper-92%, the total amount of CGs per kg of fresh material is 1.15mg for tomato and 0.25mg for pepper.

The World Food and Agricultural Organization (FAO) reported an annual average world production of 160 million tons of tomato and 30 million tons of pepper between 2010 and 2013. The obtained figures indicate an annual natural production of over 1900 tons of CGs worldwide from only these plant sources. The reported average world consumption is 136 Million tons of tomato and products, and the obtained figures indicate an annual global consumption of CGs of over 1500 tons.

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CONCLUSION The caffeoyl glucoses present in a variety of Solanaceae plant family members of great economic importance were successfully profiled and quantified with the use of the LC-MSn hierarchical key for the individual isomer identification (Jaiswal et al 2014) - constructed based on pure synthetic standards. Individual isomer regio- and stereochemical assignment of caffeoyl glucoses was possible, this being an important addition to the results of previous reports, which left stereo- and regiochemistry and even the identity of the hexose open. Therefore the, importance of synthetic work in the field of natural compounds is once more highlighted by the present study. However, the number of fully characterized caffeoyl glucoses found in the investigated extracts account for less than a quarter of the total number of isomers giving a pseudomolecular ion of m/z 341 in negative ion mode. This reveals a greater complexity of isomers than previously anticipated and draws attention towards the need for development of new hierarchical LC-MSn schemes allowing complete characterization of the remaining unidentified isomers. Quantitative data for caffeoyl glucoses were provided for the first time.

ACKNOWLEDGEMENTS IT support from Dr. Abhinandan Shrestha is greatly acknowledged. Excellent technical support from Ms Anja Müller is acknowledged.

Statement: This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

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Table 6.1. Caffeoyl glucose individual isomer content of investigated fresh tomato, tomato based products and various pepper samples in mg/100g dried material.

2-β-CG 2-α-CG 3-β-CG 3-α-CG 6-β-CG 6-α-CG Total No. Sample Name (Origin) [mg/100g] [mg/100g] [mg/100g] [mg/100g] [mg/100g] [mg/100g] [mg/100g]

1 Tomato (Romania) 2.06 18.88 20.94 2 Yellow RomaTomato 1.71 21.96 23.67 3 Cherry Tomato(Netherland) (Netherland) 2.28 27.18 29.46 4 Cherry Tomato (Italy) 1.41 22.74 24.14 5 Tomato (Italy) 1.33 12.44 13.77 6 Kumato Tomato (Spain) 1.13 30.02 31.15 7 Cherry Tomato (Marocco) 1.12 16.38 17.5 8 Tomato puree (Germany) 3.02 3.84 2.59 2.98 3.69 37.23 50.32 9 Organic tomato puree 2.9 2.65 2.47 4.53 39.05 48.7 10 Home-made(Germany) tomato juice 4.52 32.33 36.86 11 Tomato ketchup(Romania) (Germany) 0.69 5.21 5.9 12 Tomato ketchup (Germany) 0.87 4.59 5.46 13 Red Chilli (Netherland) 0.31 0.31 0.31 0.34 1.26 14 Red Chilli (India) 15 Red Chilli (Spain) 0.41 0.89 1.48 1.58 4.37 16 Green Chilli (Turkey) 0.65 0.74 1.62 0.26 1.93 2.19 7.39 17 Organic bell pepper-red 0.44 0.65 0.98 1.31 3.39 18 Organic(Netherland) bell pepper -green 0.68 0.62 1.2 1.01 3.51 Organic(Netherland) bell pepper- yellow 19 0.51 0.41 0.76 0.73 2.41 (Germany) 20 Organic bell pepper-red 0.42 0.46 0.73 0.91 2.52 21 Bell(Germany) pepper-red 0.42 0.42 Bell pepper(Spain)-yellow 22 0.32 0.32 (Spain) 23 Charlie cultivar pepper 0.29 0.29 0.55 0.21 0.69 0.68 2.7 Sivri cultivar(Turkey) pepper 24 1.42 1.16 1.73 0.59 1.09 1.09 7.08 (Turkey)

25 Aubergine (Spain)

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Fiorentino, A., D'Abrosca, B., Pacifico, S., Mastellone, C., Scognamiglio, M., & Monaco, P. (2009). Identification and assessment of antioxidant capacity of phytochemicals from kiwi fruits. Journal of Agricultural and Food Chemistry, 57(10), 4148-4155.

Hanson, K. R., & Zucker, M. (1963). Biosynthesis of chlorogenic acid and related conjugates of hydroxycinnamic acids - chromatographic separation and characterization. Journal of Biological Chemistry, 238(3), 1105-1115.

Jaiswal, R., Karakoese, H., Ruehmann, S., Goldner, K., Neumueller, M., Treutter, D., & Kuhnert, N. (2013). Identification of phenolic compounds in plum fruits (prunus salicina L. and prunus domestica L.) by high-performance liquid chromatography/tandem mass spectrometry and characterization of varieties by quantitative phenolic fingerprints. Journal of Agricultural and Food Chemistry, 61(49), 12020-12031.

Jaiswal, R., Matei, M. F., Glembockyte, V., Patras, M. A., & Kuhnert, N. (2014). Hierarchical key for the LC-MSn identification of all ten regio- and stereoisomers of caffeoylglucose. Journal of Agricultural and Food Chemistry, 62(38), 9252-9265.

Koeppen, B. H., & Herrmann, K. (1977). Flavonoid glycosides and hydroxycinnamic acid- esters of blackcurrants (ribes nigrum) - phenolics of fruits 9. Zeitschrift Fur Lebensmittel- Untersuchung Und-Forschung, 164(4), 263-268.

Macheix, J. (1977). Biosynthesis of glucose esters of hydroxycinnamic acids from uridine diphosphate-glucose and free acids. Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences Serie D, 284(1), 33-36.

Marin, A., Ferreres, F., Tomas-Barberan, F. A., & Gil, M. I. (2004). Characterization and quantitation of antioxidant constituents of sweet pepper (capsicum annuum L.). Journal of Agricultural and Food Chemistry, 52(12), 3861-3869.

Matei, M. F., Jaiswal, R., Patras, M. A., & Kuhnert, N. (2016). LC-MSn study of the chemical transformations of hydroxycinnamates during yerba mate (ilex paraguariensis) tea brewing. Food Research International, 90, 307-312.

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Moco, S., Bino, R. J., Vorst, O., Verhoeven, H. A., de Groot, J., van Beek, T. A., . . . de Vos, C. H. R. (2006). A liquid chromatography-mass spectrometry-based metabolome database for tomato. Plant Physiology, 141(4), 1205-1218.

Runeckles, V. C., & Woolrich, K. (1963). Tobacco polyphenols .1. the biosynthesis of O- glucosides and O-glucose esters of hydroxycinnamic acids. Phytochemistry, 2(1), 1-6.

Schuster, B., & Herrmann, K. (1985). Hydroxybenzoic and hydroxycinnamic acid derivatives in soft fruits. Phytochemistry, 24(11), 2761-2764.

Singh, A. P., Luthria, D., Wilson, T., Vorsa, N., Singh, V., Banuelos, G. S., & Pasakdee, S. (2009). Polyphenols content and antioxidant capacity of eggplant pulp. Food Chemistry, 114(3), 955-961.

Vallverdu-Queralt, A., Jauregui, O., Di Lecce, G., Andres-Lacueva, C., & Lamuela-Raventos, R. M. (2011). Screening of the polyphenol content of tomato-based products through accurate-mass spectrometry (HPLC-ESI-QTOF). Food Chemistry, 129(3), 877-883.

Vallverdu-Queralt, A., Jauregui, O., Medina-Remon, A., Andres-Lacueva, C., & Lamuela- Raventos, R. M. (2010). Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 24(20), 2986-2992.

Whitaker, B. D., & Stommel, J. R. (2003). Distribution of hydroxycinnamic acid conjugates in fruit of commercial eggplant (solanum melongena L.) cultivars. Journal of Agricultural and Food Chemistry, 51(11), 3448-3454.

Winter, M., & Herrmann, K. (1986). Esters and glucosides of hydroxycinnamic acids in vegetables. Journal of Agricultural and Food Chemistry, 34(4), 616-620.

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Chapter 7. Undestanding the fragmentation of glucose in mass spectrometry

Maria A. Patras, Javier Gonzalez, Juan Z. Davalos and Nikolai Kuhnert

Manuscript in preparation

Abstract:

The fragmentation mechanism of D-glucose was investigated in detail by two different fragmentation techniques, namely CID and IRMPD using all six 13C labeled isotopomers and all six 2H labeled isotopomers. For both CID and IRMPD energy resolved measurements were carried out. Individual fragmentation pathways were studied by multiple reactions monitoring fragmentation mode. Additionally we have developed an HPLC-tandem MS method to separate the anomers of D-glucose using a HILIC column and investigated their fragmentation patterns individually. Experimental results have in parts been rationalized by additional computational work. The results allowed a detailed formulation of the complex fragmentation mechanism of D-glucose. Additionally, an unprecedented isotope effect has been observed for the first time in IRMPD tandem mass spectrometry, in which fragmentation efficiency depends on the site of 13C labeling within the analyte allowing indirect insight into charge site regiochemistry in gas phase ions. The results have immediate consequences for the full structure analysis of complex carbohydrates and form the basis of a comprehensive formalism allowing carbohydrate structure elucidation.

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INTRODUCTION:

Glucose is one of nature’s most important molecules. It serves as an energy source for any living organism and constitutes an essential building block in all carbohydrate polymers used by living organisms as structural molecules, energy storage forms and carbohydrates employed incell-cell communication. Furthermore it constitutes a vital building block for posttranslational modification of proteins, modification of lipids and defines the function of numerous crucial secondary metabolites by glycosylation [1-3]. Most carbohydrates and carbohydrate related structures such as glycoproteins, glycolipids, glycosylated secondary metabolites or oligosaccharides are characterized since over two decades using mass spectrometry based techniques [4-20]. Aim of the structural characterization is always to establish the full chemical structure, acquiring information on the type of carbohydrate building block being present, acquiring information on the regiochemistry or linkage isomerism, acquiring information on the stereochemistry of the building blocks used and finally acquiring information on the stereochemistry at the anomeric centers. Such structural characterization work is always carried out by determining the full high resolution mass of the analyte in question followed by detailed fragmentation studies using tandem mass spectrometry. While the correct identification of the individual building blocks used must be considered as relatively straightforward, determination of aspects of stereo- and regiochemistry is more problematic, and the number of reports on assigning stereochemistry of carbohydrates by MS is low. Good progress has been achieved in the interpretation of fragment spectra which has been summarized in several review articles [9- 11,20-24]. However, a comprehensive framework for elucidating all aspects of carbohydrate structure by mass spectrometry is still missing. Frequently, aspects of stereochemistry can only be determined by techniques such as methylation or acetylation analysis using derivatisation followed by GC-MS analysis or in rare cases where sufficient amounts of pure material are available by NMR-spectroscopy. Analysis of carbohydrate structures involves fragmentations of the individual monosaccharide building blocks themselves. It is surprising that MS is still hardly being exploited in carbohydrate structure analysis when considering that subtle differences in fragmentation patterns of stereoisomeric hexoses e.g. glucose, mannose and galactose have been reported on numerous occasions [4,25-30]. March and co-workers[25] reported that different hexoses including glucose, mannose, galactose and fructose can be distinguished based on the differences in fragment ion signal intensity ratios of m/z 161 to 119 ranging from one (for mannose) to five (for fructose). Glucose shows a signal intensity ratio of 1.2. From these

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry reports, it becomes obvious that both fragmentation pathways leading to these diagnostic fragment ions must be sensitive towards hexose stereochemistry.

Moreover, although very limited in number, there have been studies which reported assignment of stereochemistry of the monomeric units of linear oligosaccharides by MS solely [31,32]. Ion mobility-MS has been also reported as a powerful tool for carbohydrate sequencing, as it can discriminate between epimeric glycans and glycoproteins [33]. Differences in the fragmentation of the two anomeric forms of hexose-glycolaldehyde anions have as well been reported [32]. It is even more surprising to note that despite all efforts and advances in carbohydrate MS, the fragmentation mechanisms of the most simple hexose building blocks, most notably glucose has only been studied on rare occasions [28] and no exhaustive mechanistic study using all isotopomers exists in the literature. Differences in the fragmentation of non-derivatised α-(D)- glucose and β-(D)-glucose have as well never been studied, reason thereof being the increased difficulty of isolating individual anomers which are in permanent equilibrium with one another. From this fact it becomes evident that a thorough understanding of the fragmentation mechanism of glucose will form the basis of a comprehensive framework allowing full structure elucidation of carbohydrate regio- and stereochemistry.

In this contribution we have studied the fragmentation mechanism of D-glucose in detail using seven 13C labeled isotopomers and all six 2H labeled isotopomers (Figure 7.1). We have investigated the fragmentation mechanism using two different fragmentation techniques, namely collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). CID experiments have been carried out using a triple quadrupole ion trap mass spectrometer and IRMPD measurements using a Fourier transform- ion cyclotron resonance (FT-ICR) mass spectrometer. For both CID and IRMPD energy resolved measurements were carried out. Additionally we have developed an HPLC-tandem MS method to separate and study both anomers of D-glucose individually. Experimental results have in parts been rationalized by additional computational work. The results allow a detailed formulation of the fragmentation mechanism of D-glucose. The results have immediate consequences for the full structure analysis of complex carbohydrates and form the basis of a comprehensive formalism allowing carbohydrate structure elucidation. Additionally we have for the first time observed in IRMPD tandem mass spectrometry an unprecedented isotope effect, in which fragmentation efficiency depends on the site of 13C labeling within the analyte allowing indirect insight into charge site regiochemistry in gas phase ions.

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2 2H-C-1 H-C-2 2H-C-3

2H-C-5 2H-C-4 2 H2-C-6

13 13 13 C-1 C-2 C2-1,2

13C-3 13C-4 13C-5

Figure 7.1: Structures of the thirteen glucose isotopomers employed in the study

MATERIALS AND METHODS

Chemicals 13 13 13 13 13 13 13 13 2 2 2 2 C labeled ( C-1, C-2, C-3, C-4, C-5, C and C2-1,2) and H labeled ( H-1, H-2, H- 2 2 2 3, H-4, H-5 and H2-6,6) glucose derivatives were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Isotope incorporation was determined using 13C-NMR spectroscopy, 1H-NMR spectroscopy and mass spectrometry. Data on isotope incorporation are provided in the supplementary information. HPLC grade acetonitrile and methanol used were purchased from ROTH (Karlsruhe, Germany). Taxifolin was purchased from Sigma-Aldrich (Steinheim, Germany).

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Sample preparation: Individual solutions of each D-Glucose isotopomer were prepared in a mixture of water/acetonitrile (1/9 v/v) at a concentration of 0.1mg/mL. The solutions were subject to both LC-MS measurements on the ion trap and direct infusion measurements on the Q-ToF.

LC-MS analysis of glucose isotopomers LC separations were performed on an Agilent 1100 Series system (Agilent, Karlsruhe, Germany) comprising a binary pump and an auto sampler with 100μL loop using a 100x3mm i.d NUCLEODUR HILIC column (Macherey-Nagel, Düren, Germany). The binary solvent system comprised of solvent A -water and solvent B-acetonitrile. 92% B was delivered isocratically for 10 minutes at a flow rate of 600μL/min. The LC system was coupled to an ion trap mass spectrometer (HCT Ultra, Bruker Daltonics, Bremen, Germany) fitted with an ESI ionization chamber, operating in full scan, auto MSn negative ion mode to generate fragment ions. As necessary, MRM (multiple reaction monitoring) mode was also employed to study different fragmentation pathways. The MS conditions: capillary temperature of 365 °C, drying gas flow rate of 10 L/min, and a nebulizer pressure of 50 psi-have been optimized using a solution of D-glucose in water/acetonitrile (1/9 v/v) with concentration of 0.1mg/mL.

Taxifolin epimerization and subsequent LC-MS analysis of the epimeric mixture A 1mg/mL solution of methanolic Taxifolin ((2R,3R)-3,3′,4′,5,7-Pentahydroxyflavanone) was incubated for 30 minutes at 150⁰C to produce a mixture of epimers. The resulting dry residue was dissolved in methanol and subjected to LC-MS analysis. The epimers were separated on the same LC system previously described using a 5µm particle size reversed phase C18 column (250mm x 3mm inner diameter). The binary solvent system consisted of A water and B acetonitrile. The gradient used was: 0–2 min 10% B, 2–29 min 50% B, 29– 35 min 80% B, 35–40min 100% B, and 40–65 min 100% B. The LC system was coupled to a quadrupole ion trap mass spectrometer (Bruker Daltonics, HCT Ultra) using an ESI ionization chamber. Tandem MS spectra were acquired in auto MS2 mode (smart fragmentation) using a ramping of the collision energy.

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Data analysis Data were acquired and processed using Data Analysis 4.0 software package (Bruker Daltonics).

RESULTS AND DISCUSSION

The aim of this contribution is to provide detailed insight into the fragmentation mechanism of both anomers of glucose. The resulting information should be used to identify those fragmentation pathways that are sensitive towards changes of regio- and stereochemistry allowing the generation of predictive rules for structure elucidation of more complex carbohydrate derivatives.

For this purpose we studied the fragmentation of all mono- 13C labeled derivatives and 2H labeled derivatives of D-glucose (structures shown in scheme 1) using a variety of mass spectrometric methods. The labeled derivatives were obtained commercially. All measurements were carried out using electrospray ionization in negative and positive ion mode. In the positive ion mode [M+Na]+ ions were always clearly observed, however, as previously reported no useful fragment spectra could be obtained. In negative ion mode the spectra were always dominated by the [M-H]- ions that following isolation yielded good fragment spectra.

Firstly we obtained data using a direct infusion of an aqueous solution using a quadrupole ion trap instrument. These experiments provided MS2 and MS3 fragment spectra of isotopically labeled compounds. The fragment spectra yielded from the [M-H]- ion at m/z 180 or 181 a small set of fragment ions that were further studied by MS3 tandem mass spectrometry. Energy resolved data using collision induced dissociation (CID) with helium as the collision gas were acquired, however as typical in an ion trap instrument due to the difficulty in maintaining a constant pressure in the collision cell and the inability to carry out adequate pressure measurements no further quantitative conclusions were derived from these data. In a second line of experiments again direct infusion of aqueous solutions were measured in a ESI- triple quadrupole instrument. In comparison to the ion trap data more fragment ions were observed in general and energy resolved data using CID were acquired at a constant pressure of 0.040 Torr using argon as a collision gas. Quantitative data were derived from this data set. Finally energy resolved data were acquired using an ESI-FT-ICR mass spectrometer using infrared multi photon dissociation (IRMPD) fragmentation. For energy resolved data both the laser power was varied at constant laser pulse duration intervals and the laser pulse irradiation time 144

Chapter 7. Understanding the fragmentation of glucose in mass spectrometry at constant laser power. In general it was observed that the latter resulted in better quality data. Using IRMPD fragmentation in general the largest number of fragment ions was observed if compared to the other two techniques. Additionally the use of an FT-ICR-MS allowed high resolution mass measurements of all fragment ions and therefore unambiguous determination of all molecular formulas of all fragment ions. Finally an HPLC method using a hydrophobic liquid interaction chromatography (HILIC) technique was developed to separate both anomers of D-glucose chromatographically and acquire tandem MS data of both anomers separately.

In general the following observations were made:

1. All tandem MS methods yielded high quality fragment spectra in the negative ion mode. 2. All tandem MS spectra using all MS methods applied gave consistent information with respect to isotopic incorporation into fragment ions. Due to the differences in energy transfer to the selected precursor ions relative intensities of fragment ions versus parent ions did vary slightly. Similarly, slight variations in ratios of isotopically labeled and non-labeled fragment ions were observed. 3. Since IRMPD is known to be an isotope-selective technique [34-38], in IRMPD data precursor ions displayed a significant variation in ease of fragmentation depending on the regiochemistry of isotope incorporation. This effect has to the best of our knowledge never been observed in tandem mass spectrometry. E.g. it is much more difficult to fragment 13C-3 and 13C-4 in comparison to all other derivatives. We call this effect the” labelleing suppressing fragmentation effect” (LSFE).

For the interpretation of data the following assumptions were made and the following considerations need to be taken into account:

1. The regiochemistry of ionization and therefore the detailed structure of the precursor ions are unknown. D-glucose has five OH functionalities that are all chemically distinct. Ionization could occur regioselectively producing only one distinct [M-H]- ion or non- selectively producing a mixture of ions at defined ratios. One ion might be more reactive than another ion and the reactivity rather than the relative ratio of ions will determine the fragment spectrum. Within the data discussion we assume certain ion regiochemistries in order to rationalize distinct fragmentation pathways. We will show that different regiochemistries of ionization sites will lead to distinct fragmentation pathways. In order to gain additional insight into ion stability density functional theory (DFT) calculations were performed to support the experimental data (Table 7.1).

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2. For rationalization of fragmentation mechanisms we opted to use classical organic chemistry mechanistic formalisms suggesting stepwise ionic reaction mechanisms. The driving force in all cross ring fragmentations (CRF) can in these cases be traced down enthalpically to the formation of two new C=O bonds from one C-O and one C-C bond. 3. In all fragmentation mechanisms we assume that H+ transfer is rapid in the gas phase and occurs most likely intramolecularly and stable ions with a negative charge located at oxygen are the main reaction intermediates. Rapid intramolecular H+ transfer has been observed in the MS fragmentation of protonated peptides [40-42].

