Carotenoids Volume 1A: Isolation and Analysis

Edited by G. Britton S. Liaaen-Jensen H. Pfander

Birkhäuser Verlag Basel • Boston • Berlin

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Contents

International Advisory Board and List of Contributors XXI

Foreword: The late Otto Isler XXVI

Preface to the Series XXIX

Editors' Notes XXXIII

Chapter 1 History: 175 Years of Carotenoid Chemistry

Conrad H. Eugster 1 A. The First Tentative Steps 1 B. The Classical Era: Establishment of the Basic Stnicture 1 C. The Link between Carotenoids and Vitamin A 4 D. Rapid Progress towards the Present 4

1. Chromatographie Separation 5 2. Spectroscopic and other physical methods 5 3. Infrared and Raman spectroscopy 6 4. Mass spectrometry 6 5. NMR spectroscopy 7

E. An Extra Dimension: Stereochemistry 7 1. E/Z Isomerism 7 2. Absolute configuration: chirality 8

F. Synthesis 10 G. Conclusions 10 References 11

Chapter 2 Carotenoids Today and Challenges for the Future

George Britton, Synn0ve Liaaen-Jensen and Hanspeter Pfander 13 A. Natural Occurrence and Distribution of Carotenoids 13 B. Other Natural Pigments 15 C. Chemosystematics 15 D. Structural Diversity 15 E. Structure Elucidation and Analysis 17 F. Properties 18 G. Partial Synthesis 18 H. Total Synthesis 18 I. Industrial Manufacture 19 J. Biosynthesis 20

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K. Metabolism L. Carotenoid Metabolites M. Biological Functions, Actions and Uses

1. Colour and coloration 2. Photosynthesis and photoprotection 3. Nutrition and health

a) Vitamin Aand retinoids b) Carotenoids and protection against disease

N. Need for Basic Research and an Interdisdplinary Approach

Chapter 3 Structure and Nomenclature

Basil C. L. Weedon and Gerard P. Moss A. Introduction B. Structure

1. The basic carotenoid skeleton 2. Structural features and diversity

a) The chromophore b) Oxygen functions c) Carbon skeleton

i) Non-isoprenoid end groups ii) Cyclopentyl end groups iii) Higher carotenoids iv) Apocarotenoids and norcarotenoids

C. Stereochemistry 1. Geometrical isomerism

a) Nomenclature b) Relative stability c) Occurrence of relatively stable isomers d) Sterically highly hindered isomers e) Assignment of geometrical configuration

2. Optical isomerism a) Basic principles b) Chiral carotenoids c) Analysis of mixtures of stereoisomers d) Determination of absolute configuration

3. Conformational isomerism a) Basic principles b) The acyclic polyene chain c) Cyclic carotenoid end groups d) Ring-chain conformation: the 6,7-bond e) Examples

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D. Specification of Absolute Configuration 45 1. The Sequence Rule 46

a) Sub-rule 1 46 b) Sub-rule 2 47 c) Chiralaxes 47 d) Prochirality 48 e) Examples 48

Example 1. Zeaxanthin (119) 48 Example 2. ß,e-Carotene (7) 50 Example 3. Capsorubin (413) 51 Example 4. Violaxanthin (259) 52 Example 5. The allenic end group 53

E. NomenclatureofCarotenoids:IUPAC-IUBRules 54 Rule Carotenoid 1. Class of Compound 54 Rule Carotenoid 2. The stem name 55 Rule Carotenoid 3. Specific names; end group designations 56 Rule Carotenoid 4. Numbering of carotenoid hydrocarbons 57 Rule Carotenoid 5. Norcarotenoids and secocarotenoids 58 Rule Carotenoid 6. Changes in hydrogenation level 59 Rule Carotenoid 7. Oxygenated derivatives 60 Rule Carotenoid 8. Numbering of oxygenated derivatives 62 Rule Carotenoid 9. Retro nomenclature 64 Rule Carotenoid 10. Apo nomenclature 65 Rule Carotenoid 11. Higher carotenoids 66 Rule Carotenoid 12. Stereochemistry 66 Rule Carotenoid 13. Trivial names 69

References 69

Chapter 4 Chemical Derivatization: Microscale Tests for the Presence of Common Functional Groups in Carotenoids

Conrad H. Eugster 71 A. Introduction 71 B. Chemical Tests for Common Functional Groups 71

1. Esters 72 2. Carbonyl functions 73

a) Reduction of aldehydes and ketones 73 b) Reduction of carboxy groups 73

3. Hydroxy groups 74 a) Acetylation of primary and secondary hydroxy groups 74 b) Silylation of tertiary hydroxy groups 74 c) Oxidation of allylic primary and secondary hydroxy groups 75

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d) Methylation of hydroxy groups 76 e) Epoxides; furanoid rearrangement 76