Theoretical calculations of ionization energies Ionization energies of individual glucose hydroxyl groups were calculated using the ADF (Amsterdam Density Functional) software. As shown in Table 7.1, the anomeric proton presents the lowest ionization energy and therefore highest acidity, which is in line with the findings of previous studies [43-45]. However, considering the high voltage (potential difference of 4000V) applied to the ion source, it is fair to expect a non-selective ionization producing a mixture of isomeric [M-H]- ions, each of them being the starting point for a unique fragmentation pathway. Table 7.1: Theoretical Ionization energies of the five chemically distinct hydroxyl groups of the glucose molecule

Energy (kcal/mol) Ionization energy (kcal/mol)

α-glucopyranose -3690.04 - (1-O-α- -3620.71 69.33 neutral (2-O-α- -3618.04 72 glucopyranose)- (3-O-α- -3588.28 101.76 glucopyranose)- (4-O-α- -3609.23 80.81 glucopyranose)- (6-O-α- -3592.5 97.54 glucopyranose)- β-glucopyranose -3691.49 - glucopyranose)- (1-O-β- -3621.81 69.68 neutral (2-O-β- -3614.63 76.86 glucopyranose)- (3-O-β- -3588.35 103.14 glucopyranose)- (4-O-β- -3612.86 78.63 glucopyranose)- (6-O-β- -3597.49 94 glucopyranose)-

glucopyranose)-

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

Water loss fragmentations (neutral losses of 18Da, 36 Da, 54 Da)

Fragment ions at m/z 162, 161 (neutral loss of H2O or HOD)

IRMPD fragmentation:

2H-C-1 and 2H-C-2 labelled derivatives both show [M-H]- ions at m/z 180.1 and fragment ions at m/z 161 (-HOD) and m/z 162 (-H2O), which is indicative of water loss at C-1 and C-2 2 occurring in parallel with water loss at other positions. H2C-6 labelled glucose shows no incorporation of the deuterium in the neutral loss, indicating that no water is lost at C-6 from the [M-H]- ion.

Ion trap (CID) fragmentation:

The auto MSn ion mode ion trap fragmentation spectra show incorporation of the deuterium 2 label into the water loss for all the deuterated isotopomers except H2C-6 which gives ions of m/z 163 exclusively.

The first conclusion that we could draw from the experimental data is that water loss from the - - [M-H] occurs unselectively, leading to a complex mixture of [M-H-H2O] isobaric ions, each of them being the starting point of parallel fragmentation cascades leading to more complex mixtures of smaller isobaric fragments.

The second important conclusion that we could draw, which applies to both IRMPD and CID fragmentation is that water loss occurs exclusively from the cyclic form, since 2H-C-1 was found to incorporate the isotope label in the water loss and water loss including the proton at C-1 can only occur from the cyclic form. This conclusion is further confirmed by the absence of the water loss including the protons at C-6, since this water loss could only occur from the open chain form, through the hydroxyl at C-5.

- All the eight possible [M-H-H2O] isomeric/isobaric product ions are individually represented in Figure 7.2 (Structures A-H) as pairs of the respective tautomeric keto/enols (where applicable). The position of the negative charge remains open, as we assume that intramolecular H+ transfer occurs very rapidly among the different hydroxyl moyeties.

Four potential mechanisms could operate (exclusively or in parallel) for loss of water: firstly an anti- E2 type mechanism, secondly an a syn-E2 type mechanism, thirdly an E1 type mechanisms

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry producing an oxonium ion from either anomer and finally an 1,1 elimination mechanism producing a carbene intermediate followed by a 1,2 H- or D- shift.

We could exclude the hypothesis of either anti- E2 or syn-E2 mechanisms operating exclusively, based on our results, since an anti- E2 elimination mechanism operating exclusively should only produce ion B1 (Figure 7.2) and a syn-E2 elimination mechanism operating exclusively should not produce water elimination products from the β anomer. However, our HPLC-MS results indicate water elimination products from both anomers and involving protons attached to all carbon atoms except C-6. In order to get an insight into the water loss fragmentation, we employed a mixture of taxifolin ((2R,3R)-3,3′,4′,5,7-Pentahydroxyflavanone) and its epimer ((2R,3S)-3,3′,4′,5,7- Pentahydroxyflavanone) as model compounds for the study of water elimination upon MS fragmentation. Both The chromatographic method employed provided baseline separation of the epimers and therefore allowed fragmentation of the individual species. (2R,3R): (2R,3S) ratio was 10:1 (confirmed by NMR spectroscopy). Taxifolin can only eliminate water through a syn-E2 type mechanism or a E1 type mechanism, whereas its epimer can only eliminate water through an anti-E2 type mechanism or a E1 type mechanism.

Upon fragmentation of their [M-H]- ions (m/z 303), both epimers produced the water elimination product of m/z 285 as the base peak. Therefore, the results show that water loss occurs regardless of the stereochemical orientation of the two leaving groups, which hints towards the hypothesis of an E1 type mechanism.

Fragment ions at m/z 144, 143 (neutral loss of two H2O or HOD)

IRMPD fragmentation: All deuterium labeled derivatives labelled derivatives show fragment ions at corresponding to neutral losses of 36 Da (H2O + HOD) and 37Da (2*H2O). This indicates unselective water loss 2 occurring at multiple sites in the molecule. H2C-6 labelled glucose, which was not found to lose the first unit of water from C-6, shows fragments at m/z 144 and m/z 145 indicating that one alternative for loss of the second unit of water involves C-6.

Ion trap fragmentation: All deuterium labelled derivatives show [M-H]- ions at 180.1 and fragment ions at m/z 143 3 2 - (-H2O - HOD) and m/z 144 (-H2O)2. Targeted MS experiments on the MS [M-H-H2O] product

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry ions of m/z 162 show incorporation of the deuterium label in the neutral loss generating peaks 3 2 at both m/z 144 (-H2O) and m/z 143 (-HOD). Targeted MS experiments on the MS [M-H- - 2 H2O] product ions of m/z 163 from H2C-6 shows fragments at m/z 144 and m/z 145. Therefore, all the results indicate that, just like the loss of the first water molecule, loss of a second water - molecule occurs unselectively. Since the [M-H-H2O] products exists as keto-enol tautomers, we have hypothesized that the second water loss from each structure involves preferentially the proton in the α position to the ketone, which is more acidic than the rest. Moreover, the resulting - 2-en-one structures are energetically favored due to their conjugation. For the three [M-H-H2O] ions which are not ketones (Figure 7.2, Structures G and H) we have hypothesized that the second water loss will preferentially involve the proton from α position to the double bond, as - the resulting [M-H-2H2O] ion will also be stabilized by conjugation. All proposed structures - - of the resulting [M-H-H2O] and most abundant [M-H-2*H2O] ions are presented in Figure 7.2.

Cross-ring Fragmentations

Fragment ion at m/z 150 and 149 (neutral loss of CH2O)

Ion trap fragmentation: 13C-2, 13C-3, 13C-4 and 13C-5 labelled glucose show no incorporation of a 13C-label into the neutral loss and result in fragment ions at m/z 150. 13C-1 and 13C-6 show label incorporation 2 into the neutral loss yielding fragments at m/z 149 and 150. Similarly H2C-6 labelled glucose shows from the precursor [M-H]- ion of m/z 181.1 fragment ions of m/z 151 (neutral loss of 30 13 Da) and m/z 149 (neutral loss of 32 Da). C2-1,2 labeled glucose also shows from the precursor [M-H]- ion of m/z 181.1 fragment ions at m/z 151 (neutral loss of 30 Da) and 150 (neutral loss of 31 Da).

Both IRMPD and CID fragmentation results suggest that the loss of a CH2O molecule occurs in parallel at C-1 and C-6 from the open chain form. Consequently, using the Domon and Costello nomenclature [46] two alternative cross ring fissions take place, namely 0, 1A and 5, 6A, leading to structures I and J respectively (Figure 7.3).

IRMPD fragmentation 13C-2, 13C-3, 13C-4 and 13C-5 labelled glucose show no incorporation of a 13C-label into the neutral loss and result in fragment ions at m/z 150. 13C-1 and 13C-6 in contrast show label

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry incorporation into the neutral loss yielding fragments at m/z 149 and 150 in a ratio of 3:4 and 2 - 1:1 respectively. Similarly H2C-6 labelled glucose shows from the precursor [M-H] ion of m/z 13 181.1 fragment ions of m/z 151 (neutral loss of 30 Da) and m/z 149 (neutral loss of 32 Da). C2- 1,2 labeled glucose also shows from the precursor [M-H]- ion of m/z 181.1 fragment ions at m/z 151 (neutral loss of 30 Da) and 150 (neutral loss of 31 Da). 2H-C-1 does not show peaks corresponding to the losses of 30 or 31 Da. 2H-C-2 does not show incorporation of the 2H label into the neutral loss, generating only the peak of m/z 150.

Fragment ion at m/z 120, 119 (neutral loss of C2H4O2)

IRMPD fragmentation:

13C-3 and 13C-4 labelled glucose show no isotope incorporation into the neutral loss and result exclusively in fragment ions of m/z 120. In contrast 13C-1, 13C-2, 13C-5, 13C-6 and 13C-1-13C-2 show incorporation of the label into the neutral loss with fragment ions at m/z 120 and 119.

Ion trap fragmentation.

Auto MSn spectra of all investigated isotopically labeled glucoses show peaks of m/z 120 and m/z 119 with relatively high intensity. Just like in the case of IRMPD fragmentation, in the ion trap fragmentation 13C-3, 13C-4, 2H-C-3 and 2H-C-4 labelled glucose do not show isotope 13 13 13 13 13 2 incorporation into the neutral loss, whereas C-1, C-2, C-5, C-6 and C2-1,2, H-C-1, 2 2 2 H-C-2, H-C-5 and H2-C-6 show incorporation of the isotope label into the neutral loss.

Using the Domon and Costello nomenclature [46] the results clearly indicate that two distinct fragmentation pathways tale place in parallel producing the two carbon neutral fragments 0, 2 4, 5 C2H4O2, namely A and A cross ring fragmentations (Figure 7.3). The results do not show 2, 4A, 3, 5A or 1, 3A fragmentations as frequently assigned in the literature based on chemical intuition rather than experimental evidence.

- Investigating the pathway [M-H-CH2O] - CH2O

Additionally we have also investigated the possibility of a neutral loss of 60 Da occurring through two consecutive fragmentations, each producing a neutral loss of 30 Da (CH2O). Therefore, we have performed targeted MS3 fragmentations on the relatively low intensity [M- - H-CH2O] ions corresponding to each isotopomer.

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

3 - 13 MS fragmentation of the [M-H-CH2O] (Figure 7.3, Structure I) of m/z 151 of C2-1,2 13 generates low intensity peaks of m/z 121 (loss of CH2O from C-5) and m/z 120 (loss of CH2O 3 13 - from C-1). MS fragmentation of the [M-H- CH2O] (Figure 7.3, Structure J) of m/z 150 of 13 13 C2-1,2 generates peaks of m/z 120 (loss of CH2O from C-6) and m/z 119 (loss of CH2O from 3 - 13 C-2). MS fragmentation of the [M-H-CH2O] (Figure 7.3, Structure I) of m/z 150 of C-1 13 3 generates peaks of m/z 120 (loss of CH2O) and m/z 119 (loss of CH2O). In contrast MS 13 - 13 fragmentation of the [M-H- CH2O] (Figure 7.3, Structure J) of m/z 150 of C-1 generates 3 peaks of m/z 119 (loss of CH2O) with very low intensity. MS fragmentation of the [M-H- - 13 13 CH2O] of m/z 150 of C-2 and C-5 generates peaks of m/z 120 (loss of CH2O) and m/z 119 13 (loss of CH2O), indicative of a second CH2O fragment loss from C-2 or C-5. 3 13 - 13 MS fragmentation of the [M-H- CH2O] (Figure 7.3, Structure I) of m/z 149 of C-6 generates peaks of m/z 119 (loss of CH2O).

3 - 2 MS fragmentation of the [M-H-CH2O] of m/z 150 (Figure 7.3, Structure I) from H-C-1 generates peaks of m/z 120 (loss of CH2O from C-5) and m/z 119 (loss of CHDO from C-1). MS3 fragmentation of the [M-H-CHDO]- of m/z 149 (Figure 7.3, Structure J) from 2H-C-1 3 - generates peaks of m/z 119 (loss of CH2O). MS fragmentation of the [M-H-CH2O] of m/z 150 2 from H-C-2 generates peaks of m/z 120 (loss of CH2O) and m/z 119 (loss of CHDO from C- 3 2), indicating as well the loss of a second CH2O fragment from C-2 or C-5. MS fragmentation - 2 of the [M-H-CHDO] of m/z 149 from H-C-2 generates peaks of m/z 119 (loss of CH2O). Conclusively minor pathways resulting in a total neutral loss of 60 Da occur through two successive losses of CH2O. Structures of the four possible isobaric ions of m/z 119 are presented in Figure 7.3 together with the fragmentation pathways leading to each of them.

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Chapter 7.

OH OH OH

O OH O O OO

-H+ -H+

HO HO HO

Understanding the fragmentation of glucose in mass spectrometry OH A1 OH A2 C1 C2 C2 C3 H2O lost from ( H-OH ) H2O lost from ( H-OH )

OOHOOHO OH

OH OH OH OH -H+ O O O O HOOHOOHHO OH + + + -H -H -HOH

H2H1 HO OH HO O O O

C4 C3 OH -H2O lost from ( H-OH ) OH OH B2 OH -H2O lost from B1 C5 C6 C2 C1 C3 C4 ( H-OH ) H2O lost from ( H-OH ) H2O lost from ( H-OH )

O H O H O H OH OH O H OH O O H

152 O O H O O H O OH O OH O O H + O OH + - H - H -H+ - H +

+ O H -H HO OH HO O O O H HO OH O H O G 1 C1 C 2 G 2 OH H O lost from H O lost from C3 C2 2 H O lost from(C2H-OHC3) 2 H2O lost from ( H-OH ) (C5H-OHC4 ) 2 (C3H-OHC4 )

OH OH O H O H OH OH O OH O OH O O O H O OH O OH -H+ - H + - H + -H+ HO HO H O O HO OH O OH D1 F 2 F1 OH O O D 2 -H O lost from (C4H-OHC3 ) C3 C2 H O lost from -H O from (C3H-OHC2) 2 H2O lost from ( H-OH ) 2 2 (C2H-OHC1 )

OH OH OH OH

O OH O O OH O -H+ -H+

OH OH OH O

OH O E1 O E2 O

C3 C4 C2 C1 H O lost from ( H-OH ) H2O lost from ( H-OH ) 2

- - Figure 7.2: Structures of the possible [M-H-H2O] ions and the most abundant [M-H-2*H2O] ions resulting from each of them (water loss site is indicated under each structure)

Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

O OH OH

6 6 6

5 OH O 5 5 O 5 O 1 1 H HO O HO O 4 1 4 4 4 HO O HO 2 -CH O from C6 2 3 2 3 3 2 -CH2O from C1 3 2 HO OH HO OH HO O HO O m/z 179 m/z 149 m/z 179 m/z 149 I J OH OH 6 OH 6 5 O 5 O 1 O HO OH 4 5 6 OH 4 1 O O 4 3 HO O 4 1 2 3 2 3 neutral loss m/z 119 O 3 2 neutral loss 2 1 K O OH 5 6 HO OH m/z 179 HO OH m/z 119 m/z 179 HO OH O OH L

Additional minor pathways:

5 O O H O 1 1 O O HO O 4 6 6 4 5 O -CH O from C5 2 4 2 2 3 5 OH HO I 3 5 O HO OH 4 HO OH m/z 119 4 HO 3 2 m/z 149 HO -CH O from C6 L 2 3 2 3 2 HO O J m/z 119 H+ rearrangement HO O HO O m/z 149 m/z 149 N

5 O O 5 H+ rearrangement 4 H 1 4 HO O HO -CH O from C1 6 3 2 2 3 2 HO OH O HO O HO O 5 m/z 119 m/z 149 4 H O HO 4 5 6 M -CH O from C2 OH 2 3 3 2 m/z 119 O O O K m/z 149

Figure 7.3: Mechanistic pathways for the loss of 30 Da and 60 Da from the [M-H]- ion.

Fragment ion at m/z 90, 89 (neutral loss of (CH2O)3 )

IRMPD fragmentation: 13C-3 and 13C-4 labelled glucose show no isotope incorporation into the neutral loss and result exclusively in fragment ions of m/z 90 due to the Isotope labelling effect?. In contrast 13C-1, 13C-2, 13C-5, 13C-6 and 13C-1-13C-2 show incorporation of the label into the neutral loss with fragment ions at m/z 90 and 89. Accordingly the neutral loss of (CH2CO)3 must occur via a two

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

step process. No intact three carbon fragment of (CH2CO)3 is lost in IRMPD fragmentation but alternatively a two carbon fragment (CH2CO)2 from C-1-C-2 or C-5-C-6 followed by a one carbon fragment (CH2CO) from C-6 or C-1 respectively.

Ion trap fragmentation In contrast to the results of the IRMPD fragmentation, in ion trap fragmentation all 13C labeled samples show isotope incorporation resulting in fragments of m/z 89 comparable in intensity with the fragments of m/z 90. 13C-1, 13C-2, 13C-3 and 13C-4, 13C-5, 13C-6, 2H-C-1, 2H-C-2, all produce peaks at m/z 89 and m/z 90 from upon the MS2 fragmentation of their [M-H]- ions. MS2 - 13 fragmentation of the [M-H] ion on m/z 181 from C2-1,2 produced peaks at m/z 91 (neutral 13 2 - loss of C3H6O3) and m/z 89 (neutral loss of C2CH6O3). MS fragmentation of the [M-H] ion 2 2 on m/z 181 from H2-C-6 gives peaks at m/z 91 (neutral loss of C3H6O3), m/z 90 (C3H5 H1O3) 2 and m/z 89 (neutral loss of C3H4 H2O3). Using the Domon and Costello nomenclature, the results clearly indicate that two distinct fragmentation pathways tale place in parallel producing 0, 3 4, 6 the three carbon neutral fragment C3H6O3, namely A and A cross ring fragmentations (Figure 7.4).

O OH O 6 OH 1 1 5 OH HO 5 5 2 O 4 3 2 4 3 4 O O + 6 + 6 2 1 O OH O HO OH 3 O -CH2O HO OH -CH2O O -H2O O O 5 1 4 1 2 O 2 3 O 5 O m/z 59 O m/z 71 4 m/z 59 6 m/z 71 O Figure 7.4: Mechanistic pathways for the loss of 90 Da from the [M-H]- ion.

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Combined fragmentations

Fragment ion at m/z 132 and 131 (neutral loss of H2CO and H2O)

IRMPD fragmentation:

13C-2, 13C-3, 13C-4 and 13C-5 labelled glucose show no incorporation of a 13C-label into the neutral loss and result in fragment ions at m/z 132. 13C-1 and 13C-6 in contrast show label 2 incorporation into the neutral loss yielding fragments at m/z 131 and 132. Similarly H2C-6 labelled glucose shows from a precursor ion at m/z 181.1 fragment ions at m/z 133 and 131.

Consequently for the loss of CH2O two alternative fragmentation pathways exist with loss of C-1 and alternatively at C-6 followed by loss of water from the arising fragment.

Ion trap fragmentation

- The fragmentation pathway giving rise to [M-H-CH2O-H2O] ions is the sum of two individual fragmentations, namely the loss of one formaldehyde molecule and the loss of one water moleule. The order of the fragmentations giving rise to the ions of m/z 132 and m/z 131 was - - investigated by targeted experiments on the [M-H-H2O] and the [M-H-CH2O] ions.

- Targeted fragmentation of [M-H-H2O] ions: 3 - 2 MS fragmentation of the [M-H-H2O] ion of m/z 163 from H2C-6 generates ions at m/z 133 3 - (-CH2O from C-1) and m/z 131 (-CD2O from C-6). MS fragmentation of the [M-H-H2O] ion 2 of m/z 162 from H-C-1 generates peaks at both m/z 132 (-CH2O from C-6) and m/z 131 (loss 3 - 2 of CHDO from C-1). MS fragmentation of the [M-H-H2O] ion of m/z 162 from H-C-2 generates peaks at both m/z 132 (-CH2O from C-6) and m/z 131 (-CHDO from C-1, via the 3 - rearrangement of the hydrogen attached to C-2). MS fragmentation of the [M-H-H2O] ions of m/z 162 from 13C-3, 13C-4 and 13C-5 generates peaks at m/z 132 exclusively. MS3 fragmentation of the [M-H-HDO]- ion of m/z 161 from 2H-C-1 (Figure 7.2, Structure A1) 3 - generates peaks at m/z 131 (-CH2O from C-6). MS fragmentation of the [M-H-HDO] ion of 2 m/z 161 from H-C-2 (Figure 7.2, Structures B and D) generates peaks at m/z 131 (-CH2O from C-1 and C-6). MS3 fragmentation of the [M-H-HDO]- ions of m/z 161 from 2H-C-3 (Figure 7.2, Structures D1 and E1) generates peaks at m/z 131 and m/z 133. MS3 fragmentation of the [M-H-HDO]- ions of m/z 161 from 2H-C-4 (Figure 7.2, Structure F1) generates peaks at m/z 131. MS3 fragmentation of the [M-H-HDO]- ions of m/z 161 from 2H-C-5 (Figure 7.2, Structures G1 and H1) generates peaks at m/z 133 exclusively, through the loss of 28 Da (CO moiety). 155

Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

3 - 13 MS fragmentation of the [M-H-H2O] ions of m/z 162 from C-1 generates peaks at both m/z 13 3 - 132 (-CH2O from C-6) and m/z 131 (- CH2O from C-1). MS fragmentation of the [M-H-H2O] 13 ion of m/z 162 from C-6 generates peaks at both m/z 132 (-CH2O from C-1) and m/z 131 (- 13 3 - 13 CH2O from C-6). MS fragmentation of the [M-H-H2O] ion of m/z 163 from C2-1,2 13 generates peaks at both m/z 133 (-CH2O from C-6) and m/z 132 (- CH2O from C-1). - 13 13 13 13 Fragmentation spectra of the [M-H-H2O] ions of C-2, C-3, C-4 and C-4 glucoses did not show isotope incorporation in the neutral loss and therefore produced exclusively ions of m/z 132. All these results show that one of the pathways involves firstly the loss of a water - molecule from the [M-H] ion, followed by the loss of a CH2O molecule from C-1 or C-6.

- Targeted fragmentation of [M-H-CH2O] ions: 3 - 2 2 2 2 MS fragmentation of the [M-H-CH2O] of m/z 150 from H-C-1, H-C-2, H-C-3, H-C-4 and 2 2 3 H-C-5 generates peaks of m/z 132 (loss of H2O) and m/z 131 (loss of H HO). MS - 2 fragmentation of the [M-H-CH2O] of m/z 151 from H2-C-6 (Figure 2, Structure J) generates 3 peaks of m/z 133 (loss of H2O) and m/z 132 (loss of HDO). MS fragmentation of the [M-H- CHDO]- of m/z 149 (Figure 7.3, Structure J) from 2H-C-1 generates only peaks of m/z 131 (loss 3 2 - of H2O). MS fragmentation of the [M-H-C H2O] of m/z 149 (Figure 7.3, Structure I) from 2 H2-C-6 generates only peaks of m/z 131 (loss of H2O).

3 13 - 13 MS fragmentation of the [M-H- CH2O] (Figure 7.3, Structure I) of m/z 149 of C-6 3 13 - generates peaks of m/z 131 (loss of H2O). MS fragmentation of the [M-H- CH2O] (Figure 13 3 7.3, Structure J) of m/z 149 of C-1 generates peaks of m/z 131 (loss of H2O). MS 13 - 13 fragmentation of the [M-H- CH2O] (Figure 7.3, Structure J) of m/z 149 of C-1 generates 3 13 - peaks of m/z 131 (loss of H2O). MS fragmentation of the [M-H- CH2O] (Figure 7.3, Structure 13 3 J) of m/z 150 of C2-1,2 generates peaks of m/z 132 (loss of H2O). MS fragmentation of the - 13 [M-H-CH2O] (Figure 7.3, Structure I) of m/z 151 of C2-1,2 generates peaks of m/z 133 (loss of H2O). All the results suggest that the second pathway for generating ions of m/z 132 involves firstly the selective loss of CH2O from C-1 or C-6 followed by the unselective loss of a water molecule.

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

Fragment ion at m/z 114, 113 (neutral loss of 66 Da, 2*H2O and CH2O)

IRMPD fragmentation: 13 13 13 2 2 C-1, C-6, C2-1,2, H-C-2 and H2-C-6 show incorporation of the isotope labels into the neutral losses. 13C-2, 13C-3, 13C-4 and 13C-5 don’t show incorporation of the isotope labels into the neutral loss and 2H-C-1 does not show ions at either m/z 114 or m/z 113. These results suggest also the loss of the CH2O molecule from C-1 and C-6.

Ion trap fragmentation MS2 spectra of 13C-1, 13C-2, 13C-3, 13C-4 and 13C-5 don’t show incorporation of the isotope 13 label into the neutral loss, generating only peaks at m/z 114. C2-1,2 as well shows no incorporation of the isotope label into the neutral loss, generating only peaks of m/z 115. 2H-C- 2 13 2 1, H-C-2, C-6 and H2-C-6 show incorporation of the isotope labels into the neutral loss.

In theory, two fragmentation pathways could generate a total neutral loss of 66 Da, namely: first the loss of water followed by the loss of a CH2O unit or first loss the loss of the CH2O unit followed by loss of water. In order to identify the possible pathways leading to a final neutral loss of 66 Da, targeted experiments were performed for the species showing isotope incorporation.

Investigating the pathway: [M-H -CH2O]-(H2O)2 3 3 13 - Targeted MS fragmentation of the MS [M-H- CH2O] (Figure 7.3, Structure I) of m/z 149 from 13C-6 generates peaks of m/z 113. Targeted MS3 fragmentation of the MS3 product ion 13 - 13 [M-H- CH2O] (Figure 7.3, Structure J) of m/z 149 from C-1 generates peaks of m/z 113. 3 2 - Targeted MS fragmentation of the MS [M-H-CH2O] ion (Figure 7.3, Structure I) of m/z 150 13 3 2 - from C-1 generates peaks of m/z 114. Targeted MS fragmentation of the MS [M-H-CH2O] 2 (Figure 7.3, Structure I) of m/z 150 from H-C-1 generates peaks of m/z 114 (loss of 2*H2O) 3 2 - and m/z 113 (loss of H2O and HDO). Targeted MS fragmentation of the MS [M-H-CH2O] ion (Figure 7.3, Structure J) of m/z 149 from 2H-C-1 generates peaks of m/z 113. Targeted MS3 3 - 2 fragmentation of the MS [M-H-CH2O] of m/z 150 from H-C-2 generates peaks of m/z 114 3 2 - and m/z 113. Targeted MS fragmentation of the MS [M-H-CH2O] (Figure 7.3, Structure J) 2 3 of m/z 151 from H2-C-6 generates peaks of m/z 115 and m/z 114. Targeted MS fragmentation 2 - 13 of the MS [M-H-CH2O] ion (Figure 7.3, Structure J) of m/z 151 from C2-1,2 generates 3 2 13 - peaks of m/z 115. Targeted MS fragmentation of the MS [M-H- CH2O] ion (Figure 7.3, 13 Structure I) of m/z 150 from C2-1,2 generates peaks of m/z 114. Conclusively, one pathway

157

Chapter 7. Understanding the fragmentation of glucose in mass spectrometry accounting for a total neutral loss of 66 Da is achieved by loss of two water molecules from the - [M-H-CH2O] ions (Figure 7.3, Structures I and J).

- Investigating the pathway [M-H-2*H2O] -CH2O 3 - 13 13 MS fragmentation of the [M-H-2*H2O] ion of m/z 144 from C-1 and C-6 generates ions 3 - 13 of m/z 114 and m/z 113. MS fragmentation of the [M-H-2*H2O] ion of m/z 144 from C-2, 13 13 13 3 - C-3, C-4 and C-5 generates ions at m/z 114. MS fragmentation of the [M-H-2*H2O] ion 13 of m/z 145 from C2-1,2 generates ions at m/z 115 and m/z 113.Therefore, the second fragmentation pathway accounting for a total neutral loss of 66 Da is initiated by the loss of two consecutive water molecules from the [M-H]- ions followed by the cross ring fission.

Fragmentation differences between α and β anomers All previous studies on hexose MS fragmentation have employed direct infusion analysis of aqueous mixtures of both anomers infused concomitantly. Therefore, the resulting fragment spectra are superimpositions of the spectra of both hexose anomers. In an aqueous infusion of glucose, the relative ratio of α versus β is known to be 34:66 [39]. In our chromatography experiment, the relative α versus β ratio at the solvent composition at which separation occurred was found to be 50:50. The ratio was determined independently using NMR spectroscopy and was found to be 45:55 in acetonitrile/water 92/8. Generally, no outstanding differences were observed between the relative intensities of fragmentation products corresponding to individual anomers. However, individual anomers of 2H-C-1 and 2H-C-2 show slight differences between the relative intensity ratios of the fragment ions of m/z 162 and m/z 161. The lower polarity α anomer is expected to elute before the more polar β anomer from a HILIC column using 92% acetonitrile as isocratic eluent. The α and β 2 anomers of H-C-1 presented ratios of the product ions relative intensities %I m/z 162 / %I m/z 161 of 10/6 and 10/4 respectively. The α and β anomers of 2H-C-2, presented ratios of the product ions relative intensities m/z 162/ m/z 161 of 10/4 and 10/6 respectively. Chromatograms and MS2 spectra of the two selected isotopomers are presented in Figure 7.5.

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

CONCLUSIONS

The present study has proven that the fragmentation of gluose in mass spectrometry is far more complex than previously thought. The results show the formation of several isobaric fragment ions which coexist in the gas phase giving complex fragment spectra. Fragmentations involving loss of water molecules from the [M-H]- ions have shown to occur unselectively from the cyclic - pyranose form and eight different [M-H-H2O] isobaric ions contributing to the overall intensity of the peak at m/z 161 were identified. Cross ring fissions on the other hand occur with a certain degree of selectively from both the pyranose ring structure and the open chain form. Two - - different [M-H-CH2O] ions, four different [M-H-C2H4O] ions and two different [M-H- - C3H6O3] ions were identified. Mixed fragmentations have shown to occur both by cross ring - - fission of the [M-H-H2O] or [M-H-2*H2O] and by water loss fragmentations from the [M-H- - - CH2O] ions and the [M-H-C2H4O] ions. We have so far shown that the fragmentation pathway leading to cross ring fragmentations is determined by the regiochemistry of anion formation. From this it can be tentatively concluded that if one site for anion formation is not available the corresponding fragmentation pathway will be shut down. We suggest to use this effect in regiochemical assignment of interglycosidic linkages. Additionally, for the first time in literature, the glucose anomers were separated by HPLC on a HILIC column and their fragmentation was studied individually.

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

Figure 7.5: Total ion chromatograms and MS2 spectra of individual anomers of 2H-C-1 (up) and 2H-C-2(down)

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Chapter 7. Understanding the fragmentation of glucose in mass spectrometry

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164

Chapter 8. Conclusions

The overarching aim of the present work was the better understanding of the isomeric complexity of the secondary metabolome of dietary plants with particular focus on flavonoids and glycosylated hydroxycinnamic acids using various mass spectrometry-based analytical tools. The first part of this study dealt with the investigation of cocoa polyphenols using both a bottom-up and a top-down approach. An insight into the great chemical complexity of cocoa was achieved by ultra-high resolution FTICR-MS analysis of crude aqueous methanolic extracts of fermented cocoa beans of different origins. With around 11 000 signals present in the spectra, cocoa polyphenolic extract has proven to be one of the most complex organic mixtures ever analyzed (of comparable complexity with organic environmental samples). The automated algorithms for peak detection and molecular formula assignment embedded in the software packages offer a good high-throughput method for handling, comparing and visualizing of mass spectral data. Furthermore, molecular formulae could be divided into different compound classes, depending on their elemental composition. Moreover, although the total number of signals was comparable between the two origins investigated, the class plot diagrams and the van Krevelen data representation revealed a significant difference in the abundance and intensity of the nitrogen containing class of compounds, thus proving to be valuable tools for the rapid investigation and comparison of the chemical profiles of complex mixtures.

Additionally, an HPLC-MSn method revealing a greater isomeric complexity than previously reported for cocoa (32 resolved isomeric peaks in the base peak chromatogram) was developed, which allowed for the assignment of fourteen novel compounds not previously reported in this plant source, mainly glycosylated (both O-glycosylated and C-glycosylated) and sulphonated flavan-3-ols, on the basis of their molecular formulae and fragmentation patterns. Additionally 12-hydroxy jasmonic acid sulfate, which was proposed as a marker of fermentation induced stress, was identified here for the first time. The fragmentation pattern of dideoxyclovamide- previously identified in cocoa by isolation and NMR investigation-were described for the first time. Due to lack of reference standards, the absolute regio- and stereochemical identity of some of the assigned compounds could not be assigned unambiguously.

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Chapter 8. Conclusions

The second part of the present work dealt with complete characterization of individual regioisomeric hydroxycinnamoyl glucoses and their profiling and quantification in dietary relevant plant materials. Most importantly, regioisomeric esters were successfully selectively synthesized and subsequently used for developing a LC-MS based hierarchical scheme, which allows their unambiguous assignment based on their relative elution from a reversed-phase chromatographic column and their distinct fragmentation patterns. Their fragmentation mechanisms were elucidated and it was concluded that, just like in the case of chlorogenic acids, hydroxycinnamoyl glucose regioisomers present distinct fragmentation patterns due to different arrays of intramolecular bonds, which activate certain leaving groups and thus favor certain fragmentation patterns. Homologous hydroxycinnamic acid-glucose regioisomers were shown to present identical fragmentation patterns. The developed LC-MSn scheme makes it possible to profile all ten ester regioisomers of any hydroxycinnamic acid-glucose pair without compound isolation or the need of reference standards. The targeted MSn mode allows for detection of even trace amounts of the respective species present in the extract (limit of detection was determined to be 0.1µg/mL). The present work will serve as the theoretical background and framework for many future profiling studies of a wide variety of plant sources, studies which would be difficult to carry out in the absence of such a tool, given the fact that hydroxycinnamoyl glucoses are not commercially available and that not many laboratories involved in plant profiling research do not have the capacities required for their synthesis. After development of our method, 20 different species of edible plants were investigated for their hydroxycinnamoyl-glucose profiles, namely: strawberries (Fragaria ananassa), raspberries (Rubus idaeus), blueberries (Vaccinium corymbosum), blackberries (Rubus fruticosus), red currants (Ribes rubrum), black currants (Ribes nigrum), lingonberries (Vaccinium vitis-idaea), gooseberries (Ribes uva-crispa), purple chokeberries (Aronia melanocarpa), elderberries (Sambucus melanocarpa), cranberries (Vaccinium oxycoccos), goji berries (Lycium chinense), sea buckthorn (Hippophae rhamnoides), açai berries (Euterpe oleracea), sour cherries (Prunus cerasus), pomegranate (Punica granatum), tomatoes (Solanum lycopersicum), peppers (Capsicum spp), chillies (Capsicum annuum,) and aubergines (Solanum melongena). Furthermore, an LC-MS quantification method was developed and validated and individual caffeoyl-glucoses were quantified in over 40 different samples. The present work provided the first set of caffeoyl-glucose quantitative data in literature. Taking into account the individual

166

Chapter 8. Conclusions contents of the investigated species and the worldwide production of the most significant of these crops, the data show an annual global consumption of over 1500 tons of caffeoylglucoses. The most abundant isomer in all Solanaceae vegetables was found to be 6-O-caffeoylglucose whereas in berries 1-O-caffeoylglucose was found to be the most prominent isomer. Furthermore, all the plant extracts were profiled for their hydroxycinnamic acid-O-glycosides content using either synthetic standards (where available) or the so called surrogate standards: plant extracts which have previously been reported to contain the respective species exclusively (species identified by isolation and NMR analysis). The improved HPLC method has revealed a greater complexity of isomers than ever reported in all the investigated samples (up to 40 isomeric peaks present in one sample). The hydroxycinnamoyl-glucose derivatives account, depending on the sample, for between 30-50% of the total number of isomeric species present in the extract. At this stage, on the basis of their fragmentations these compunds could only be tentatively assigned as either ester isomers of hydroxycinnamoyl hexoses different from glucose or as O-hexosides of hydroxicinnamic acids, with a hexose different from glucose. Complete structural characterization is at this point not possible in the absence of pure synthetic standards. Taking into account the also the results obtained from the analysis of the cocoa extracts, that show multiple isomeric species which can at this stage not be identified unambiguously due to the lack of reference standards, I would like to point out the importance of synthetic preparative work in the field of natural products for the unequivocal characterization of isomers. Hydroxycinnamoyl hexoses, just like chlorogenic acids are fortunate examples of isomers which can easily be differentiated on the basis of their fragmentation patterns. For the vast majority of other classes of compounds, this behavior is not encountered. This constitutes the reason why mass spectrometry has so long been considered an isomer blind technique. For isomeric species showing identical fragmentation patterns, assignment could be carried out based on the isomers’ relative elution times from the chromatographic columns. Once available as pure synthetic standards, the structures could be characterized down to the stereochemical level by NMR spectroscopy and their elution behavior could be further studied. Additionally, experimental results could be backed up with in silico studies regarding the dipole moments of individual isomers, the strength of the interaction forces between the compound and the stationary material of the column and the mobile phase. Thus, theoretical models predicting elution order of isomers could be developed and employed in tentative assignment of unknown compounds.

167

Chapter 8. Conclusions

Therefore, it seems like the better the methods for analysis become, the more questions arise waiting to be answered by future work bringing together preparative organic synthesis, HPLC- MSn analytics and molecular modelling techniques. I believe that the present work is a significant piece in the large puzzle depicting the picture of isomeric complexity in plants and I hope that it will contribute to arousing the scientific curiosity of future reasearchers and prompt them to engage in projects aiming at answering the questions still left open. For nine out of the ten caffeoylglucose isomers, the most abundant fragment ions are formed by the breakage of the bonds within the sugar moiety, greatly dependent on the regio-and stereochemistry of the O-acyl bond. Moreover different isomeric C- and O-glycosides of flavan-3-ols present in the cocoa extracts could easily be distinguished on the basis of their characteristic fragmentation patterns. Prompted by the results of the first parts of this work and taking into consideration that a detailed mechanistic study of the fragmentation of glucose in mass spectrometry is missing from literature, the third part of the present work was dedicated to the in-depth study of the fragmentation mechanism of D-glucose, employing all 13C and all 2H labeled isotopomers. Additionally, a HPLC-tandem MS method able to separate the anomers of D-glucose using a HILIC column was developed and their fragmentation patterns were investigated individually for the first time in literature. The results have proven to be unexpectedly complex, which constitutes a very good example of how simple molecules are often disregarded in terms of their potential complexity and therefore not sufficiently studied. Results show that for glucose several isobaric fragment ions coexist in the gas phase giving complex fragment spectra. Fragmentations involving loss of water molecules from the [M-H]- ions have shown to occur unselectively from the cyclic - pyranose form and eight different [M-H-H2O] isobaric ions contributing to the overall intensity of the peak at m/z 161 were identified. Cross ring fissions on the other hand occur selectively - from both the pyranose ring structure and the open chain form. Two different [M-H-CH2O] - - ions, four different [M-H-C2H4O] ions and two different [M-H-C3H6O3] ions were identified. Fragmentation pathways leading to individual cross ring fissions depend on the regiochemistry of the negative charge and this effect can have direct application in assignment of glycosidic linkage regiochemistry of larger molecules. Furthermore, for the first time in literature, an HPLC-tandem MS method was developed to separate the anomers of D-glucose using a HILIC column and their fragmentation patterns were - investigated individually. Slight differences were observed in the intensities of the [M-H-H2O] ions involving either the H+ or the HO- groups at the anomeric center. The results have direct

168

Chapter 8. Conclusions implications for tentative assignment of the anomeric center regiochemistry in larger more complex glycosylated derivatives. The present study employed thirteen different isotopomeric glucose samples. However, due to resource limitations, it could not be extended to involve other natural-product relevant isotopically labeled sugars like galactose manose or arabinose. Complementary detailed studies of the fragmentation of other epimeric hexoses or pentoses would further contribute to a better understanding of these mechanisms and would form the basis of a comprehensive formalism allowing complete and unambiguous carbohydrate structure elucidation based solely on MS techniques, which would be a very effective, high throughput analysis tool readily available for any analytical purpose.

169

Appendix 1

Apendix 1. Supporting information for Chapter 4

1 Figure A1.1. H NMR spectrum of 3-O-caffeoylglucoses (5 and 6) in methanol-d3.

170

Appendix 1

Figure A1.2. 1H NMR spectrum of 3-O-(3,4-dimethoxy)cinnamoylglucoses (13 and 14) in methanol-d3.

171

Appendix 1

Figure A1.3. 1H NMR spectrum of 6-O-(3,4-dimethoxy)cinnamoylglucoses (15 and 16) in methanol-d3.

Table A1.1. High Resolution Mass Spectral (HRMS) Data of Regio- and Stereoisomers of Caffeoylglucose 1-10 from Yerba Maté.

No. Caffeoylglucose [M-H+] m/z Theor. m/z Exp. Error (ppm)

1 α-1-CG C15H18O9 341.0878 341.0883 -1.3

2 β-1-CG C15H18O9 341.0878 341.0891 -3.9

3 α-2-CG C15H18O9 341.0878 341.0889 -1.1

4 β-2-CG C15H18O9 341.0878 341.0884 -1.8

5 α-3-CG C15H18O9 341.0878 341.0893 -4.5

6 β-3-CG C15H18O9 341.0878 341.0876 0.6

7 α-4-CG C15H18O9 341.0878 341.0869 2.7

8 β-4-CG C15H18O9 341.0878 341.0880 -0.2

9 α-6-CG C15H18O9 341.0878 341.0878 0.1

10 β-6-CG C15H18O9 341.0878 341.0889 0.7

172

Appendix 2

Appendix 2: Supporting Information for Chapter 5

Table A2.1: High resolution and tandem MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of strawberry methanolic extract

HR m/z Ret. Peak Error MS2(341) MS3 MS 4 Assignment value of Time no. (ppm) m/z(%) m/z(%) m/z (%) [M-H]- (min) 203(8), 179(100), 1 α-1-CG 341.0896 -5.1 25.3 135(100) 161(32), 135(8) 281(5), 251(5), 203(8),

2 C15H18O9 341.0880 -0.5 26.8 179(100), 161(25), 135(100) 135(9) 323(12), 281(100), 221(29), 179(100), 3 β-6-CG 341.0876 0.5 29.5 251(71), 221(31), 135(100) 135(15) 179(93), 135(11) 323(8), 281(100), 221(43), 179(100), 4 α-6-CG 341.0881 -0.9 35.4 251(56), 221(24), 135(100) 135(16) 179(41) Caffeic acid 5 341.0872 1.9 37.2 179(100), 135(15) 135(100) 3-O-β-glucose Caffeic acid 6 341.0896 -5.1 39.9 179(100), 135(16) 135(100) 4-O-β-glucose

C15H18O9 323(23), 281(16), 177(100), 159(21), 159(19), 7 (gluconic acid- 341.0896 -5.1 41.1 195(100), 177(48), 129(80), 111(10), 149(10), hexose) 163(12) 99(12) 129(100)

173

Intens. x106

0 0 10 20 30 40 50 60 70 80 90 Time [min] Strawberry Romania total extract alternating MS3.D: EIC 325.0 -All MS

Appendix 2

Intens.

174 -MS, 21.1min #1004 324.9 x106

144.8162.8 470.9 05 x10 162.8 -MS2(324.9), 21.1min #1005 6 144.9 4 186.8 2 119.1 204.8 234.8 264.8 0 x104 -MS3(325.1->162.8), 21.2min #1006 118.9 2 0 x105 -MS, 27.8min #1346 217.8 324.9 261.9 577.0595.0 0.5 461.0 173.8 286.8305.9 357.0 430.9 519.0 539.1 130.9 230.8 648.9 718.0 759.1 781.4 867.1 889.2 0.0 4 -MS2(324.9), 27.8min #1347 x10 162.3 1 118.9 100.9 322.7 0 -MS3(325.0->162.3), 27.8min #1349 1000 100.9 500 112.8 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A2.1: Ion trap extracted ion chromatogram at m/z 325 (negative ion mode) from Strawberry and MS2-3 fragmentation spectra of individual peaks.