C. Preparationof Diastereoisomeric Derivatives 78 References 80

Chapter 5 Isolation and Analysis

Katharina Schiedt and Synn0ve Liaaen-Jensen 81 A. Properties and General Precautions 81

1. Properties 81 a) Colour 81 b) Stability 81 c) Solubility 82

2. General precautions 82 a) Atmosphere: exclusion of oxygen 82 b) Antioxidants 82 c) Temperature 83 d) Light 83 e) Avoidance of acid or alkali 83

3. Purification of solvents, adsorbents and reagents 83 4. Required laboratory equipment 84

B. Artifacts 84 1. Definitions 84 2. Production of artifacts in biological material before extraction 85 3. Production of artifacts during extraction and work-up 85

a) Geometrical isomers 85 b) Allenic isomerization 86 c) Epoxide-furanoid oxide rearrangement 86 d) Artifact formation catalysed by base 86

i) Ester hydrolysis 86 ü) Aldol condensation 86 iii) Dehydration 88 iv) Retro-&\do\ cleavage 88 v) Other alkali reactions 89

e) Air oxidation 90 f) Reactions on active surfaces 90

C. Extraction and Isolation 91 1. Biological material 91

a) Introduction 91 b) Storage 91

i) Deep-freezing 91 ii) Air-drying 91 iii) Lyophilization 92

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iv) Re-moistening of dried samples 92 2. Disruption and homogenization 92

a) Homogenization of plant and animal tissues 92 b) Dehydration of fresh or frozen animal material 92 c) Disruption of microbial cells 93

3. Extraction 93 4. Removal of water and solvents 94 5. Saponification 94

a) Normal saponification 95 i) Carotenol esters 95 ii) Esters of carotenoid carboxylic acids 95

b) Anaerobic saponification 95 c) Enzymic hydrolysis 97

6. Isolation and purification 97 a) Strategy 97 b) Factors determining choice of method 98

i) Quantity 98 ii) Properties 98 iii) Contaminants 98

c) Non-chromatographic methods: Removal of colourless impurities 98 i) Precipitation 98 ii) Partition or phase Separation 100 iii) Crystallization 101

7. Identification 102 a) Minimum identification criteria 102 b) Geometrical isomers 102 c) Füll characterization, structure elucidation 103

D. Quantitative Analysis 103 1. Principles 103 2. Spectrophotometric determination of a pure carotenoid 103 3. Calculation 104 4. Determination of absorption coefficients 105 5. Carotenoid content in natural extracts and fractions 105 6. Quantitative analysis by HPLC 107

References 107

Chapter 6 Chromatography: Part I. General Aspects

Hanspeter Pfander 109 A. Introduction 109 B. Theoretical Principles and Definitions 109

1. Liquid-solid chromatography 112 2. Liquid-liquid chromatography 113

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3. Bonded-phase chromatography C. Strategy References

Chapter 6 Chromatography: Part II. Column Chromatography

Kurt Bernhard A. Introduction

1. Definitions 2. Scope and limitations

B. Equipment 1. Columns 2. Stationary phases

C. Practica! Approach 1. General aspects 2. Column packing

a) Slurry packing b) Snow-in packing c) Dry packing

3. Separation D. Examples

References

Chapter 6 Chromatography: Part III. Thin-Layer Chromatography

Katharina Schiedt A. Introduction B. Advantages of TLC

1. General aspects 2. Monitoring of reactions 3. Radioactive samples

C. Apparates and Materials 1. Plates

a) General b) Preparation of plates in the laboratory c) Impregnation

i) Reversed-phase (RP) chromatography ii) Incorporation of citric acid

d) Washing 2. Developing Chambers 3. Solvents

D. Practical Aspects 1. Sample size and application

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2. Development and detection 3. Elution

E. Strategy and Applications 1. Preliminary tests 2. General strategy for biological extracts 3. Separation of different classes of carotenoids

a) Carotenes b) Hydroxycarotenoids c) 5,6-Epoxidesand5,8-epoxides d) Carotenoids containing the 3-hydroxy-4-oxo-ß end group (e.g. astaxanthin)

or the 3-hydroxy-4-oxo-2-nor-ß end group (e.g. actinioerythrol) e) Acetylenic astaxanthin analogues f) Apocarotenoids g) Glycosides and glycosyl esters h) Sulphates i) E/Zisomers j) Optical isomers k) Samples containing Chlorophyll or bacteriochlorophyll

References

Chapter 6 Chromatography: Part IV. High-Performance Liquid Chromatography

Hanspeter Pfander and Reto Riesen A. Introduction B. Apparates for HPLC

1. Pumps 2. Sample injectors 3. Pre-columns 4. Columns 5. Detectors 6. Photodiode-array detector

C. The Chromatogram 1. General features 2. Display of data from photodiode-array detection

D. Practica! Advice for HPLC 1. General remarks 2. Stationary phase: columns

a) General considerations b) Selection of column c) Packing a column d) Testing a column