Appendix 2

Figure A2.2: High- resolution negative ion mode EIC at m/z 325.0929.+/- 0.002 (blue), m/z 355.1035+/- 0.002 (red),and 385.1140.+/- 0.002 (green) from strawberry

175

Intens. x105 8

0 0 10 20 30 40 50 60 70 80 90 Time [min] Appendix 2 Strawberry Romania total extract alternating MS3.D: EIC 309.0 -All MS

176

Intens. 308.9 -MS, 17.1min #814 x105 262.6 206.8

174.9 362.9 464.1 575.9 0 x105 206.8 -MS2(308.9), 17.1min #815 262.9 100.9 160.8178.9 288.8 0 4000 160.8 -MS3(309.0->206.8), 17.1min #817 2000 113.0 142.9 178.8 0 x104 100.9 -MS2(262.6), 17.1min #816 130.9 243.8 160.8 217.8 0 6 -MS, 31.9min #1543 x10 308.9 354.9 146.8 206.8 261.8 408.9 466.0 521.1 567.0 665.1 0 x105 308.9 -MS2(354.9), 31.9min #1544 146.9 206.8 0 x105 146.8 -MS2(308.9), 31.9min #1545 288.8 0 102.9 -MS3(309.0->146.8), 31.9min #1547

0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.3: Extracted ion chromatogram at m/z 309 (negative ion mode) from Strawberry and MS2-3 fragmentation spectra of individual peaks.

Intens. x105 8

0 0 10 20 30 40 50 60 70 80 90 Time [min] Strawberry Romania total extract alternating MS3.D: EIC 385.0 -All MS

Appendix 2

Intens. -MS, 50.1min #2421 385.0 x105 260.9 2 217.8 493.1 130.9 173.9 304.9 331.9 356.9 411.0 453.0 543.0 177 0 5 -MS2(385.0), 50.1min #2422 x10 222.9 2 264.9 0 x104 -MS3(385.2->222.9), 50.1min #2424 178.9 2 124.9 0 5 -MS, 57.4min #2768 x10 385.0 4 217.8 2 261.9 130.9 174.8 304.9 331.9 357.9 426.9445.1 499.0 0 5 -MS2(385.0), 57.4min #2769 x10 246.8 1 222.9 276.9 324.9 0 6000 -MS3(385.2->246.9), 57.5min #2771 228.8 4000 148.8 174.7 2000 130.8 160.8 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.4: Ion trap negative ion mode extracted ion chromatogram at m/z 385 from Strawberry and MS2-3 fragmentation spectra of individual peaks

Appendix 2

Table A2.2: High resolution and tandem MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of raspberry

HR Ret. Peak m/z value Error MS2(341) MS 3 MS 4 Assignment time no. of [M- (ppm) m/z(% intensity ) m/z(%) m/z (%) (min) H]- 147(100), 1 α-2-CG 341.0866 3.6 21.5 323(13), 203(100), 179(14) 175(100) 131(16), 119(74) 323(16), 233(13), 195(39), 2 C15H18O9 341.0865 3.7 22.8 119(100) 179(14), 163(100) 323(6), 293(12), 281(13),

3 C15H18O9 341.0875 0.5 24.1 251(48), 233(77), 203(27), 135(100) 179(100), 161(9), 135(16) 203(9), 179(100), 161(34), 4 α-1-CG 341.0868 3.0 25 135(100) 135(11) 317(5), 251(5), 203(6), 5 C15H18O9 341.0866 3.4 26.3 135(100) 179(100), 161(29), 135(13) 323(10), 281(82), 251(74), 161(15), 6 C15H18O9 341.0876 0.5 29 233(22), 221(21), 179(100), 135(100) 135(27)

7 C15H18O9 341.0881 -0.9 32.9 233(11), 179(100), 135(17) 135(100)

323(8), 281(100), 251(44), 221(34), 8 α-6-CG 341.0871 2.1 34.9 221(20), 203(9), 179(47), 179(100), 135(100) 135(11) 161(5), 135(7)

Caffeic acid 9 341.0870 2.3 36.7 179(100), 135(11) 135(100) 3-O-β- glucose 221(14), 281(100), 253(4), 233(5), 161(16), 10 C15H18O9 341.0868 3.0 38.8 179(10) 119(100), 113(31) Caffeic acid 11 341.0864 4.1 39.6 179(100), 135(19) 135(100) 4-O-β- glucose

178

Appendix 2

Table A2.3: High resolution (HR) and tandem MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of blueberry

HR m/z Ret. Peak Error MS2(341) MS3 Assignment value of time no. (ppm) m/z(%) m/z(%) [M-H]- (min) 313(95), 281(31), 251(59), 223(21), 1 C19H17O6 341.1033 -0.7 15.9 125(100) 197(100), 125(53)

2 C22H13O4 18.1 323(39), 239(7), 153(100), 123(23) 123(100) 3 α-1-CG 341.0875 0.8 24.9 203(10), 179(100), 161(35), 135(9) 135(100)

4 C15H18O9 341.0873 1.4 28.6 179(100), 135(8) 135(100) Caffeic acid 5 341.0882 -1.3 36.4 179(100), 135(18) 135(100) 3-O-β-glucose Caffeic acid 6 341.0878 0.0 39.5 179(100), 135(14) 135(100) 3-O-β-glucose

Figure A2.5. MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), m/z 355.1035+/- 0.002 (green), and 385.1140.+/- 0.002 (blue) from blueberry

179

Appendix 2

Intens. Blueberry Germany total extract alternating MS4.D: EIC 325.0 -All MS x106 a

1.0

0.8

0.6

0.4

0.2

0.0 Intens. Blueberry Germany total extract alternating MS4.D: EIC 355.0 -All MS x106 b 1.5

1.0

0.5

0.0 10 20 30 40 50 60 Time [min]

Figure A2.6: Ion trap negative ion mode extracted ion chromatograms at: a) m/z 325; b) m/z 355 from blueberry

Table S4: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of blackberry

Peak HR m/z value of Error Ret. Time MS2(341) MS 3 Assignment no. [M-H]- (ppm) (min) m/z(%) m/z(%)

1 C15H18O9 341.0861 4.9 23.2 179(100), 135(15) 135(100)

2 C15H18O9 341.0880 -0.5 24.4 179(100), 135(15) 135(100)

3 C15H18O9 341.0886 -2.3 26.9 179(100), 135(15) 135(100)

4 C15H18O9 341.0879 -0.1 34.4 179(100), 135(15) 135(100)

5 C15H18O9 341.0865 3.8 37.6 179(100), 135(15) 135(100)

180

Appendix 2

Figure A 2.7: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), m/z 355.1035+/- 0.002 (green), and 385.1140.+/- 0.002 (blue) from blackberry

181

Appendix 2

Table S5: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of redcurrant

HR Ret. Peak m/z value Error MS2(341) MS3 MS4 Assignment Time no. of (ppm) m/z(%) m/z(%) m/z(%) (min) [M-H]- 287(5), 261(14), 201(75),

1 C15H18O9 341.0864 4.1 17.7 305(100) 219(100), 177(100), 179(76) 123(46)

2 C15H18O9 341.0888 -2.8 20.9 179(100), 135(19) 135(100) 203(8), 179(100), 3 C15H18O9 341.0858 5.9 24.9 135(100) 161(35), 135(9) 203(7), 179(100), 4 α-1-CG 341.0855 6.9 26.3 135(100) 161(31), 135(8)

5 C15H18O9 341.0863 4.4 28.7 179(100), 135(11) 135(100)

C15H18O9 323(7), 195(82), 6 (gluconic acid 341.0865 3.9 29.8 179(10), 163(100), 119(100) hexose conjugate) 119(11)

C15H18O9 323(9), 195(100), 177(8), 159(60), 85(23), 7 (gluconic acid 341.0864 4.2 30.7 181(44), 163(82), 129(100), 57(100) hexose conjugate) 119(11) 111(13)

C15H18O9 323(5), 293(23), 177(10), 159(78), 85(100), 8 (gluconic acid 341.0860 5.2 31.6 195(100), 179(12), 129(100), 57(23) hexose conjugate) 163(72), 129(13) 111(12) 294(24), 188(14),

9 C15H18O9 341.0875 0.8 32.8 179(100), 161(12), 135(100) 135(19) 188(100), 294(100), 188(59), 10 C15H18O9 341.0868 2.9 33.8 161(29), 161(39) 188(161) 135(100), 11 C15H18O9 341.0883 -1.5 36.6 179(100), 135(9) 121(11)

12 C15H18O9 341.0872 1.8 39.6 179(100), 135(15) 135(100)

182

Appendix 2

Figure A 2.9. MicrOTOF negative ion mode extracted ion chromatogram at m/z 325.0929.+/- 0.002 (brown), m/z 355.1035+/- 0.002 (green), and 385.1140.+/- 0.002 (blue) from red currant

183

Intens. Red currant Mohamed's garden total extract alternating MS3.D: EIC 325.0 -All MS x106

1.5

1.0

0.5

0.0 Appendix 2 10 20 30 40 50 60 Time [min]

184 Intens. 162.9 371.0 -MS, 16.5min #781 x105 0 x105 162.8 -MS2(371.0), 16.5min #782

0 x104 118.9 -MS2(162.9), 16.6min #783

0 5 -MS, 18.8min #886 x10 162.8 325.0 0 x105 162.8 -MS2(325.0), 18.8min #887

0 x104 119.0 -MS2(162.8), 18.8min #888

0 x105 325.0341.0 -MS, 20.8min #983

0 x104 144.9 -MS2(325.0), 20.9min #985 2 0 4000 -MS3(341.1->178.9), 20.9min #986 2000 0 400 -MS3(325.1->144.9), 20.9min #987 200 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.10: Ion trap negative ion mode extracted ion chromatogram at m/z 325 and MSn fragmentation spectra from redcurrant

Intens. Red currant Mohamed's garden total extract alternating MS3.D: EIC 385.0 -All MS x105

2.0

1.5

1.0

0.5

0.0 10 20 30 40 50 60 Time [min]

Appendix 2 Intens. -MS, 22.3min #1053 x105 385.0 431.1 261.9 597.0 218.8 465.0 579.0 129.9 174.8 286.9304.9 336.0 357.9 401.0 487.0 530.9 552.5 725.0 755.1773.1 185 0 x104 -MS2(385.0), 22.3min #1054 222.8 2

204.8 246.8 163.9 189.8 264.8 324.9 366.9 0 4000 -MS3(385.3->222.9), 22.4min #1056 163.8 2000 148.7 178.8 207.8 0 x105 -MS, 23.1min #1092 431.1 597.1 579.0 1 217.8 261.8 611.1 173.8 287.9305.9 331.9 357.9 400.9 465.0 647.1 743.1 773.1 0 x105 -MS2(431.1), 23.1min #1093 385.0 0.5 204.9222.8 0.0 6000 -MS3(431.2->385.0), 23.1min #1095 152.9 204.8 4000 222.8 2000 112.9 137.9 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.11: Ion trap negative ion mode extracted ion chromatogram at m/z 385 and MSn fragmentation spectra from redcurrant

Appendix 2

Figure A 2.12. MicrOTOF negative ion mode EIC at m/z 355.1035+/- 0.002 (green), m/z 325.0929.+/- 0.002 (orange) and 385.1140.+/- 0.002 (blue) from blackcurrant

186

Intens. Black currant direct juice alternating MS4.D: EIC 325.0 -All MS x105

0 Appendix 2 10 20 30 40 50 60 Time [min]

187 Intens. 162.8 -MS2(370.9), 17.7min #853 x105 0 2000 -MS3(325.1->162.8), 17.8min #856

0 x105 162.8 -MS2(324.9), 20.0min #958

0 x104 -MS3(325.1->162.9), 20.0min #960

0 x105 144.9 -MS2(324.9), 22.3min #1068

0 -MS3(325.1->144.9), 22.3min #1070 100 0 x104 264.8 -MS2(324.9), 24.5min #1176

0 -MS3(324.8->264.9), 24.6min #1178 2000 0 x104 234.8 -MS2(324.9), 27.0min #1292 2 0 x104 -MS3(325.0->234.8), 27.0min #1294

0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.13: Negative ion mode extracted ion chromatogram at m/z 325 and MSn fragmentation spectra from blackcurrant

Appendix 2

Figure A 2.14. MicrOTOF negative ion mode EIC at m/z 325.0929+/- 0.002 (brown), m/z 355.1035.+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from gooseberry

188

Appendix 2

Table A2.6: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of gooseberry

HR m/z Ret. Peak Error MS2(341) MS3 MS 4 Assignment value of time no. (ppm) m/z(%) m/z(%) m/z(%) [M-H]- (min) 323(100), 233(24), 203(24), 233(100), 221(22), 1 β-3-CG 341.0894 -4.8 15.6 179(32) 203(14), 189(38) 157(18), 147(100), 2 β-2-CG 341.0885 -2.2 17.7 323(11), 203(100), 175(100) 129(26) 305(15), 233(100), 323(100),305(59), 233(35), 3 α-3-CG 341.0874 1.2 18.3 203(10), 189(23), 203(72), 179(64) 179(10)

4 C15H18O9 341.0898 -5.8 19.2 323(11), 203(100) 175(100)

5 C15H18O9 341.0897 -5.6 21.1 203(10), 179(100), 135(11) 135(100) 157(14), 147(100), 6 α-2-CG 341.0879 -0.2 21.7 323(9), 203(100), 175(100) 131(18), 119(25) 323(94), 233(39), 203(100), 7 β-4-CG 341.0873 1.5 23 175(100) 179(41), 161(4), 135(9) 323(19), 281(26), 251(23), 8 α-4-CG 341.0870 2.3 24.1 233(37), 203(100), 179(77), 175(100) 161(14), 135(15) 203(10), 179(100), 161(33), 9 α-1-CG 341.0874 1.2 25.4 135(100) 135(10)

10 C15H18O9 341.0876 0.4 26.6 203(6), 179(100), 161(29), 135(9) 135(100)

323(11), 281(100), 251(64), 221(30), 179(100), 11 β-6-CG 341.0877 0.3 29.5 135(100) 221(20), 179(68), 135(5) 135(12)

323(8), 281(6), 251(5), 195(64), 12 C15H18O9 341.0876 0.4 30.3 119(100) 179(10), 163(100), 119(11)

323(5), 195(100), 177(10), 177(19), 159(100), 13 C15H18O9 341.0882 -1.0 32 129(100), 111(41) 163(55), 119(7) 129(86)

14 C15H18O9 341.0882 -1.0 33.3 179(100), 135(15) 135(100) 135(5), 121(100), 15 C15H8O9 341.0864 4.2 34.8 281(6), 251(7), 179(100), 109(20) 323(14), 281(100), 251(68), 221(36), 179(100), 16 α-6-CG 341.0874 1.0 35.5 135(100) 221(28), 179(37), 135(13) 323(10), 281(11), 251(5), 177(22), 159(50), 17 C15H18O9 341.0873 1.5 36.4 57(100) 195(100), 163(50) 129(100), 111(22) Caffeic acid-3- 18 341.0871 2.1 37.3 179(100), 135(8) 135(100), 121(41) O-β-glucose Caffeic acid-4- 19 341.0897 -5.4 40 179(100), 135(10) 135(100) O-β-glucose 323(34), 195(100), 177(32), 177(20), 159(100), 141(12), 129(100), 20 C15H18O9 341.0876 0.4 40.3 163(21) 129(57), 111(10)

189

Intens. Gooseberry Mohamed's garden total extract alternating MS4.D: EIC 325.0 -All MS x105

0 Appendix 2 10 20 30 40 50 60 Time [min]

190 Intens. 371.0 467.0 -MS, 18.0min #852 x105 162.9 261.9 613.0 131.0 217.8 283.1 304.9 325.0 403.1 521.0 576.5 651.1 694.4 897.0 0 x104 162.8 -MS2(371.0), 18.0min #853 2 118.9 208.8 324.9 0 -MS3(371.1->162.9), 18.0min #855 2000 118.9 0 x105 324.9 -MS, 20.3min #960 162.8 577.0 217.8 261.8 288.9 350.9 393.0 438.9 464.9 486.9505.0 609.0 651.1 0 x105 162.8 -MS2(324.9), 20.3min #961 119.0 0 x104 119.0 -MS2(162.8), 20.3min #962

0 x105 324.9 465.0 -MS, 22.5min #1064 447.0 130.9 178.8 218.8 261.9 305.9 367.0 478.9 509.0 551.1 578.9 609.0 04 x10 144.9162.8 -MS2(324.9), 22.5min #1065 186.8 2 119.0 204.8 234.8 264.9 0 6000 118.9 -MS3(325.1->162.9), 22.5min #1067 4000 2000 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.15: Ion trap negative ion mode extracted ion chromatogram at m/z 325 and MSn fragmentation spectra from gooseberry

Intens. Gooseberry Mohamed's garden total extract alternating MS4.D: EIC 355.0 -All MS x105 8

0 Appendix 2 10 20 30 40 50 60 Time [min]

191 Intens. 192.8 -MS, 21.3min #1007 x105 354.9

401.0 217.9 261.8 130.9 174.8 305.9 331.9 381.0 423.0 446.9465.0 500.9 535.0 557.1 592.9 625.0 653.0 711.0 04 x10 192.8 -MS2(354.9), 21.3min #1008 4 2 133.9 04 x10 148.8 -MS2(192.8), 21.3min #1009 2 177.8 133.9 0 -MS3(192.9->147.8), 21.4min #1011 2000 133.8 1000 100.8 0 x105 -MS, 23.2min #1104 355.0 465.0 4 447.0 2 192.8 217.8 261.8 500.9 525.0 577.0 611.0 05 x10 192.8 -MS2(355.0), 23.3min #1105 1 174.9 216.8 133.9 234.8 294.9 0 x104 -MS3(355.1->192.8), 23.3min #1107 133.9 2 148.9 177.7 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A 2.16: Negative ion mode extracted ion chromatogram at m/z 355 and MSn fragmentation spectra from gooseberry

Appendix 2

Table A2.7: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of lingonberry

HR m/z Ret. Peak Error MS2(341) MS3 MS 4 Assignment value of time no. (ppm) m/z(%) m/z(%) m/z (%) [M-H]- (min) 323(11), 203(100), 1 β-2-CG 341.0894 -4.8 17.9 175(100) 133(100) 179(19) 2 α-2-CG 341.0884 -1.6 21.9 323(14), 203(100) 175(100) 133(100) 3 α-1-CG 341.0897 -5.5 25.5 179(100), 161(24) 135(100)

4 C15H17O9 341.0887 -2.2 29.3 179(100), 135(14) 135(100) 323(10), 281(100), 221(30), 179(100), 5 β-6-CG 341.0890 -3.6 29.6 251(15), 221(15), 135 135(12) 179(40), 135(5)

281(100), 251(53), 221(25), 179(100), 6 α-6-CG 341.0887 -2.7 35.7 135 221(15), 179(55), 135(5) 135(10)

Caffeic acid-3- 7 341.0890 -3.6 37.4 179(100), 135(14) 135(100) O-β-D-glucose

Figure A2.17: MicrOTOF negative ion mode EIC at m/z m/z 325.0929.+/- 0.002 (black), 355.1035+/- 0.002 (red) and 385.1140.+/- 0.002 (green) from lingonberry

192

Intens. Lingon Berry total extract alternatig MS3.D: EIC 325.0 -All MS x106 1.00 0.75 a 0.50 0.25 0.00 Intens. Lingon Berry total extract alternatig MS3.D: EIC 355.0 -All MS x106 1.5 b 1.0

0.5

0.0 Intens. Lingon Berry total extract alternatig MS3.D: EIC 385.0 -All MS x105

4 c Appendix 2

193 10 20 30 40 50 60 Time [min]

Intens. 192.8 355.0 -MS, 30.2min #1479 x106 0 x105 192.8 -MS2(355.0), 30.2min #1480 2 0 6000 160.8 -MS2(192.8), 30.3min #1481 4000 2000 0 x105 354.9 575.0 -MS, 31.9min #1558 2 0 x105 308.9 -MS2(354.9), 31.9min #1559 0 x104 -MS3(355.2->308.9), 31.9min #1561 2 0 x105 192.8 -MS2(355.0), 33.1min #1619 0 4000 -MS3(355.2->192.8), 33.2min #1621 2000 06 x10 431.1 465.0 -MS, 23.2min #1134 0 x105 385.0 -MS2(431.1), 23.2min #1135 2 0 x104 -MS3(431.4->385.1), 23.2min #1137 0 100 200 300 400 500 600 700 800 900 1000 m/z Figure A2.18: Negative ion mode extracted ion chromatogram at m/z 355 and MSn fragmentation spectra from lingonberry

Appendix 2

Table A2.8: High resolution and MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of aronia juice

HR Ret. Peak Error MS2(341) MS3 MS4 Assignment m/z value time no. (ppm) m/z (% ) m/z (%) m/z (%) of [M-H]- (min) 147(80), 1 β-2-CG 341.0884 -1.8 17.9 323(19), 203(100) 175(100) 119(72)

2 C15H18O9 341.0875 0.5 18.7 281(18), 251(100) 179(100) 135(100) 281(21), 251(100), 233(42), 175(100), 3 C15H17O9 341.0871 2.2 24.6 203(100) 203(20), 179(65) 161(74) 4 α-1-CG 341.0863 4.4 25.7 179(100) 135(100) 323(22), 281(100), 251(79), 221(30), 5 β-6-CG 341.0876 0.5 29.8 135(100) 221(18), 179(79) 179(100) 187(100), 6 C15H17O9 341.0888 -2.9 33.2 294(100) 161(100) 161(67) 323(10), 281(100), 251(69), 251(42), 7 α-6-CG 341.0877 0.4 35.5 135(100) 221(21), 179(79) 179(100) Caffeic acid 8 341.0873 1.5 37.4 179(100) 135(100) 3-O-β-glucose 137(100), 9 C15H17O9 341.0869 2.6 43.1 323(100) 165(100) 121(27)

194

Appendix 2

Figure A2.19: MicrOTOF negative ion mode EIC at m/z m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from aronia juice

195

Intens. Aronia crude alternating MS3.D: EIC 325.0 -All MS x106 4 a

0 Intens. Aronia crude alternating MS3.D: EIC 385.0 -All MS x106 b 0.8

0.6

0.4

Appendix 2 0.2

0.0 10 20 30 40 50 60 Time [min]

196

Intens. 325.0 -MS, 20.1min #1111 6 x10 651.4 119.0 162.9 361.0 393.1 465.1 515.2 673.3 0 x106 162.9 -MS2(325.0), 20.1min #1112 119.1 05 x10 119.0 -MS3(325.2->162.9), 20.2min #1114

0 x107 431.2 -MS, 24.4min #1381 421.8 353.0 0 x106 385.1 -MS2(431.2), 24.4min #1382 2 160.9 205.0223.0 05 x10 152.9 -MS3(431.4->385.1), 24.4min #1384 2 204.9222.9 113.1 138.0 187.0 0 x105 385.1 435.1 -MS, 27.1min #1541 4 353.1 409.1 461.3 2 113.0 143.8 180.9 217.8 245.9 280.9 325.0 520.2 565.4 616.3 641.8 705.2724.2 751.3 797.5 833.4 859.8 904.2 944.2 997.2 0 x105 266.9 -MS2(385.1), 27.1min #1542 2 248.9 04 x10 113.0 -MS3(385.2->266.9), 27.1min #1544 248.9 130.9 156.9174.8 206.9 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.20: Ion trap negative ion mode extracted ion chromatogram at: a) m/z 355 and b) m/z 385 and MSn fragmentation spectra from aronia juice

Appendix 2

Table A.2.9: High resolution and MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of elderberry juice

High resolution Retention Peak Error MS2(341) MS 3 MS 4 Assignment m/z value time no. (ppm) m/z(% intensity ) m/z(% intensity) m/z(% intensity ) of (min) [M-H]- α-3- 305(100), 1 341.0896 -5.1 18.4 323(100),203(10) 197(100) Caffeoylglucose 261(53), 239(94) 195(48), 2 C15H17O9 341.0879 -0.3 20.5 119(100) 163(100) 195(100), 177(19), 3 C15H17O9 341.0892 -4.1 21.8 163(43) 159(100), 129(62) 299(54), 281(43), 203(100), 4 C15H17O9 341.0889 -3.1 24.6 251(100), 203(100) 175(44), 161(18) 233(42), 179(67)

5 C15H17O9 341.0898 -5.8 29.4 179(100) 135(100) 195(85), 6 C15H17O9 341.0865 3.7 30.5 119(100) 163(100) 195(100), 177(27), 159(92), 7 C15H17O9 341.0873 1.4 32.5 129(100) 163(72) 129(100)

8 C15H17O9 341.0893 -4.5 33.4 179(100) 135(100) 323(33), α-6- 9 341.0892 -4 35.6 281(100), 221(20), 179(100) 135(100) Caffeoylglucose 251(96), 179(33) Caffeic acid 10 341.0894 -4.7 37.3 179(100) 135(100) 3-O-β-glucose 323(36), 177(25), 159(92), 11 C15H17O9 341.0897 -5.7 40.3 195(100), 129(100) 129(100) 163(22) 323(43), 177(16), 12 C15H17O9 341.0896 -5.2 41.7 195(100), 129(100) 159(100), 129(86) 163(22)

13 C15H17O9 341.0893 -4.5 42.7 193(100) 149(100) 134(100)

197

Appendix 2

Figure A.2.21: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from elder berry

198

Intens. Elderberry crude alternating MS3.D: EIC 325.0 -All MS x107 1.00 a 0.75 0.50 0.25 0.00 Intens. Elderberry crude alternating MS3.D: EIC 355.0 -All MS x106 b 2

0 Intens. Elderberry crude alternating MS3.D: EIC 385.0 -All MS x106 c 1.0

0.5 Appendix 2

0.0 10 20 30 40 50 60 Time [min]

199 Intens. -MS, 20.0min #1163 x106 325.0 651.3 357.0 2 162.9 403.1 439.1 465.1 505.2 537.3 673.4 701.4 0 x106 -MS2(325.0), 20.0min #1164 162.8 2 119.0 0 x105 -MS3(325.1->162.9), 20.1min #1166 1 119.0

0 x105 -MS, 30.0min #1793 385.1 6 481.2 4 461.3 507.3 543.3 367.1 433.3 525.3 2 180.8 218.7 261.9 290.0 323.0 575.3 613.4631.6 652.5 711.6 736.4 781.3 805.3 868.5 894.4 988.4 0 x105 -MS2(385.1), 30.0min #1794 262.9 2 121.0 230.9 0 -MS3(385.4->262.9), 30.1min #1796 4000 113.0 2000 142.8 168.9 230.8 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.22: Negative ion mode extracted ion chromatogram at: a) m/z 325 and b) m/z 355 and c) m/z 385 and MSn fragmentation spectra from elderberry juice

Appendix 2

Table A.2.10: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of cranberry juice

High Retention Peak resolution Error MS2(341) MS 3 Assignment time no. m/z value (ppm) m/z(% intensity ) m/z(% intensity) (min) of [M-H]-

1 C15H17O9 341.0873 1.3 18.1 323(52), 153(100) 123(100)

2 C15H17O9 341.0866 3.6 20.9 179(100) 135(100)

3 C15H17O9 341.0861 5.0 25 179(100) 135(100)

4 C15H17O9 341.0860 5.2 26.4 179(100) 135(100)

5 C15H17O9 341.0863 4.3 28.6 179(100) 135(100)

6 C15H17O9 341.0866 3.6 32.8 179(100) 135(100) 7 Caffeic acid 3-O-β-glucose 341.0868 2.9 36.5 179(100) 135(100) 8 Caffeic acid 4-O-β-glucose 341.0865 3.9 39.6 179(100) 135(100)

Intens. Cranberry juice 20pc MeOH fraction final method targeted.D: EIC 325.0 -All MS x107 a 1.5

1.0

0.5

0.0 Intens. Cranberry juice 20pc MeOH fraction final method targeted.D: EIC 355.0 -All MS x106

2.0 b

1.5

1.0

0.5

0.0 Intens. Cranberry juice 20pc MeOH fraction final method targeted.D: EIC 385.0 -All MS x107 c 1.0

0.8

0.6

0.4

0.2

0.0 10 20 30 40 50 60 Time [min]

Figure A.2.23: Ion trap negative ion mode extracted ion chromatogram at: a) m/z 325 and b) m/z 355 and c) m/z 385 from cranberry juice

200

Appendix 2

Figure A.2.24: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from açai berry juice

201

Intens. Acai total alternating MS3.D: EIC 355.0 -All MS x105

0 10 20 30 40 50 60 Time [min] Appendix 2

202 Intens. 331.0 355.0 -MS, 11.6min #583 x105

120.9 160.8 180.8 214.9 255.1 288.9 371.0 401.1 449.1 481.2 509.2 548.3 569.2 593.4 657.3 765.8 800.4 883.2

0 x104 248.9 -MS2(355.0), 11.7min #584 2 160.9 190.9208.8 0 -MS3(355.1->248.9), 11.7min #586 200 100.9 0 x106 463.1 -MS, 17.1min #853 355.1 180.9 218.9 260.9 337.0 385.1 513.1 577.3 597.3 0 x104 191.4 -MS2(355.1), 17.1min #855 2 172.9 336.9 0 600 172.8 -MS3(355.1->190.9), 17.2min #857 400 109.1 146.8 200 0 x106 425.1 -MS, 19.1min #953 355.0 210.9 321.0 385.0 541.4 611.3 0 x104 190.8 -MS2(355.0), 19.1min #955 4 2 172.9 208.8 337.0 0 -MS3(355.1->190.9), 19.2min #957 400 128.8 170.7 200 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.25: Negative ion mode extracted ion chromatogram at m/z 355 and from açai berry juice

Intens. Acai total alternating MS3.D: EIC 385.0 -All MS x106

1.25

1.00

0.75

0.50

0.25

0.00 Appendix 2 10 20 30 40 50 60 Time [min]

203 Intens. 385.1 -MS, 13.3min #663 x105 419.1 160.8 202.9 262.9 290.8 339.0 439.2 461.2 481.2 517.1 578.2 617.5 637.4 671.2 0 x105 190.8 -MS2(385.1), 13.3min #664 129.0 172.8 264.9 294.9 355.0 0 2000 -MS3(385.1->190.9), 13.3min #666 128.9 172.7 111.1 146.9 0 5 -MS, 15.3min #763 x10 385.0391.1 2 101.0 130.9 160.8 180.9 216.8 255.0 283.1 304.9 343.0 423.1 445.1 474.2493.2511.1 547.1 589.2 609.4 680.9 701.4 768.4 861.8 933.9 0 x104 190.8 -MS2(385.0), 15.3min #765 2 208.8 366.9 04 x10 123.0 -MS3(391.4->194.8), 15.3min #766 134.8 164.9 0 x105 373.1385.0 -MS, 16.3min #813 513.3 115.0133.0 160.8 180.9 202.9 240.9 261.8 355.0 411.2 445.2 473.1 541.2 571.3 607.3 643.3 681.4 730.4 772.7 795.3 843.3 889.4 911.3 949.3 0 x104 222.9 -MS2(385.0), 16.3min #815 2 129.0 204.9 366.9 04 x10 204.8 -MS3(385.1->222.9), 16.4min #817 110.9 186.8 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.26: Negative ion mode extracted ion chromatogram at m/z 385 and from açai berry juice

Appendix 2

Table A.211: High resolution and MS2-3 data from extracted ion chromatogram at m/z 341 (negative ion mode) of goji berry juice

High resolution Retention MS2(341) MS 3 MS 4 Peak Error Assignment m/z value time m/z m/z m/z no. (ppm) of [M- (min) (% intensity ) (% intensity) (% intensity ) H]- 323(100), 233(100), 233(24), 221(22), 1 β-3-CG 341.0886 -2.2 15.6 203(24), 203(14), 179(32), 189(38) 157(18), 323(11), 2 β-2-CG 341.0891 -3.9 17.7 175(100) 147(100), 203(100) 129(26) 157(14), 323(9), 147(100), 3 α-2-CG 341.0885 -2.2 21.7 175(100) 203(100) 131(18), 119(25) 203(10), 179(100), 4 α-1-CG 341.0897 -5.4 25.4 135(100) 161(33), 135(10)

5 C15H18O9 341.0898 -5.9 29.2 179(100) 135(100)

6 C15H18O9 341.0887 -2.5 33.3 179(100) 135(100) 323(14), 281(100), 221(36), 7 α-6-CG 341.0896 -5.1 35.6 251(68), 179(100), 135(100) 221(28), 135(13) 179(37), Caffeic acid 8 341.0889 -3.9 37.2 179(100) 135(100) 3-O-β-glucose Caffeic acid 9 341.0898 -5.9 40 179(100) 135(100) 4-O-β-glucose

204

Appendix 2

Figure A 2.27: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from goji berry juice

205

Intens. Goji berry alternating MS3.D: EIC 325.0 -All MS x106

0 Appendix 2 10 20 30 40 50 60 Time [min]

206 Intens. 325.0 -MS2(533.3), 11.5min #657 x106 0 x106 -MS3(533.6->325.0), 11.5min #659

0 x105 325.0 -MS2(523.2), 13.8min #807 4 2 0 x105 -MS3(523.7->325.0), 13.9min #809

0 x105 324.9 -MS2(651.4), 17.8min #1059 2 0 x105 -MS3(651.7->325.0), 17.8min #1061

0 x106 162.9 -MS2(324.9), 20.0min #1205

0 x105 -MS3(325.0->162.9), 20.0min #1207

0 x105 144.9 -MS2(325.0), 22.3min #1359 4 2 0 2000 -MS3(325.2->144.9), 22.3min #1361 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.28: Negative ion mode extracted ion chromatogram at m/z 325 and MS2-3 from goji berry juice

Intens. Goji berry alternating MS3.D: EIC 385.0 -All MS x106

2.0

1.5

1.0

0.5

0.0 Appendix 2 10 20 30 40 50 60 Time [min]

207 Intens. -MS, 25.9min #1588 x106 385.1 613.3

547.4 178.9 222.8 286.9 355.1 411.2 445.1 467.2 515.3 583.3 645.3 676.3 703.3 753.4 805.4 909.5 0 x106 -MS2(385.1), 25.9min #1590 222.9 0.5 164.0 0.0 x104 -MS3(385.3->222.8), 26.0min #1592 207.8 2 163.9 0 x105 -MS, 31.1min #1888 2 385.1 486.3 162.9 261.9 419.1 515.3 119.1 344.1 437.1 461.2 541.3 577.3 216.8 289.0 612.3 637.4 669.4 689.4 719.4 753.4 792.8 817.5 839.4 863.4882.7900.3918.8 947.3 983.3 0 x104 -MS2(385.1), 31.1min #1889 176.9 4 2 285.9 339.0 366.1 0 -MS3(385.2->176.9), 31.2min #1891 200 144.9

0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.29: Negative ion mode extracted ion chromatogram at m/z 385 and MS2-3 from goji berry juice

Appendix 2

Figure A.2.30: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from sea buckthorn berry juice

208

Intens. Buckthorn alternating MS3.D: EIC 325.0 -All MS x106

0 Appendix 2 10 20 30 40 50 60 Time [min]

209 Intens. -MS, 20.1min #1075 x106 325.0

2 651.3

361.0 162.9 288.9 393.1 465.1 491.2 673.3 0 x106 -MS2(325.0), 20.1min #1076 162.8 1 119.0 0 x105 -MS3(325.1->162.9), 20.1min #1078 1 119.0

0 x106 -MS, 22.5min #1202 325.0 1 371.1 162.8 190.8 353.0 393.1 457.2 479.1 515.2 609.3 0 x105 -MS2(325.0), 22.5min #1203 144.9 2 162.9 186.8 119.1 204.9 234.9 264.9 0 -MS3(325.2->144.9), 22.5min #1205 2000 117.0 1000 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.31: Negative ion mode extracted ion chromatogram at m/z 325 and MS2-3 from sea buckthorn berry juice

Intens. Buckthorn alternating MS3.D: EIC 355.0 -All MS x106

0.8

0.6

0.4

0.2

Appendix 2

0.0 10 20 30 40 50 60 Time [min]

210

Intens. -MS, 23.2min #1242 x105 447.1 4 3 355.0 2 401.1 529.1 551.2 1 511.0 623.6 648.2 732.2 114.9132.9 157.8 184.8 214.9 259.9 308.9 331.2 484.9 585.5 672.4 784.3 804.6 840.2 931.6 0 x104 -MS2(355.0), 23.2min #1244

192.8 4 216.9 191.9 2 134.0 159.9 234.9 264.9 295.0 0 -MS3(355.0->192.3), 23.2min #1246

133.9 4000

2000 148.9 175.8 672.0 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.32: Negative ion mode extracted ion chromatogram at m/z 355 and MS2-3 from goji sea buckthorn berry juice

Intens. Buckthorn alternating MS3.D: EIC 385.0 -All MS x106

2.5

2.0

1.5

1.0

0.5

Appendix 2

0.0 10 20 30 40 50 60 Time [min]

211

Intens. 385.1 431.2 -MS, 23.6min #1262 x106 351.1 166.9 190.9 223.0 260.9 288.0 319.0 453.2 517.3 553.3 702.5 0 x105 222.9 -MS2(385.1), 23.6min #1263 204.9 246.9 163.9 266.9 294.9 325.0 04 x10 163.9 -MS3(385.3->223.0), 23.6min #1265 2 148.9 178.9 207.8 0 x105 385.1 449.1 -MS, 26.4min #1412 2 288.9 427.2 585.4 114.9 146.9 190.9 216.9 264.9 329.0 353.1 467.1485.3 529.3 605.4 634.6 669.2 727.3 771.3 795.0 889.8 981.7 0 x104 263.5 -MS2(385.1), 26.4min #1414 325.0 2 121.1 222.9 294.9 04 x10 220.9 -MS3(385.3->263.5), 26.4min #1416 113.1 139.0 168.9 06 x10 385.1 785.4 -MS, 30.2min #1632 218.8 261.9 288.9 333.0 353.0 418.2 461.2 487.2 517.2 541.3 623.4 669.4 755.3 823.3 0 x105 262.9 -MS2(385.1), 30.2min #1633 2 121.0 230.9 04 x10 113.0 -MS3(385.3->262.9), 30.2min #1635 142.9 168.9 188.8 230.9 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.33: Negative ion mode extracted ion chromatogram at m/z 385 and MS2-3 from goji sea buckthorn berry juice

Appendix 2

Table A.2.12: High resolution and MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of sour cherry juice

High resolutio Retention MS2(341) MS 3 Peak Error MS4 m/z Assignment n m/z time m/z m/z no. (ppm) (% intensity ) value of (min) (%intensity ) (% intensity) [M-H]- 323(11), 157(18), 147(100), 1 β-2-Caffeoylglucose 341.0876 0.7 17.9 175(100) 203(100) 129(26) 281(37), 207(9), 2 C15H17O9 341.0886 -2.3 18.7 135(100) 251(100) 179(100) 233(100), 189(100), 3 C15H17O9 341.0886 -2.3 24.2 174(100), 161(56) 179(86) 171(25), 251(33), 251(100), 4 C15H17O9 341.0897 -5.4 24.8 203(100) 175(100), 161(38) 233(81), 179(94) 5 α-1-Caffeoylglucose 341.0879 -0.2 25.7 179(100) 135(100) 281(100), 251(65), 221(34), 6 β-6-Caffeoylglucose 341.0890 -3.6 29.7 135(100) 221(23), 179(100) 179(49)

7 C15H17O9 341.0895 -5.0 33.6 179(100) 135(100) 281(28), 8 C15H17O9 341.0894 -4.8 34.4 179(100) 135(100) 251(100) 281(31), 9 C15H17O9 341.0894 -4.8 35.1 179(100) 135(100) 251(100) 281(100), 251(52), 221(26), 10 α-6-Caffeoylglucose 341.0884 -1.9 35.9 221(22), 179(100) 179(40) Caffeic acid 3-O-β- 11 341.0879 -0.2 37.5 179(100) 135(100) glucose Caffeic acid 4-O-β- 12 341.0897 -5.4 40 179(100) 135(100) glucose

212

Appendix 2

Figure A.2.34: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from Montmorency sour cherry juice

213

Intens. Cherry total alternating MS3.D: EIC 355.0 -All MS x106

Appendix 2

0 10 20 30 40 50 60 Time [min]

214

Intens. 355.0 499.2 -MS, 11.2min #624 x106 0 x105 172.9 -MS2(355.0), 11.2min #626

0 -MS3(355.1->172.9), 11.2min #628 1000 0 x106 477.2 -MS, 12.6min #704 355.0 0 x105 172.9 -MS2(355.0), 12.6min #706

0 1000 -MS3(355.0->172.9), 12.6min #708

06 x10 192.9 355.0 -MS, 33.7min #1988 0 x106 192.8 -MS2(355.0), 33.7min #1989

0 x105 148.9 -MS2(192.9), 33.7min #1990 2 0 6000 -MS3(193.0->148.9), 33.7min #1992 4000 2000 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.35: Negative ion mode extracted ion chromatogram at m/z 355 and MS2-3 from Montmorency sour cherry juice

Intens. Cherry total alternating MS3.D: EIC 385.0 -All MS x106

2.0

1.5

1.0

0.5

Appendix 2

0.0 10 20 30 40 50 60 Time [min]

215

Intens. -MS, 27.3min #1618 x106 461.2 385.1 0.5 585.5 115.0132.9 180.9 214.9 260.8 312.0 352.9 431.2 491.3 523.1 553.2 632.8 660.4 727.5 764.3 812.2 877.3 905.4 926.3 978.3 0.0 x105 -MS2(385.1), 27.3min #1620 266.9 2 248.9 0 x104 -MS3(385.2->266.9), 27.4min #1622 2 113.0 248.9 128.9 156.9174.9 0 x106 -MS, 34.9min #2058 385.0 1 533.3 433.1 577.3 115.0 160.7 180.9 216.8 273.9 323.0 361.1 487.2 611.5 631.3 674.5 714.4 755.4 0 x105 -MS2(385.0), 34.9min #2059 340.9 4 2 296.9 0 x105 -MS3(385.2->341.1), 34.9min #2061 1 296.9 216.9 280.9 176.9 322.9 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.36: Negative ion mode extracted ion chromatogram at m/z 385 and MS2-3 from Montmorency sour cherry juice

Appendix 2

Table A.2.13: High resolution and MS2-4 data from extracted ion chromatogram at m/z 341 (negative ion mode) of pomegranate juice

High Retention MS2(341) MS 3 MS 4 Peak resolution Error Assignment time m/z m/z m/z no. m/z value of (ppm) (min) (%intensity ) (% intensity) (% intensity ) [M-H]- 179(69), 161(100), 143(32), 125(15), 1 C15H18O9 341.0891 -3.8 17.8 143(57), 131(27), 85(100) 113(100), 101(25) 119(82), 113(50), 101(51) α-1- 2 341.0894 -4.7 25.6 179(100) 135(100) Caffeoylglucose 323(10), 281(100), 3 C15H18O9 341.0894 -4.7 29.8 251(45), 179(100) 135 251(69), 221(21), 179(79) 323(10), α-6- 281(100), 4 Caffeoyl- 341.0898 -5.7 35.8 251(42), 179(100) 135 251(69), 221(21), glucose 179(79)

5 C15H18O9 341.0885 -2.0 37.5 179(100) 135(100)

216

Appendix 2

Figure A.2.37: MicrOTOF negative ion mode EIC at m/z 325.0929.+/- 0.002 (brown), 355.1035+/- 0.002 (green) and 385.1140.+/- 0.002 (blue) from pomegranate juice

217

Intens. Pomegranate alternating MS3.D: EIC 325.0 -All MS x106

10 20 30 40 50 60 Time [min] Appendix 2

Intens.