137 138 138 138 139 140 140 140 141

141 141 141 142 143 143 143 144 144

145 145 146 146 148 149 149 150 151 151 151 153 153 153 155 155 155 156 157

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e) Column regeneration 157 f) Equilibration 158

3. Mobile phase 158 4. Quantitative analysis 159

a) General aspects 159 b) External-standard calibration 160 c) Internal-standard calibration 160

5. Semi-preparative separations 161 E. Examples of the Separation of Carotenoids by HPLC 161

1. Introduction 161 2. Test chromatograms 162 3. Overview chromatogram 162 4. Separation of carotenes (hydrocarbons) 164 5. Separation of hydroxycarotenoids 168 6. Fatty acid esters 173 7. Separation of EIZ isomers 174

a) ß,ß-Carotene (3) 175 b) ß,e-Carotene (7) 175 c) Other carotenes 176 d) Ketocarotenoids 177 e) Hydroxycarotenoids 179 f) Spirilloxanthin (166) 179 g) Fucoxanthin (369) and neoxanthin (234) 179 h) C50 Carotenoids 180 i) Apocarotenoids 180

8. Separation of optical isomers 182 a) Carotenes 182 b) Hydroxycarotenoids 183 c) Ketocarotenoids 186

9. Semi-preparative separations 187 References 187

Chapter 6 Chromatography: Part V. Supercritical-Fluid Chromatography

Hanspeter Pfander and Urs Niggli 191 A. Supercritical Fluids 191 B. Supercritical-fluid Chromatography (SFC) 193

1. Principle of Separation 193 2. Instrumentation 193 3. Stationary phases 193 4. Mobile phases 193 5. Detectors 194

C. Applications in Carotenoid Analysis 194

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D. Further Applications of Supercritical Fluids 197 1. Supercritical-fluid extraction (SFE) 197 2. Microcrystal formation 197

E. Outlook 197 References 198

Chapter 7 Worked Examples of Isolation and Analysis 199

A. Introduction 199 B. Isolation of Reference Carotenoids 200

Example 1: Higher Plants George Britton 201 A. Introduction 201 B. Extraction of Carotenoids from Leaves 203

1. General procedures: choice of material and avoidance of artifacts 203 2. Extraction 203 3. Saponification 205 4. Chromatography 206

a) Column chromatography 206 b) TLC 207 c) Identification 208 d) HPLC 208

5. Carotenoids from bleached or stressed leaves 209 C. Carotenoids from Fruits and Flowers 210

1. Procedure: Isolation of carotenes from tomato fruit 210 a) Summary 210 b) Extraction 210 c) Chromatography 210 d) Identification 212

2. Isolation of reference carotenoids from other fruits and flowers 213 References 214

Example 2: Macroalgae and Microalgae Jarle Andri Haugan, Torunn Aakermann and Synn0ve Liaaen-Jensen 215 A. Introduction 215 B. Carotenoids of Brown Seaweeds 217

1. Strätegy 217 2. HPLC analysis of fresh brown algae 218

a) Procedure 218 3. Geometrical isomers of fucoxanthin 219

a) Procedure 219 4. Preparative isolation from brown algae 220

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a) Procedure 221 b) Identification 222

C. Carotenoids of Dinoflagellates 222 1. Preparative isolation from a dinoflagellate bloom 222

a) Procedure 224 b) Identification 224

References . 225

Example 3: Bacteria George Britton and Reto Riesen 227 A. Introduction 227 B. Carotenoids of Phototrophic Bacteria 227

1. Occurrence and distribution 227 2. Strategy 228 3. Procedure 228

a) Extraction 228 b) Saponification 229 c) Chromatography 229 d) HPLC 232 e) Identification 232

C. Carotenoids of Non-phototrophic Bacteria 234 1. Occurrence and distribution 234 2. Procedure: Isolation of C50 acyclic carotenoids from Halobacterium 235

a) Extraction 235 b) HPLC 237 c) Identification 237

References 237

Example 4: Invertebrate Animals Aryl carotenoids from the marine sponge Suberites sericeus

Takao Matsuno 239 A. Introduction 239 B. Procedure 240

1. Extraction and isolation 240 2. Characterization of the carotenoids 241 3. Identification 241 4. Quantitative composition 242

References 242

Example 5: Fish Isolation of astaxanthin and its metabolites from skin of Atlantic salmon (Salmo salar)