218 -MS, 13.8min #774 x105 325.1 483.2 2 556.2 632.3 783.2 110.9 178.9 214.8 292.9 367.1 390.1 453.1 515.2 707.2 888.2 142.9 254.9 675.9 751.6 821.3 915.1 951.2 971.2 0 x104 -MS2(325.1), 13.8min #775 279.0 2 0 -MS3(325.3->279.0), 13.8min #777 4000 160.9 2000 112.9 220.6 0 x106 -MS, 22.4min #1243 325.0 2 340.9 427.2 453.1 0 x105 -MS2(325.0), 22.4min #1244 4 144.9162.9 186.8 2 119.1 204.8 234.9 264.9 0 -MS3(325.2->144.9), 22.4min #1246 400 200 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.38: Ion trap negative ion mode extracted ion chromatogram at m/z 385 and MS2-3 from pomegranate juice

Intens. Pomegranate alternating MS3.D: EIC 355.0 -All MS x106

0 10 20 30 40 50 60 Time [min] Appendix 2

219 Intens. -MS, 23.2min #1283 x106 355.1 1.0

0.5 427.2 387.1 160.8178.9 214.8 475.2 525.3 669.3 785.3 0.0 x105 -MS2(355.1), 23.2min #1284 192.9 2 216.8 1 174.9 133.9 159.9 234.9 264.9 295.0 353.0 0 x104 -MS3(355.1->192.8), 23.2min #1286

133.9 2

1 148.9 177.8 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.39: Ion trap negative ion mode extracted ion chromatogram at m/z 355 and MS2-3 from pomegranate juice

Intens. Pomegranate alternating MS3.D: EIC 385.0 -All MS x106

1.0

0.8

0.6

0.4

0.2

Appendix 2

0.0 10 20 30 40 50 60 Time [min]

220

Intens. 385.1 463.2 -MS, 23.5min #1303 x105 x105 222.9 -MS2(385.1), 23.5min #1304 0 x104 -MS3(385.5->222.0), 23.6min #1306 0 x106 421.2431.2 -MS, 24.5min #1353 0 x106 385.1 -MS2(431.2), 24.5min #1354 0 x105 -MS3(431.5->385.1), 24.6min #1356 0 x105 385.1 447.1 -MS, 42.3min #2293 0 x104 222.9 -MS2(385.1), 42.4min #2295 0 x104 -MS3(385.4->223.0), 42.4min #2297

06 x10 300.8 385.1 -MS, 43.3min #2343 0 x104 222.9 -MS2(385.1), 43.4min #2345 2 0 x104 -MS3(385.3->222.8), 43.4min #2347 0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure A.2.40: Ion trap negative ion mode extracted ion chromatogram at m/z 385 and MS2-3 from pomegranate juice

Appendix 2

Table A.2.14: Statistical data for the calibration curve points:

Relative Conc. Replicate Average Absolute Relative Area STDEV STDEV (µg/mL) number area error Error (%) (%) 1 479105 2 485758 0.5 501359.5 31289.61 15644.81 3.12 6.24 3 493067 4 547508 1 1099316 1 2 1217900 1143396 64882.63 37460.01 3.28 5.67 3 1112972 1 3673610 2.5 2 3547574 3555562 114263.60 65970.12 1.86 3.21 3 3445502 1 5343930 4 2 6032538 5688234 354243.94 204522.83 3.60 6.23 3 5832562 1 7301772 5 2 7740038 7541947 222143.13 128254.39 1.70 2.95 3 7584031 1 9328249 6 2 8709655 9054053 315215.7 181989.9 2.01 3.48 3 9124256 1 12115022 7.5 2 10652388 11435279 736752.39 425364.19 3.72 6.44 3 11538426 1 16344120 10 2 15425238 15820982 472501.94 98953.12 0.63 2.99 3 15693587 1 44388646 25 2 43053467 43368599 904629.54 207973.24 0.48 2.09 3 42663684 1 65843256 40 2 63248639 64401190 1321314.97 762861.55 1.18 2.05 3 64111676

221

Appendix 2

70000000

60000000 y = 2E+06x - 568358 R² = 0.9978 50000000

40000000

Area 30000000

20000000

10000000

0 0 5 10 15 20 25 30 35 40 45 Concentration (µg/mL)

Figure A.2.41: Calibration curve

Table A.2.15: Intra and inter day statistical data for the Limit of Quantification (0.5µg/mL):

Intra-day replicate no. Area Average area STDEV Relative error (%) Relative STDEV (%) 1 479105 2 485758 501359.5 31289.61 1.63 6.24 3 493067 4 547508

Inter day replicate no. Area Average area STDEV Relative error (%) Relative STDEV (%) 1 504821.5 2 556006.5 569789.1 42506.98 2.78 7.46 3 615413 4 576698 5 596006.5

222

Appendix 2

Table A2.16: Relative standard deviation values obtained for the quantification of caffeoyl glucose individual isomers in selected samples (Sample number corresponds to the entries in Table 2 from the manuscript): Sample Sample name % STDEV % STDEV % STDEV % STDEV % STDEV number (Origin) 2-β-CG (3) 2-α-CG (4) 1-α-CG (2) 6-β-CG (9) 6-α-CG (10) Strawberry 7 2.39 5.58 3.04 (Romania) Blueberry 10 2.97 (Germany) Raspberry 12 5.17 (Germany) Red currant 15 2.81 (Garden) Gooseberry 18 6.4 6.09 3.48 (garden)

223

Appendix 3

Appendix 3: Supporting Information for Chapter 6

Table A3.1: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of fresh tomato (Sample 1)

Peak Ret. time Assignment MS2(341) m/z(% intensity) MS3 m/z (% intensity) no. (min)

261(49), 207(27), 168(50), 1 C15H18O9 17.7 279(100), 203(18), 179(8) 152(100)

2 C15H18O9 21.1 179(100), 135(17) 135(100) 3 2-α-CG 21.8 323(15), 203(100) 175(100)

4 C15H18O9 24 233(100), 189(19), 179(74), 135(6) 205(10), 189(100), 171(23) 323(8), 281(34), 251(100), 233(56), 5 C15H18O9 24.4 203(100), 161(9) 203(14), 179(97), 135(11) 298(17), 233(16), 203(7), 179(100), 6 C15H18O9 25.4 135(100) 161(28), 135(13) 251(11), 233(100), 203(19), 189(12), 7 C15H18O9 26.1 205(8), 189(100), 171(10) 179(57), 161(21), 135(7) 323(12), 281(24), 251(21), 233(59), 8 C15H18O9 26.7 135(100) 179(100), 161(10), 135(12)

9 C15H18O9 29.1 179(100), 135(12), 135(100) 6-β-CG 323(19), 281(79), 251(73), 233(47), 10 30.5 135(100) (co-eluting) 221(20), 189(9), 179(100), 135(16) 281(11), 251(10), 233(11), 179(100), 11 C15H18O9 33.3 135(100) 135(16) 323(10), 281(100), 251(72), 221(23), 12 6-α-CG 35.4 221(34), 179(100), 135(15) 179(40)

13 C15H18O9 37.2 179(100), 135(14) 135(100)

14 C15H18O9 39.9 179(100), 135(14) 135(100)

15 C15H18O9 40.7 179(100), 135(16) 135(100)

224

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Table A3.2: MSn data from extracted ion chromatogram at m/z 341 of tomato puree (Sample 8)

Peak Ret. time MS2(341): MS3: Assignment no. (min) m/z(% intensity) m/z(% intensity)

261(11), 233(100), 219(6), 1 3-β-CG 15.3 323(100), 233(56), 203(29), 179(28) 189(5) 2 2-β-CG 17.5 323(18), 203(100) 175(100) 323(100), 261(6), 233(55), 203(39), 305(7), 261(8), 233(100), 3 3-α-CG 18.1 179(26) 189(11)

4 C15H18O9 20.8 179(100), 135(13) 135(100) 5 2-α-CG 21.5 323(9), 203(100) 175(100)

6 C15H18O9 23.6 233(100), 189(22), 179(68), 135(11) 205(11), 189(100), 171(18) 323(8), 281(30), 251(100), 233(53), 7 C15H18O9 24 203(100), 177(14) 203(36), 179(94), 161(7), 135(19) 323(15), 281(20), 251(26), 233(85), 8 C15H18O9 24.6 135(100) 203(71), 179(100), 161(14), 135(17) 298(8), 233(11), 203(10), 179(100), 9 C15H18O9 25 135(100) 161(28), 135(11) 323(23), 281(72), 251(85), 233(100), 10 C15H18O9 26.4 135(100) 198(25), 179(96),161(11),135(14)

11 C15H18O9 28.7 179(100), 135(10) 135(100) 323(18), 281(81), 251(87), 233(29), 12 6-β-CG 30.2 135(100) 179(100), 161(5), 135(9) 281(18), 251(12), 233(6), 221(5), 13 C15H18O9 32.8 135(15) 179(100), 135(15) 323(11), 281(100), 251(68), 221(25), 14 6-α-CG 34.9 221(36), 179(100), 135(11) 179(39)

15 C15H18O9 36.7 179(100), 135(12) 135(100)

16 C15H18O9 39.7 179(100), 135(18) 135(100)

17 C15H18O9 40.5 179(100), 135(13) 135(100)

225

Appendix 3

Table A3.3: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of homemade tomato juice (Sample 10)

Peak Ret.time MS2(341) MS3 MS4: Assignment no. (min) m/z(% intensity) m/z (%intensity) m/z (% intensity) 305(5), 233(100), 215(71), 206(41), 189(100), 323(100), 233(59), 203(42), 1 3-β-CG 15.6 189(14), 161(4), 174(35), 161(52), 123(49), 179(34), 135(5) 123(3) 109(44) 323(12), 305(4), 203(100), 2 2-β-CG 17.8 175(100), 157(11), 147(100), 119(23) 179(12), 161(15), 323(100), 305(11), 261(4), 305(6), 261(8), 3 3-α-CG 18.3 233(62), 203(67), 179(36), 233(100), 189(7) 135(6) 233(5), 203(5), 179(100), 4 C15H18O9 21.3 135(100) 135(19) 158(30), 147(100), 133(15), 5 2-α-CG 22 323(10), 233(6), 203(100) 175(100) 129(18), 103(39) 233(100), 189(19), 179(80), 215(5), 205(16), 174(100), 171(67), 161(80, 6 C15H18O9 24.1 135(10) 189(100), 171(26) 159(19), 146(17), 143(19) 323(9), 281(36), 251(100), 203(100), 179(9), 7 C15H18O9 24.5 233(59), 203(48), 179(93), 175(100), 161(60) 177(18), 161(9) 161(9), 135(18) 323(16), 298(34), 281(16),

8 C15H18O9 25.2 251(24), 233(85), 203(71), 135(100) 179(100) 298(10), 233(15), 203(8), 9 C15H18O9 25.5 135(100) 179(100), 161(36), 135(15) 323(43), 296(15), 281(71),

10 C15H18O9 27 251(100), 221(34), 179(86), 179(100) 135(100) 161(16), 135(11) 323(11), 281(23), 251(17),

11 C15H18O9 27.6 233(95), 189(17), 179(100), 135(100) 135(45)

226

Appendix 3

Table A3.3 (continued): MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of homemade tomato juice (Sample 10)

Peak Ret.time MS2(341) MS4 m/z Assignment MS3 m/z (%intensity) no. (min) m/z(% intensity) (% intensity) 323(13), 251(20), 233(88),

12 C15H18O9 27.8 189(22), 179(100), 161(11), 161(9), 135(100) 135(46)

13 C15H18O9 29.4 281(6), 179(100), 135(12) 135(100) 323(18), 281(80), 251(85), 14 6-β-CG 30.7 233(10), 221(26), 179(100), 135(100) 135(7) 281(9), 251(7), 179(100), 15 C15H18O9 33.6 135(100) 135(18) 323(14), 281(56), 251(100),

16 C15H18O9 34.2 233(13), 221(30), 179(61), 179(100), 135(6) 135(100) 135(7) 323(8), 281(100), 251(79), 149(12), 17 6-α-CG 35.7 221(37), 179(100), 135(12) 221(12), 179(50) 135(100)

18 C15H18O9 37.5 179(100), 135(13) 135(100)

19 C15H18O9 40.1 179(100), 135(13) 135(100)

20 C15H18O9 40.9 298(14), 179(100), 135(16) 135(100)

227

Appendix 3

Table A3.4: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of tomato ketchup (Sample 11)

Ret. Peak MS2(341): MS3: Assignment Time nr m/z(% intensity) m/z (% intensity) (min) 323(94), 261(18), 233(100), 215(100), 203(57), 189(92), 177(87), 1 C15H18O9 18 203(28), 179(39) 123(72) 233(6), 203(6), 179(100), 2 C15H18O9 20.9 135(100) 135(11) 3 2-α-CG 21.5 323(14), 203(100), 179(13) 175(100) 281(24), 251(95), 233(60),

4 C15H18O9 24 203(38), 179(100), 161(11), 135(100) 135(16) 323(17), 281(90), 251(100), 5 C15H18O9 26.4 179(100), 135(12) 221(23), 179(89), 161(8), 135(7)

6 C15H18O9 28.7 179(100), 135(11) 135(100) 323(25), 281(70), 251(100), 7 C15H18O9 30.1 179(100), 135(8) 221(26), 179(74), 161(40) 323(5), 281(9), 251(7), 179(100), 8 C15H18O9 32.8 135(100) 135(15) 323(9), 281(100), 251(69), 9 6-α-CG 35.2 221(45), 179(100), 135(15) 221(18), 179(40)

10 C15H18O9 36.6 179(100), 135(13) 135(100)

11 C15H18O9 39.6 179(100), 135(15) 135(100) 323(15), 298(16), 207(8), 12 C15H18O9 40.5 161(32), 135(100) 179(100), 161(10)

228

Appendix 3

Table A3.5: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of Red Chilli of Dutch origin (Sample 13)

Ret. Peak MS2(341): MS3: MS4: Assignment Time no. m/z(% intensity) m/z (% intensity) m/z(% intensity) (min) 1 2-β-CG 17.4 321(25), 295(30), 203(100) 175(100) 2 2-α-CG 21.6 323(11), 280(15), 203(100) 175(100) 339(10), 294(53), 251(6), 3 C15H18O9 23.2 135(100) 179(100), 135(32) 339(100), 321(23), 219(80), 321(27), 219(100), 4 C15H18O9 25 191(100) 205(70), 179(19) 205(97), 191(10) 339(21), 281(8), 251(6), 161(9), 149(9), 5 C15H18O9 26.3 203(20), 179(100), 161(35), 135(100), 89(36) 135(15) 281(100), 251(60), 221(20), 221(17), 179(100), 6 6-β-CG 29 135(100) 179(44) 135(6) 339(10), 295(11), 265(6), 7 C15H18O9 32.9 135(100) 179(100), 135(24) 339(21), 293(35), 281(100), 221(40), 179(100), 8 6-α-CG 34.9 265(17), 251(61), 221(9), 135(17) 179(35) 341(100), 309(13), 241(37), 9 C15H18O9 41.8 181(17), 97(100) 97(27)

10 C15H18O9 42.3 341(100), 241(44), 97(22) 97(100)

229

Appendix 3

Table A3.6: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of Red Chilli of Indian origin

Ret. Peak MS3: m/z MS4: Assignment time MS2(341): m/z(% intensity) no. (% intensity) m/z(% intensity) (min) 308(27), 281(20), 341(22), 325(11), 203(41), 274(34), 251(17), 1 C15H18O9 17.9 167(100), 143(7) 225(6), 194(23), 166(44), 151(100) 339(56), 323(43), 311(65), 2 C15H18O9 19.7 191(100), 180(20) 241(86), 211(21), 199(100), 299(51), 241(100), 211(17), 223(100), 181(54), 3 C15H18O9 21.3 179(74), 161(11) 97(59)

4 C15H18O9 29.5 179(100), 135(12) 135(100) 177(57), 175(20), 251(33), 205(100), 5 C15H18O9 30.8 295(100), 205(5), 161(100), 161(96) 119(23)

6 C15H18O9 33.7 179(100), 135(14) 135(100)

7 C15H18O9 37.7 179(100), 135(16) 135(100) 341(100), 241(89), 181(6), 223(38), 205(24), 8 C15H18O9 41.9 151(8), 97(46) 198(8), 97(100) 341(100), 241(84), 151(7), 223(26), 125(14), 9 C15H18O9 42.3 97(45) 97(100)

230

Appendix 3

Table S7: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of Green Chilli (Sample 16)

Ret. Peak Assignment time MS2(341): m/z(% intensity) MS3: m/z(% intensity) no. (min)

1 3-β-CG 15.4 323(100), 233(32), 230(31), 179(33) 305(8), 261(8), 233(100), 189(19),

2 2-β-CG 17.5 323(15), 203(100), 175(100)

3 3-α-CG 18.1 323(100), 233(65), 203(71), 179(43) 305(9), 361(10), 233(100), 189(13)

4 2-α-CG 19.1 323(16), 203(100) 175(100)

339(18), 293(30), 281(15), 251(9), 221(16), 5 C15H18O9 20.3 203(41), 179(66), 161(100) 339(43), 293(41), 265(18), 203(100), 179(99), 161(76), 143(56), 131(23), 119(6), 6 C15H18O9 21.1 161(11), 143(16), 89(100)

7 C15H18O9 21.7 323(10), 203(100) 175(100) 339(41), 293(28), 203(12), 179(100), 161(4), 8 C15H18O9 23.3 135(100) 135(13) 323(22), 281(41), 269(22), 251(95), 233(66), 9 C15H18O9 24.3 135(100) 203(41), 203(58), 179(100), 161(12), 135(7) 339(14), 321(7), 219(22), 205(17), 203(10), 10 C15H18O9 25.2 135(100) 179(100), 161(36), 135(12)

11 C15H18O9 26.6 203(8), 179(100), 161(26), 135(8) 135(100)

12 C15H18O9 29.2 323(12), 281(100), 251(70), 221(26), 179(42) 221(45), 179(100), 135(11)

323(20), 281(100), 251(45), 221(19), 179(47), 13 C15H18O9 30.1 221(28), 179(100), 135(14) 135(7)

14 6-β-CG 30.3 323(32), 281(100), 251(80), 221(16), 179(57) 221(29), 179(100), 135(13)

323(20), 294(19), 281(100), 251(60), 221(12), 15 C15H18O9 32.2 221(11), 179(100) 179(57),

16 C15H18O9 33.1 179(100), 135(12) 135(100)

17 6-α-CG 35.2 323(12), 281(100), 251(67), 221(25), 179(38) 221(33), 179(100), 135(14)

18 C15H18O9 37.1 179(100), 135(13) 135(100)

19 C15H18O9 39.9 179(100), 135(14) 135(100)

20 C15H18O9 41.8 341(100), 241(19), 97(11) 227(28), 97(100)

21 C15H18O9 42.2 341(100), 241(22), 97(12) 227(36), 151(10), 97(87) 319(15), 280(20), 257(100), 241(24), 213(31), 213(60), 195(33), 177(100), 167(18), 22 C15H18O9 43 177(37), 97(53) 97(62)

231

Appendix 3

Table A.3.8: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of Organic bell pepper (Sample 19)

Ret. Peak time no. Assignment (min) MS2(341): m/z (% intensity) MS3: m/z(% intensity) 1 2-β-CG 17.6 323(7), 203(100), 179(5) 175(100) 2 2-α-CG 21.8 323(19), 203(100), 179(5) 175(100)

3 C15H18O9 23.4 323(24), 179(100), 135(12) 135(100) 4 6-β-CG 30.4 323(13), 281(100), 251(45), 221(15), 179(65) 221(31), 179(100)

5 C15H18O9 33.3 281(11), 251(4), 179(100), 135(16) 135(100) 323(19), 293(61), 281(86), 251(49), 221(12), 6 6-α-CG 35.3 179(33) 221(31), 179(100) 7 42.4 341(100), 241(28) 97(100)

8 C15H18O9 43.9 295(24), 179(100), 161(79), 135(70), 101(20) 161(8), 135(100)

Table A.3.9: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of conventional bell pepper (Sample 22)

Ret. Peak Assignment Time MS2(341): m/z(% intensity) MS3: m/z(% intensity) nr (min) 1 2-α-CG 21.8 323(6), 203(100), 175(100)

2 C15H18O9 23.3 340(8), 294(15), 203(5), 179(100), 161(5), 135(20) 135(100) 340: 339(100), 321(30), 219(100), 205(85), 179(51), 3 C15H18O9 25.3 191(100) 161(12), 135(5) 341(6), 326(20), 309(25), 281(4), 229(6), 203(13), 4 C15H18O9 26.6 135(100), 119(8) 179(100), 161(23), 135(9)

5 C15H18O9 29.1 179(100), 135(7) 135(100)

6 C15H18O9 33.2 179(100), 135(12) 135(100)

7 C15H18O9 39.8 298(9), 179(100), 135(11) 135(100)

8 C15H18O9 41.9 341(100), 241(16), 97(10) 139(27), 97(100)

9 C15H18O9 42.3 341(53), 241(17), 97(100) 241(100), 139(27), 97(65) 161(14), 135(100), 123(64), 10 C15H18O9 43.9 339(56), 179(100), 161(70), 135(61), 113(17), 106(32), 79(39)

232

Appendix 3

Table A3.10: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of banana pepper (Sivri cultivar)

Ret. Peak MS2 (341): MS 3 : MS 4 : Assignment Time no. m/z (% intensity) m/z(% intensity) m/z (% intensity) (min) 215(65), 205(60), 323(100), 261(5), 233(53), 305(5), 261(8), 1 3-β-CG 15.6 189(75), 161(69), 203(31), 179(23) 233(100), 189(16) 123(100), 109(25) 2 2-β-CG 17.7 323(12), 203(100) 203(6), 175(100) 158(33), 147(100) 215(79), 205(40), 323(100), 261(4), 233(53), 305(6), 233(100), 3 3-α-CG 18.3 189(76), 161(100), 203(42), 179(27), 189(10) 123(95), 109(93) 157(26), 147(100), 4 C15H18O9 19.2 323(19), 203(100), 203(7), 175(100) 119(24) 297(28), 241(48), 221(100), 221(30), 219(33), 5 C15H18O9 20.8 203(52), 179(12), 135(76) 193(74), 176(100) 157(13), 147(100), 6 2-α-CG 21.8 323(10), 203(100), 203(5), 175(100) 119(27) 323(23), 297(10), 203(100), 7 C15H18O9 22.3 175(100) 147(100) 179(8) 261(10), 233(100), 323(100), 261(5), 233(58), 215(100), 205(28), 8 C15H18O9 23 219(7), 203(8), 203(43), 179(26) 189(83), 161(58), 123(34) 189(13) 323(12), 294(33), 203(14), 9 C15H18O9 23.4 135(100) 179(100), 135(13) 323(27), 281(31), 251(64),

10 C15H18O9 24.3 233(48), 203(70), 179(100), 135(100) 161(8), 135(17) 339(89), 321(27), 281(5),

11 C15H18O9 25.4 219(100), 205(88), 179(93), 191(100) 173(100) 161(28), 135(10) 203(24), 179(100), 161(28), 12 C15H18O9 26.7 135(100) 135(12)

323(11), 281(100), 251(69), 221(45), 179(100), 135(100) 13 6-β-CG 29.5 221(20), 179(63) 135(9)

Ret. Peak MS2(341): MS3: MS4: Assignment time no. m/z(% intensity) m/z (% intensity) m/z (% intensity) (min) 233

Appendix 3

323(30), 295(0), 281(82), 14 C15H18O9 30.5 179(100), 135(6) 135(100) 251(97), 221(33), 179(76),

15 C15H18O9 33.3 179(100), 135(13) 135(100)

323(11), 281(100), 251(69), 221(34), 179(100), 16 6-α-CG 35.4 135(100) 221(23), 179(43) 135(9)

323(24), 281(15), 251(22),

17 C15H18O9 36.6 233(14), 203(29), 179(100), 135(100) 161(10), 135(8)

18 C15H18O9 37.2 179(100), 135(11) 135(100)

19 C15H18O9 37.5 179(100), 135(15) 135(100)

20 C15H18O9 40 179(100), 135(15) 135(100) 223(100), 211(9), 21 41.9 341(100), 241(28), 97(16) 186(12), 165(7), 151(15), 97(85) 223(32), 151(7), 22 42.3 341(100), 241(21), 97(11) 139(9), 97(100) 257(100), 223(11), 177(23), 213(100), 177(82), 23 43 97(21) 169(25), 97(85)

234

Appendix 3

Table A3.11: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of banana pepper (Charlie cultivar)

Ret. Peak MS2 (341): MS 3 : Assignment time no. m/z(%intensity) m/z(%intensity) (min)

261(8), 233(100), 1 3-β-CG 15.6 323(100), 233(43), 203(33), 179(25) 189(16), 2 2-β-CG 17.7 323(12), 203(100) 203(13), 175(100)

3 3-α-CG 18.2 323(100), 233(61), 203(72), 179(33) 233(100), 189(12)

4 C15H18O9 19.2 323(12), 279(5), 203(100) 175(100) 5 2-α-CG 21.9 323(12), 203(100) 175(100)

6 C15H18O9 23.2 323(32), 233(7), 203(26), 179(100), 161(9), 135(12) 135(100)

323(22), 281(46), 251(59), 233(65), 203(62), 179(100), 7 C15H18O9 24.4 135(100) 161(7), 135(15)

8 C15H18O9 25.4 321(10), 281(6), 219(43), 205(34), 179(100), 161(39), 135(10) 135(100)

9 C15H18O9 26.7 203(14), 179(100), 161(34), 135(10) 135(100)

221(30), 179(100), 10 6-β-CG 29.5 323(11), 281(100), 281(69), 221(25), 179(46) 135(11)

11 C15H18O9 33.4 179(100), 135(13) 135(100) 221(36), 179(100), 12 6-α-CG 35.6 323(11), 281(100), 251(75), 221(21), 179(42) 135(11)

13 C15H18O9 37.4 179(100), 135(11) 135(100) 161(33), 143(30), 319(62), 283(47), 227(7), 179(100), 161(17), 143(21), 135(57), 131(14), 14 C15H18O9 40 119(22), 125(14), 119(19), 89(100) 227(49), 181(8), 15 41.9 341(100), 241(20), 97(14) 151(8), 97(100) 227(45), 205(5), 16 42.3 341(100), 241(24), 97(9) 181(5), 139(17), 97(100)

341(76), 257(100), 241(23), 210(47), 192(9), 177(77), 213(100), 177(90), 17 43.1 161(20), 130(8), 97(18) 97(51)

235

Appendix 3

Table A.3.12: MSn data from extracted ion chromatogram at m/z 341 (negative ion mode) of aubergine

Ret. Peak MS3: Assignment Time MS2(341): m/z(% intensity) no. m/z(% intensity ) (min) 1 23.9 328(43), 233(38), 181(100), 166(48) 166(100), 135(37) 166(36), 159(20), 2 C15H18O9 24.4 251(43), 233(78), 203(13), 179(100), 135(28) 135(100)

3 C15H18O9 25.3 233(7), 203(10), 179(100), 161(34), 135(11) 135(100)

4 C15H18O9 26.7 309(18), 299(23), 233(30), 203(8), 179(100), 161(9) 161(9), 135(100)

5 C15H18O9 29.3 281(21), 251(9), 233(21), 189(4), 179(10)

6 C15H18O9 33.4 179(100), 135(14) 135(100)

7 C15H18O9 37.4 179(100), 135(13) 135(100)

236

Appendix 3

Table A3.13: Statistical data for the calibration curve points:

Relative Relative Concentration Replicate Average Absolute Area STDEV Error STDEV (µg/mL) number area error (%) (%) 0.5 1 479105 501359.5 31289.61 15644.81 3.12 6.24 2 485758 3 493067 4 547508 1 1 1099316 1143396 64882.63 37460.01 3.28 5.67 2 1217900 3 1112972 2.5 1 3673610 3555562 114263.60 65970.12 1.86 3.21 2 3547574 3 3445502 4 1 5343930 5688234 354243.94 204522.83 3.60 6.23 2 6032538 3 5832562 5 1 7301772 7541947 222143.13 128254.39 1.70 2.95 2 7740038 3 7584031 6 1 9328249 9054053 315215.7 181989.9 2.01 3.48 2 8709655 3 9124256 7.5 1 12115022 11435279 736752.39 425364.19 3.72 6.44 2 10652388 3 11538426 10 1 16344120 15820982 472501.94 98953.12 0.63 2.99 2 15425238 3 15693587 25 1 44388646 43368599 904629.54 207973.24 0.48 2.09 2 43053467 3 42663684

237

Appendix 3

Area 50000000

45000000 y = 2E+06x - 1E+06 R² = 0.9984 40000000

35000000

30000000

25000000

20000000

15000000

10000000

5000000

0 0 5 10 15 20 25 30 Concentration (μg/mL)

Figure A3.1: Calibration curve

238

Appendix 3

Table A 3.14: Intra and inter day statistical data for the Limit of Quantification (0.5µg/mL):

Intra-day replicate Average Standard Relative error Relative STDEV Area number area deviation (%) (%)

1 479105

2 485758 501359.5 31289.61 1.63 6.24 3 493067

4 547508

Inter day replicate Average Standard Relative error Relative STDEV number Area area deviation (%) (%)

1 504821.5

2 556006.5

3 615413 569789.1 42506.98 2.78 7.46

4 576698

5 596006.5

239

Appendix 3

100 90 % extracted 80 70 60 50 40 30 20 10 0

Figure A3.2: Extraction reproducibility results obtained for selected samples:

Table A3.15: Relative standard deviation values obtained for the quantification of caffeoyl glucose individual isomers in selected samples:

Relative standard deviation (%)

Sample 2-β-CG 2-α-CG 3-β-CG 3-α-CG 6-β-CG 6-α-CG Sample type number (3) (4) (5) (6) (9) (10) 1 Fresh tomato 9.03 15.92

Tomato 8 8.05 11.47 7.81 7.59 8.57 13.22 puree Tomato 11 10.05 14.17 ketchup

Red 13 9.27 8.93 10.25 13.38 pepperoni

Organic bell 19 13.36 13.63 9.42 9.09 pepper

Conventional 22 11.68 Bell pepper

240

Appendix 4

Appendix 4: Supporting Information for Chapter 7

Varian MS 12-MAR-2012 12:19:39 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C1_neg_IRMPD100-70.trans Glucose 13C1 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 9,8315 Total Intensity: 71,118 Scans: 2 Negative Ions External Calibration

144,0398 100

80 114,0288

60 180,0618 132,0397 102,0286

40 162,0508

89,0251 20 87,0093 75,0093 59,0141 149,0471

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.1: Direct infusion IRMPD spectrum of 13C-1

Table A 4.1: IRMPD data of 13C-1: Neutral loss Relative intensity m/z value Assignment (Da) (%)

13 12 - 180.0618 [ C C5H11O6] 53.17

13 12 - 162.0508 [ C C5H9O5] 18.011 (H2O) 32.54

13 12 - 144.0398 [ C C5H7O4] 36.022 (2H2O) 100.00

13 12 - 12 132.0397 [ C C4H7O4] 48.0221 (H2O+ CH2O) 50.00

12 - 13 131.0308 [ C5H7O4] 49.031 (H2O+ CH2O) 32.54

13 12 - 12 120.0388 [ C C3H7O4] 60.023 ( C2H4O2) 47.62

12 - 13 12 119.0299 [ C4H7O4] 61.0319 ( C CH4O2) 25.40

13 12 - 12 114.0288 [ C C4H5O3] 66.033 (2H2O + CH2O) 68.25

12 - 13 113.0199 [ C5H5O3] 67.0419 (2H2O + CH2O) 10.32

13 12 - 12 102.0286 [ C C3H5O3] 78.0332 (H20 + C2H4O2) 46.03

12 - 13 12 101.0197 [ C4H5O3] 79.0421 (H2O + C CH4O2) 19.05

13 12 - 12 90.034 [ C C2H5O3] 90.0278 ( C3H6O3) 18.25

12 - 13 12 89.0251 [ C3H5O3] 91.0367 ( C C2H6O3) 22.22

241

Appendix 4

Varian MS 08-MAR-2012 17:30:14 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C2_neg_IRMPD100-70.trans glucose 13C2 in H20 50 ug/ mL IRMPD 100 ms 70% Base-Peak Amplitude: 4,4324 Total Intensity: 30,653 Scans: 1 Negative Ions External Calibration

144,0389 100

132,0388 80 114,0282

120,0388 180,0604 102,0282 40

162,0495

20 89,0247 72,0197 150,0501 59,0141 75,0457

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.2: Direct infusion IRMPD spectrum of 13C-2

Table S2: IRMPD data of 13C-2: Neutral loss Relative intensity m/z value Assignment (Da) (%)

13 12 - 180.0604 [ C C5H11O6] - 45.67

13 12 - 162.0495 [ C C5H9O5] 18.0109 (H2O) 23.62

13 12 - 12 150.0501 [ C C4H9O5] 30.0103 ( CH2O) 11.02

13 12 - 144.0389 [ C C5H7O4] 36.0215 (2*H2O) 100.00

13 12 - 1 1 132.0388 [ C C4H7O4] 48 ( H2O+C H2O) 82.68

13 - 126.028 [ C C5H5O3] 54.0324 (3*H2O) 25.20

13 12 - 12 120.0388 [ C C3H7O4] 60.0216 ( C2H4O2) 45.67

12 1 - 13 12 119.0299 [ C4 H7O4] 61.0305 ( C CH4O2) 28.35

13 - 12 114.0282 [ C C4H5O3] 66.0322 (2*H2O+ CH2O) 74.80

13 - 12 102.0282 [ C C3H5O3] 78.0322 (H20+ C2H4O2) 33.07

- 13 12 101.0193 [C4H5O3] 79.0411 (H2O+ C CH4O2) 18.90

13 - 90.0336 [ C C2H5O3] 90.0268 (C3H6O3) 14.17

- 13 12 89.0247 [C3H5O3] 91.0357 ( C C2H6O3) 16.54

13 - 12 72.0197 [ C C2H3O2] 108.0407 (H2O+ C3H6O3) 10.24

242

Appendix 4

Varian MS 12-MAR-2012 10:21:45 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C3_neg_IRMPD100-70.trans Glucose 13C3 H2O 50 ug/ mL 100 ms 70% Base-Peak Amplitude: 17,1884 Total Intensity: 34,750 Scans: 1 Negative Ions External Calibration

180,0599 100

162,0494 20 144,0388 120,0384 90,0302 132,0388 81,0250 114,0281 150,0490 0

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.3: Direct infusion IRMPD spectrum of 13C-3

Table A4.3: IRMPD data of 13C-3: Neutral loss Relative intensity Assignment m/z value (Da) (%)

13 - 180.0599 [ CC5H11O6] 100.00

13 - 162.0494 [ CC5H9O5] 18.0105 (H2O) 22.05

13 - 12 150.049 [ CC4H9O5] 30.0109 ( CH2O) 3.15

13 - 144.0388 [ CC5H7O4] 36.0211 (2*H2O) 17.32

13 12 - 12 132.0388 [ C C4H7O4] 48.0211 (H2O+ CH2O) 7.87

13 12 - 12 120.0384 [ C C3H7O4] 60.0215 ( C2H4O2) 10.24

13 - 12 114.0281 [ CC4H5O3] 66.0318 (2*H2O+ CH2O) 3.15

13 - 12 90.0302 [ CC2H5O3] 90.0297 ( C3H6O3) 8.66

243

Appendix 4

Varian MS 08-MAR-2012 18:18:36 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C4_neg_IRMPD100-70.trans glucose 13C4 in H20 50 ug/ mL IRMPD 100 ms 70% Base-Peak Amplitude: 37,4365 Total Intensity: 69,887 Scans: 1 Negative Ions External Calibration

180,0620 100

162,0513 20 144,0403 120,0398 132,0400 90,0313 114,0290 150,0509 0

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.4: Direct infusion IRMPD spectrum of 13C-4

Table A4.4: IRMPD data of 13C-4: Neutral loss Relative intensity m/z value Assignment (Da) (%)

13 - 180.062 [ CC5H11O6] 100.00

13 - 1 162.0513 [ CC5H9O5] 18.0107 ( H2O) 21.26

13 - 12 1 150.0509 [ CC4H9O5] 30.0111 ( C H2O) 2.36

13 - 144.0403 [ CC5H7O4] 36.0217 (2*H2O) 14.96

13 12 - 12 132.04 [ C C4H7O4] 48.022 (H2O+ CH2O) 7.87

13 12 - 12 1 120.0398 [ C C3H7O4] 60.0222 ( C2 H4O2) 8.66

13 - 12 1 90.0313 [ C2CH5O3] 90.0307 ( C3 H6O3) 5.51

244

Appendix 4

Varian MS 12-MAR-2012 15:09:58 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C5_neg_IRMPD100-70.trans Glucose 13C5 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 14,3242 Total Intensity: 92,461 Scans: 2 Negative Ions External Calibration

144,0397 100

80 180,0616 132,0395

60 114,0287 162,0507 119,0360 40

101,0252 20 89,0251 150,0504 72,0201 57,0145 0

60 80 100 120 140 160 180 200 Mass/ Charge

Figure A4.5: Direct infusion IRMPD spectrum of 13C-5

Table A4.5: IRMPD data of 13C-5: Relative intensity m/z value Assignment Neutral loss (%)

13 - 180.0616 [ C C5H11O6] 77.9528

13 - 1 162.0507 [ CC5H9O5] 18( H2O) 46.4567

13 12 1 150.0504 CC4H9O5 30( C H2O) 15.7480

13 1 144.0397 CC5H7O4 36(2* H2O) 100.0000

13 12 1 1 132.0395 C C4H7O4 48( H2O+C H2O) 67.7165

13 126.086 C C5H5O3 54(3*H2O) 20.4724

13 12 12 1 120.0449 C C3H7O4 60( C2 H4O2) 29.9213

13 12 119.036 C4H7O4 61( C CH4O2) 36.2205

13 12 114.0287 CC4H5O3 66(2*H2O+ CH2O) 53.5433

13 12 102.0341 CC3H5O3 78(H20+ C2H4O2) 14.1732

13 12 101.0252 C4H5O3 79(H2O+ C CH4O2) 21.2598

13 90.034 CC2H5O3 90(C3H6O3) 14.1732

13 89.0251 C3H5O3 91( CC2H5O3) 14.1732

13 12 72.0201 CC2H3O2 108 (H2O+ C3H6O3) 4.7244

245

Appendix 4

Varian MS 12-MAR-2012 11:39:50 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C6_neg_IRMPD100-70.trans Glucose 13C6 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 12,4948 Total Intensity: 82,242 Scans: 2 Negative Ions External Calibration

144,0401 100

180,0622

60 162,0510

119,0362 132,0398 40

101,0253 20 90,0284 150,0507 59,0142 72,0201 176,0501 0

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.6: Direct infusion IRMPD spectrum of 13C-6

Table A4.6: IRMPD data of 13C-6: Neutral loss Relative intensity m/z value Assignment (Da) (%)

13 12 - 180.0622 [ C C5H11O6] 100.00

13 12 - 162.051 [ C C5H9O5] 18.0112 (H2O) 59.63

13 12 - 150.0507 [ C C4H9O5] 30.0115 (CH2O) 13.76

13 12 - 144.0401 [ C C5H7O4] 36.0221 (2*H2O) 115.60

13 12 - 12 132.0398 [ C C4H7O4] 48.0224 (H2O+ CH2O) 45.87

12 - 13 131.0309 [ C5H7O4] 49.0313 (H2O+ CH2O) 33.94

13 12 - 126.0292 [ C C5H5O3] 54.033 (3*H2O) 20.18

13 12 - 12 120.0451 [ C C3H7O4] 60.0171 ( C2H4O2) 38.53

12 - 13 12 119.0362 [ C4H7O4] 61.026 ( C CH4O2) 44.95

13 12 - 12 102.0342 [ C C3H5O3] 78.028 (H2O+ C2H4O2) 16.51

12 - 13 12 101.0253 [ C4H5O3] 79.0369 (H2O+ C CH4O2) 23.85

246

Appendix 4

Varian MS 12-MAR-2012 15:42:27 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 13C1,2_neg_IRMPD100-70.trans Glucose 13C1,2 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 14,9386 Total Intensity: 100,250 Scans: 2 Negative Ions External Calibration

145,0435 100

115,0323 60 181,0654 133,0432

40 103,0321 163,0544

89,0251 20

75,0093 150,0514 59,0141 0

60 80 100 120 140 160 180 200 Mass/ Charge 13 Figure A4.7: Direct infusion IRMPD spectrum of C2-1,2

13 Table A4.7: IRMPD data of C2-1,2: Relative intensity m/z value Assignment Neutral loss (%)

13 12 - 181.0654 [ C2 C4H11O6] 53.54

13 12 - 163.0544 [ C2 C4H9O5] 18.011 (H2O) 33.07

13 12 - 145.0435 [ C2 C4H7O4] 36.0219 (2*H2O) 100.00

13 12 - 12 133.0432 [ C2 C3H7O4] 48.0222(H2O+ CH2O) 47.24

13 12 - 13 132.0341 [ C C4H7O5] 49.0311 (H2O+ CH2O) 30.71

13 12 - 127.0326 [ C2 C4H5O3] 54.0328 (3*H2O) 22.05

13 12 - 12 121.0504 [ C2 C2H7O4] 60.015 ( C2H4O2) 43.31

12 - 13 119.0326 [ C4H7O4] 62.0328 ( C2H4O2) 21.26

13 12 - 12 115.0323 [ C2 C3H5O3] 66.0331 (2*H2O+ CH2O) 61.42

13 12 - 12 103.0321 [ C2 C2H5O3] 78.0333 (H20+ C2H4O2) 33.86

13 12 - 12 91.0429 [ C2 CH5O3] 90.0225 ( C3H6O3) 15.75

12 - 13 12 89.0251 [ C3H5O3] 92.0403 ( C2 CH6O3) 18.11

247

Appendix 4

Varian MS 12-MAR-2012 11:02:26 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose D1_neg_IRMPD100-70.trans Glucose D1 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 36,2555 Total Intensity: 59,814 Scans: 3 Negative Ions External Calibration

180,0619 100

90,0313 20 162,0518 60,0210 108,0196 120,0410 144,0410 0

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.8: Direct infusion IRMPD spectrum of 2H-1

Table A4.8: IRMPD data of 2H-1: Neutral loss Relative intensity m/z value Assignment (Da) (%)

2 1 - 180.0619 [C6 H H10O6] 100.00

2 1 - 1 162.0518 [C6 H H8O5] 18.0101 ( H2O) 7.87

1 - 2 1 161.0429 [C6 H9O5] 19.019 ( H HO) 3.15

2 1 - 1 144.041 [C6 H H6O4] 36.0209 (2* H2O) 3.94

1 - 2 1 143.0321 [C6 H7O4] 37.0298 ( H HO+H2O) 2.83

2 1 - 1 120.041 [C4 H H6O4] 60.0209 (C2 H4O2) 3.15

1 - 1 2 119.0321 [C4 H7O4] 61.0298 (C2 H3 HO2) 2.36

2 1 - 1 90.0313 [C3 H H4O3] 90.0306 (C3 H6O) 20.47

248

Appendix 4

Varian MS 12-MAR-2012 12:48:53 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose D2_neg_IRMPD100-70.trans Glucose D2 H2O 50 ug/ mL 100 ms 70 % Base-Peak Amplitude: 4,7192 Total Intensity: 60,817 Scans: 2 Negative Ions External Calibration

119,0355 100 113,0249 180,0634 132,0416 80 143,0358 101,0248

89,0247 40 87,0091

162,0529

20 75,0090 175,1159 59,0140 150,0525 66,0212 81,0348

60 80 100 120 140 160 180 200 Mass/ Charge Figure A4.9: Direct infusion IRMPD spectrum of 2H-C-2

Table A4.9: IRMPD data of 2H-C-2: Relative intensity m/z value Assignment Neutral loss (%)