Katharina Schiedt, Stefan Bischof and Ernst Glinz 243 A. Introduction 243

B. Procedura 244 1. Extraction 244 2. Partition in hexane/dimethyl sulphoxide 244 3. Saponification of the hexane phase 247 4. Column chromatography 248 5. Thin-layer chromatography (TLC) 248 6. HPLC 249

C. Analysis and Results 251 1. Identification of carotenoids 251

a) Hexane phase 251 b) DMSO phase 251

2. Chiral analysis of zeaxanthin, idoxanthin and astaxanthin 251 a) Zeaxanthin dicarbamate and idoxanthin tricarbamate 251 b) Astaxanthin di(-)-camphanate 252

References 252

Example 6: Egg Yolk Isolation of apocarotenoids as metabolites of 3',4'-didehydro-2'-apo-ß,\j>caroten-2'-al

Katharina Schiedt, Stefan Bischof and Ernst Glinz 253 A. Introduction 253 B. Strategy 254 C. Experimental Procedure 255

1. Extraction and precipitation 255 2. Preliminary tests by derivatization 258 3. Chromatography and identification 258 4. Quantitative analysis 260

Reference 260

Example 7: Human Plasma Simultaneous determination of carotenoids, retinol and tocopherols

Willy Schüep, Denise Hess and Joseph Schierle 261 A. Introduction 261 B. Procedure 261

1. Sample preparation 261 2. HPLC 262 3. Calibration and calculation 263 4. Reliability and precision of results 263

a) Linearity 263 b) Sensitivity 264 c) Recovery 264 d) Reproducibility 264

Reference

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Example 8: Geometrical Isomers of ß,ß-Carotene A rapid routine methodfor quantitative determination

Joseph Schierle, Walter Härdi, Nicole Faccin, Urne Bühler and Willy Schüep 265 A. Introduction 265 B. Experimental Procedura 266

1. Sample processing and extraction 266 a) Extraction of ß,ß-carotene from stabilized preparations 266

i) Enzymic digestion 266 ii) Extraction with organic solvents 266

b) Extraction of ß,ß-carotene from plant materials 267 c) Extraction of ß,ß-carotene from fatty sources 267

2. HPLC 267 3. Calibration and quantitative analysis 268

a) General procedure 268 b) Calculation 270

4. Results and evaluation 270 a) Linearity and sensitivity 271 b) Reproducibility 271 c) Recovery and isomerization during the analysis 272

Example 9: Astaxanthin Determination of stabilized, added astaxanthin infishfeeds and pre-mixes

Willy Schüep and Joseph Schierle 273 A. Introduction 273 B. Procedure 273

1. Extraction and pre-purification 273 2. Analysis by HPLC 274 3. Determination of astaxanthin content in feed samples 275

i) Preparation of Standard Solution 275 ii) Analysis and calculation 276

Example 10: Carotenoid Glycosides and Glycosyl Esters Pascale Meyer, Roland Riesen and Hanspeter Pfander 277 A. Carotenoid Glycosides 277

1. Introduction 277 2. Carotenoid glycosides from Spirulina 278

a) Derivatization 278 b) Identification of the carbohydrate moiety 279 c) Cleavage of the glycosidic bond 279 d) Cleavage of the methyl glycosides 279 e) Optical rotation 279 f) Identification 279

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B. Carotenoid Glycosyl Esters 281 1. Introduction 281 2. Carotenoid glycosyl esters from Stigmas of Crocus sativus L. 281

a) Identification 281 b) Identification of the carbohydrates 282

References 282

Example 11: Carotenoid Sulphates Synn0ve Liaaen-Jensen and Sissel Hertzberg 283 A. Introduction 283 B. Special Methods for Carotenoid Sulphates 284

1. Sulphur analysis 284 2. Electrophoresis 284 3. Column chromatography 284 4. TLC and reversed-phase HPLC 284 5. Conversion of salts into free hydrogen sulphates by ion exchange 284 6. Removal of sulphate groups by acid hydrolysis 285 7. Enzymic hydrolysis 285

C. Isolation of Bastaxanthin c (420) 285 D. Isolation of Other Bastaxanthins 286 References 286

Example 12: Carotenoproteins Peter F. Zagalsky 287 A. Introduction 287 B. The Carotenoproteins of the Lobster Carapace 288 C. Purification of the Carotenoproteins of Lobster Carapace 289

1. Extraction 289 2. Separation of pigments 291

a) DEAE-cellulose chromatography: gradient elution 291 b) Further purification of crustacyanins on DEAE-cellulose with

stepwise elution 291 c) Purification of crustochrin 292 d) Purification of a-crustacyanin by FPLC (fast protein liquid

chromatography) on anion-exchanger Mono Q 292 D. Purification of Ovoverdin 294

1. Procedure 294 2. Storage 294

References 294

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Chapter8 Appendix: List of New Carotenoids

Daniel Kuli and Hanspeter Pfander 295

Index 319