2 1 - 180.0634 [C6 H H10O6] 84.25

2 1 - 1 162.0529 [C6 H H8O5] 18.0105 ( H2O) 23.62

1 - 2 1 161.044 [C6 H9O5] 19.0194 ( H HO) 18.90

2 1 - 1 150.0525 [C5 H H8O5] 30.0109 (C H2O) 11.02

2 1 - 1 144.0447 [C6 H H6O4] 36.0187 (2* H2O) 71.65

- 2 1 143.0358 [C6H7O4] 37.0276 ( H HO+H2O) 76.38

2 1 - 1 1 132.0416 [C5 H H6O4] 48.0218 ( H2O+C H2O) 77.95

1 - 1 2 1 131.0327 [C5 H7O4] 49.0307 ( H HO+C H2O) 58.27

2 1 - 1 126.0342 [C6 H H4O3] 54.0292 (3* H2O) 17.32

1 - 1 2 1 125.0253 [C6 H5O3] 55.0381 (2* H2O+ H HO) 22.05

2 1 - 1 120.0444 [C4 H H6O4] 60.019(C2 H4O2) 67.72

- 2 1 119.0355 [C4H7O4] 61.0279 (C2 H H3O2) 100.00

2 1 - 1 1 114.0338 [C5 H H4O3] 66.0296 (2* H2O+C H2O) 45.67

1 - 2 1 1 1 113.0249 [C5 H5O3] 67.0385 ( H HO+ H2O+C H2O) 88.98

249

Appendix 4

Varian MS 08-MAR-2012 15:45:03 File: E:\ Kuhnert-Jacobs Uni_Madrid\ Black Tea\ glucose 6,6D2_neg_IRMPD100-70.trans glucose 6,6 D2 in H20 50 ug/ mL IRMPD 100 ms 40% Base-Peak Amplitude: 3,4198 Total Intensity: 33,853 Scans: 1 Negative Ions External Calibration

181,0709 100

145,0491 113,0253 80

119,0360 133,0488 60 163,0599 121,0487 101,0253 40

90,0313 151,0598 20 87,0093

62,616472,5249

60 80 100 120 140 160 180 200 Mass/ Charge 2 Figure A4.10: Direct infusion IRMPD spectrum of H2-6,6

2 Table A4.10: IRMPD data of H2-6,6: Neutral loss m/z value Assignment Relative intensity (Da)

12 2 1 - 181.0709 [ C6 H2 H9O6] 100.00

12 2 1 - 1 163.0599 [ C6 H2 H7O5] 18 ( H2O) 53.17

12 2 1 - 12 1 151.0598 [ C6 H2 H7O5] 30 ( C H2O) 18.25

12 2 1 1 145.0491 C6 H2 H5O4 36 (2* H2O) 84.13

12 2 1 1 2 1 144.0402 C6 H H6O4 37 ( H2O+ H HO) 51.59

12 2 1 1 1 133.0488 C5 H2 H5O4 48 ( H2O+C H2O) 50.00

1 2 1 49 ([C H HO+ H2O]+ 12 2 1 1 2 1 132.0399 C5 H H6O4 [C H2O+ H HO]) 19.05

12 1 2 1 2 1 131.031 C5 H7O4 50 (C H HO+ H HO) 38.89

12 2 1 12 1 121.0487 C4 H2 H5O4 60( C2 H4O2) 46.03

12 1 2 1 119.036 C4 H7O4 62(C2 H2 H2O2) 57.14

12 2 1 12 114.0342 C5 H H4O3 67 (2H2O+ CH2O) 23.81

12 1 113.0253 C5 H5O3 68 79.37

12 2 1 102 C4 H H4O3 79 15.08

12 1 101.0253 C4 H5O3 80 39.68

2 1 - 90.0313 [C3 H H4O3] 91 21.43

1 - 89.0224 [C3 H5O3] 92 16.67

250

Int. x106 13C-1 3.0

2.5

2.0

1.5

1.0

0.5

1 2 3 4 5 6 7 8 9 Time [min]

Appendix 4

Int. 179.8

251 [%] 84.3 -MS, 6.1min 143.8 161.9 0 [%] 143.9 161.9 -MS2(179.8), 6.1min 89.2 114.0 120.0 132.0

0 [%] 114.0 -MS2(161.9), 6.1min 131.9 143.9 0 71.5 102.1 125.9 [%] 86.3 125.9 -MS3(179.9->143.8), 6.1min 71.5 82.4 100.0 114.0 0 [%] 96.2 -MS3(162.0->114.0), 6.1min

0 [%] 84.3 179.8 -MS, 7.3min 143.9 89.3 114.0 161.9 0 132.8 [%] 161.8 143.9 -MS2(179.8), 7.3min 89.2 120.0 71.5 114.0 125.9 131.9 149.9 0 [%] 125.9 114.0 141.7 -MS2(143.9), 7.3min 72.5 88.3 0 [%] 114.0 -MS3(179.9->161.9), 7.3min 89.2 102.1 125.9 131.9 143.8 0 [%] 81.2 97.3 -MS3(144.0->125.9), 7.3min

0 60 80 100 120 140 160 180 200 m/z

Figure A4.11: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-1.

Int. 7 x10 13C-2

1.4

1.2

1.0

0.8

0.6

0.4 0 1 2 3 4 5 6 7 8 9 Time [min]

Int. Appendix 4 179.8 -MS, 6.1min [%] 84.3 143.9 89.2

252 0 [%] 143.9 161.8 89.2 -MS2(179.8), 6.1min 114.0 119.9 125.9 131.9 149.9 0 [%] 114.0

88.3 125.9 141.8 -MS2(143.9), 6.1min 100.1 0 [%] 114.0 102.1 125.9 131.9 143.9 -MS3(179.9->161.8), 6.1min 0 [%] 86.2 69.4 -MS3(143.7->114.0), 6.1min 0 [%] 179.8 84.3 161.8 -MS, 7.2min 89.2 114.0 119.0 143.9 0 [%] 143.8 161.8 -MS2(179.8), 7.3min 89.2 114.0 120.0 131.9 149.9 0 [%] 114.0 -MS2(161.8), 7.3min 102.2 131.9 143.8 0 [%] 114.0 -MS3(179.9->161.8), 7.3min 89.2 125.9 131.9 143.9 0 [%] 96.1 -MS3(161.9->113.0), 7.3min

0 60 80 100 120 140 160 180 200 m/z

Figure A4.12: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-2.

Int. 7 x10 13C-3

1.50

1.25

1.00

0.75

0.50

1 2 3 4 5 6 7 8 9 Time [min]

Int. 179.8 [%] 84.3 161.9 -MS, 6.1min 120.0 143.9 0 114.0 [%] 161.8 Appendix 4 143.9 -MS2(179.8), 6.1min 89.2 120.0 102.1 114.0 125.9 131.9 149.9 0 253 [%] 113.9 -MS2(161.9), 6.1min 102.0 125.9 131.9 143.8

0 [%] 114.0 -MS3(179.9->161.9), 6.1min 143.9 87.2 102.1 131.9 0 [%] 86.2 96.1 -MS3(161.9->114.0), 6.1min

0 [%] 179.8 84.3 -MS, 7.3min 143.9 120.0 161.9 0 102.0 114.0 131.9 [%] 143.9 161.8 89.2 119.9 -MS2(179.8), 7.3min 72.5 102.1 114.0 131.9 149.9 0 [%] 126.0 -MS2(143.9), 7.3min 141.7 72.4 82.2 88.2 100.1 113.9 0 [%] 125.9 -MS3(179.9->143.9), 7.3min 86.2 114.0 72.5 99.1 0 [%] 97.1 -MS3(143.9->126.1), 7.3min

0 60 80 100 120 140 160 180 200 m/z

Figure A4.13: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-3.

Int. x106 13C-4

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. 179.9 -MS, 6.1min [%] 84.3 165.9 89.2 120.1 143.9 161.9 0 Appendix 4 [%] 143.9 161.9 100 90.2 120.0 -MS2(179.9), 6.1min 114.0

254 102.2 132.0 149.9 0 [%] 114.0 -MS3(179.9->161.9), 6.1min

100 131.9 88.3 102.0 125.9 143.9

0 [%] 86.3 100 95.3 -MS, 7.3min

0 [%] 179.9 100 84.3 -MS4(179.9->161.9->114.0), 6.2min 165.9 90.2 114.0 120.0 143.9 161.8 0 [%] 161.9 -MS2(179.9), 7.3min 100 90.3 120.0 143.9 102.1 114.1 131.9 0 [%] 114.1 -MS3(179.9->161.8), 7.3min 100 143.9 96.2 102.1 132.0 0 87.3 [%] -MS4(179.9->161.9->114.0), 7.3min 100 86.3 96.2

0 60 80 100 120 140 160 180 200 m/z

Figure A4.14: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-4.

Int. 7 x10 13C-5

1.2

1.0

0.8

0.6

0.4

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. 179.8 [%] 84.3 -MS, 6.0min 161.8 90.2 114.0 120.0 143.9 165.9

0 Appendix 4 [%] 143.9 90.2 161.9 -MS2(179.8), 6.0min 114.1119.0 131.9 149.9 0 255 [%] 114.0 -MS2(161.8), 6.1min 131.9 143.9 0 125.9 [%] 126.0 -MS, 7.2min 114.0 0 [%] 96.1 -MS3(179.8->143.9), 6.1min 85.3 0 [%] 84.3 179.8 161.9 -MS2(179.8), 7.3min 90.2 114.0 143.9 165.8 0 118.9 134.8 149.8 [%] 161.9 -MS3(162.0->114.0), 6.1min 90.2 114.0 120.0 143.9 71.5 102.1 131.9 149.9 0 125.9 [%] 114.0 -MS2(161.9), 7.3min 101.1 131.9 143.8 159.7 0 125.9 [%] 114.0 143.9 -MS3(179.9->161.9), 7.3min 73. 126.0 131.9 0 101.1 [%] 96.1 -MS3(161.8->114.0), 7.3min 86.2 0 60 80 100 120 140 160 180 200 m/z

Figure A4.15: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-5

Figure A4.16: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 13C-6.

Int. 7 x10 13C-6

1.0

0.8

0.6

0.4

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. Appendix 4 179.8 [%] 84.3 -MS2(179.8), 6.1min 90.2 113.0 119.0 143.9 161.9165.9

256 0 [%] 161.8 -MS, 6.1min 100 90.2 143.9 120.0 71.5 113.0 131.9 149.9 0 101.1 126.0 [%] 113.0 100 -MS3(179.9->161.9), 6.1min 143.9 87.2 101.1 131.9 0 72.5 [%]

100 95.1 -MS, 7.3min 85.2 0 [%] 84.3 179.8 -MS4(179.9->161.8->113.0), 6.2min 100 165.8 90.2 113.0 119.9 143.9 161.9 0 [%] 161.8 -MS2(179.8), 7.3min 100 90.2 143.9 113.1 118.9 131.9 149.9 0 [%] 113.0 100 -MS3(179.9->161.9), 7.3min 88.3 126.0 131.9 143.8 0 [%] -MS4(179.9->161.9->113.0), 7.3min 100 95.1 85.3 0 60 80 100 120 140 160 180 200 m/z

Int. 7 x10 1.75 13C2-1,2

1.50

1.25

1.00

0.75

0.50

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. [%] 180.8 -MS, 6.1min 100 84.3 216.8 Appendix 4 91.2 114.9119.0 144.9 162.8 0 [%]

257 144.9 100 162.9 -MS2(180.8), 6.1min 89.2 115.0 121.0 103.1107.0 126.9 132.9 150.8 0 [%]

100 115.0 126.9 -MS, 7.2min 88.3 99.1 71.5 83.3 103.2 0 [%]

180.8 -MS3(180.8->144.9), 6.1min 100 84.3

91.2 114.9 144.9 165.8 0 [%] 162.8 -MS2(180.8), 7.2min 100 144.9 91.2 115.0 121.0 103.1 109.0 126.9 132.9 150.8 0 [%] -MS3(180.8->162.8), 7.3min 100 114.9 144.8 73.4 89.2 103.1 126.9 132.9 0 60 80 100 120 140 160 180 200 m/z

2-3 13 Figure A4.17: Negative ion mode Total Ion Chromatogram and auto MS fragmentation spectra of C2-1,2.

Int. 6 x10 D-1 4

1 2 3 4 5 6 7 8 9 Time [min]

Intens. 179.8 [%] 84.3 -MS, 6.2min 161.5 89.2 113.0 119.0 165.9 0 143.9148.8 [%] 89.2 161.8 Appendix 4 118.9 143.9 -MS2(179.8), 6.2min 107.0 113.0 0 [%] 113.0 258 -MS2(161.5), 6.2min 102.0 124.9 131.9 142.9 0 [%]

59.7 71.5 -MS3(179.9->89.2), 6.2min

0 [%] 95.1 -MS3(161.5->113.0), 6.2min 85.2 0 [%] 179.8 84.3 -MS, 7.4min 161.8 89.2 119.0 0 113.0 143.9 [%] 161.5 89.2 119.0 -MS2(179.8), 7.4min 71.5 101.1 113.0 143.9 0 [%] 113.0 -MS2(161.8), 7.4min 142.9 89.1 101.1 131.9 0 [%] 112.9 -MS3(179.8->161.5), 7.4min 71.5 89.2 94.9 102.1 124.9 131.9 142.9 0 [%] 85.3 95.0 -MS3(161.8->113.0), 7.4min 0 60 80 100 120 140 160 180 200 m/z

Figure A4.18: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 2H-1

Int. 7 x10 D-2

1.50

1.25

1.00

0.75

0.50

1 2 3 4 5 6 7 8 9 Time [min]

Int. 179.8 [%] 84.3 119.0 -MS, 6.1min 89.2 113.0 143.8 161.8 215.8

0 Appendix 4 [%] 89.2 161.8 -MS2(179.8), 6.1min 119.0 143.9 160.8 102.1 113.0 132.0 148.9 0 124.9 259 [%] 101.1 -MS2(119.0), 6.2min 89.2 0 [%] 113.0

-MS3(179.9->160.9), 6.2min 101.1 131.9 142.8 0 125.9 [%] 73.4 83.2 -MS, 7.2min 55.759.7 0 [%] 179.8 84.3 -MS3(119.0->101.1), 6.2min 118.9 89.2 215.8 0 101.1 143.8 161.9 [%] 119.0 89.2 161.8 -MS2(179.8), 7.3min 113.0 142.9 59.7 71.5 101.1 107.0 131.9 148.9 0 [%] 101.1 -MS2(118.9), 7.3min 59.7 89.2 0 [%] 101.1 -MS3(179.9->118.9), 7.3min 59.7 89.2 0 [%] 83.3 -MS3(119.1->101.1), 7.3min 71.5 0 60 80 100 120 140 160 180 200 m/z

Figure A4.19: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 2H-2

Int. 7 x10 D-3

2.2

2.0

1.8

1.6

1.4

0 1 2 3 4 5 6 7 8 9 Time [min]

Appendix 4

Int. 260 179.6 [%] 112.7 -MS, 5.0min 126.7 144.6 158.6 186.6 0 [%] 88.9 143.6 161.5 113.7 -MS2(179.6), 5.0min 59.3 71.1 101.8 119.7 125.7 131.7 149.6 0 [%] 69.1 94.8 -MS2(112.7), 5.0min 71.0 82.9 0 [%] 59.3 71.1 -MS3(179.6->88.9), 5.1min

0 [%] 179.6 112.7 -MS, 6.0min 96.9 126.7 144.6 158.6 0 [%] 161.5 88.9 143.6 -MS2(179.6), 6.0min 113.7 71.2 119.7 131.6 148.6 0 [%] 69.1 -MS2(112.7), 6.0min 71.1 94.9 83.0 0 [%] 113.7 -MS3(179.7->160.6), 6.0min 71.2 88.9 98.9 131.7 142.6 0 60 80 100 120 140 160 180 200 m/z

Figure A4.20: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 2H-3

Int. 7 x10 D-4

2.1

2.0

1.8

1.6

1.4

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. 179.6 -MS, 5.1min

[%] Appendix 4 112.7 165.6 119.7 126.7 142.6 0 [%] 161.5

261 100 89.9 119.7 143.6 -MS2(179.6), 5.1min 113.7 149.6 59.3 71.1 124.7 0 [%] 113.7 100 -MS3(179.6->161.6), 5.1min

143.7 0 125.7 [%]

100 -MS2(112.7), 5.1min 57.3 69.1 95.5 0 [%] 179.6 100 112.7 -MS, 6.0min 158.6 119.7 126.7 144.6 208.5 0 [%] 161.5 100 88.9 113.7 119.6 143.6 -MS2(179.6), 6.0min 101.8 125.7 131.6 149.5 0 [%]

100 113.7 -MS3(179.7->161.6), 6.0min 88.9 101.8 125.5130.7 142.5 0 [%] 69.2 100 -MS2(112.7), 6.0min 82.985.9 94.8 55.3 73.0 0 60 80 100 120 140 160 180 200 m/z

Figure A4.21: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 2H-4

Int. 7 x10 D-5

2.2

2.0

1.8

1.6

1.4

0 1 2 3 4 5 6 7 8 9 Time [min]

Intens. 179.6 [%] 112.7 -MS, 5.1min 88.9 96.8 126.6 Appendix 4 0 119.7 [%] 143.6 100 119.7 161.6

262 88.9 -MS2(179.6), 5.1min 113.7 142.6 59.3 71.2 100.8 125.7 149.5 0 [%]

69.2 100 94.8 -MS2(112.7), 5.1min 82.9

58.3 0 [%] 81.9 88.0 -MS3(179.6->142.6), 5.1min 100 100.8 115.7 126.0 0 [%] 179.6 -MS, 5.9min 100 112.7 62.3 96.9100.7 119.6 144.6 0 [%] 143.6 119.7 161.6 100 89.8 142.6 -MS2(179.6), 6.0min 101.7 113.7 149.6 0 [%] 69.2 -MS2(112.7), 6.0min 100 94.8 67.0 97.9 0 [%]

100 115.7 -MS3(179.6->143.4), 6.0min 80.9 98.8 124.7 0 60 80 100 120 140 160 180 200 m/z

Figure A4.22: Negative ion mode Total Ion Chromatogram and auto MS2-3 fragmentation spectra of 2H-5

Int. 7 x10 D2-6,6

1.50

1.25

1.00

0.75

0.50

0 1 2 3 4 5 6 7 8 9 Time [min]

Int. [%] 180.8 -MS, 6.1min 100 84.2 216.8

Appendix 4 89.2 113.0 162.9 0 [%] 162.8 263 100 -MS2(180.8), 6.1min 143.9 89.2 113.0 121.0 178.8

59.7 72.5 101.0 108.1 125.0 130.9 150.8 0 [%]

100 112.9 -MS, 7.3min 144.9 59.6 130.9 0 91.2 101.1 [%] 180.8 100 84.3 -MS3(180.9->162.9), 6.2min

89.2 113.0 119.0 132.9 143.9 0 [%] 162.8 100 -MS2(180.8), 7.3min 89.2 112.9 121.0 144.9 71.5 102.0 108.0 125.9 132.9 150.9 0 [%]

100 113.0 -MS3(180.9->162.9), 7.3min 143.9 71.5 87.291.2 101.1 124.9 132.9 0 60 80 100 120 140 160 180 200 m/z

2-3 2 Figure A4.23: Negative ion mode Total Ion Chromatogram and auto MS fragmentation spectra of H2-6,6

Appendix 4

Int. D-1 114.0 600 -MS3(180.0->162.0), 6.2min

500

400

300 131.9

200

143.9 100 125.8

0 800 D-1 113.0 -MS3(180.0->162.0), 7.4min

600

400

131.9 200 143.9 102.0 126.0

0 60 80 100 120 140 160 m/z Figure A4.24: MS3 spectra (m/z 180162) of 2H-C-1

Int. D-1 -MS3(180.0->161.0), 6.2min 113.0

1500

1000

500 130.9 142.9 101.1 71.5 83.3 124.9

0 D-1 -MS3(180.0->161.0), 7.4min 113.0

2000

1500

1000

130.9 142.9 500

101.1 124.9 0 60 80 100 120 140 160 m/z Figure A4.25: MS3 spectra (m/z 180161) of 2H-C-1

264

Appendix 4

Int. 500 D-1 -MS3(180.0->150.0), 6.2min 131.9

400

300

200

101.0 118.9 100 89.2 112.9

0 D-1 131.9 -MS3(180.0->150.0), 7.3min

300

200

86.2 100 90.2

102.0 113.9 118.9

0 60 80 100 120 140 160 m/z

Figure A4.26: MS3 spectra (m/z 180150) of 2H-C-1

Int. D-1 -MS3(180.0->120.0), 6.2min 102.1 1500

1000

500

89.1 0 1500 D-1 -MS3(180.0->120.0), 7.3min 102.1

1250

1000

750

500

250

89.1

0 60 80 100 120 140 160 m/z Figure A4.27: MS3 spectra (m/z 180120) of 2H-C-1

265

Appendix 4

Int. 4000 D-1 -MS3(180.0->119.0), 6.3min 101.1

3000

2000

1000 89.2

0 D-1 -MS3(180.0->119.0), 7.3min 101.1

3000

2000

1000 89.2

0 60 80 100 120 140 160 m/z Figure A4.28: MS3 spectra (m/z 180119) of 2H-C-1

Int. D-1 95.9 400 -MS3(180.0->114.0), 6.1min

86.0 300

200

100

0 D-1 400 -MS3(180.0->114.0), 7.2min 86.0 95.9

300 94.9

200

100

0 60 80 100 120 140 160 m/z Figure A4.29: MS3 spectra (m/z 180114) of 2H-C-1

266

Appendix 4

Int. D-1 -MS3(180.0->113.0), 6.0min 85.3

200

150

95.1

100

0 D-1 -MS3(180.0->113.0), 7.1min 85.3

200

150

95.1

100

0 60 80 100 120 140 160 m/z Figure A4.30: MS3 spectra (m/z 180113) of 2H-C-1

Int. 4 D-1 x10 -MS3(180.0->89.0), 71.4 1.0 6.2min

0.8 59.7

0.6

0.4

0.2

4 x10 D-1 1.0 -MS3(180.0->89.0, 71.4 7.2min 59.7

0.8

0.6

0.4

0.2

0.0 60 80 100 120 140 160 m/z Figure A4.31: MS3 spectra (m/z 180189) of 2H-C-1

267