Practical Analysis of Flavorand Fragrance Materials

Practical Analysis of Flavorand Fragrance Materials

Edited by

Kevin Goodner and Russell Rouseff

A John Wiley & Sons, Ltd., Publication

This edition first published 2011 2011 Blackwell Publishing Ltd.

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Library of Congress Cataloging-in-Publication Data

Practical analysis of flavor and fragrance materials / edited by Kevin Goodner, Russell Rouseff.p. cm.

Includes bibliographical references and index.ISBN 978-1-4051-3916-8 (cloth)

1. Flavor – Analysis. 2. Flavoring essences – Analysis. 3. Flavor – Biotechnology. I. Goodner,Kevin. II. Rouseff, Russell.TP418.P73 2011664′.07–dc23

2011013226

A catalogue record for this book is available from the British Library.

Print ISBN: 978-1-405-13916-8; ePDF ISBN: 978-1-444-34314-4;oBook ISBN: 978-1-444-34313-7; ePub ISBN: 978-1-119-97521-2; mobi ISBN: 978-1-119-97522-9

Typeset in 10.5/13pt Sabon by Laserwords Private Limited, Chennai, India

Contents

Preface xiii

About the Editors xvii

List of Contributors xix

1 Overview of Flavor and Fragrance Materials 1David Rowe

1.1 Flavor Aroma Chemicals 11.1.1 Nature Identical 1

1.1.1.1 Alcohols 21.1.1.2 Acids 31.1.1.3 Esters 41.1.1.4 Lactones 51.1.1.5 Aldehydes 51.1.1.6 Ketones 6

1.1.2 Heterocycles 61.1.2.1 Oxygen-containing 61.1.2.2 Nitrogen-containing 71.1.2.3 Sulfur-containing 8

1.1.3 Sulfur Compounds 81.1.3.1 Mercaptans 91.1.3.2 Sulfides 9

1.2 Flavor Synthetics 101.3 Natural Aroma Chemicals 11

1.3.1 Isolates 121.3.2 Biotechnology 121.3.3 ‘Soft Chemistry’ 13

vi CONTENTS

1.4 Fragrance Aroma Chemicals 141.4.1 Musks 141.4.2 Amber 151.4.3 Florals 161.4.4 ‘Woodies’ 171.4.5 Acetals and Nitriles 17

1.5 Materials of Natural Origin 181.5.1 Essential Oils 18

1.5.1.1 Cold-pressing – Citrus Oils 181.5.1.2 Steam-distilled Oils 191.5.1.3 A Note on ‘Adulteration’ 20

1.5.2 Absolutes and Other Extracts 21Acknowledgments 21References 21

2 Sample Preparation 23Russell Bazemore

2.1 Introduction 232.2 PDMS 242.3 Static Headspace Extraction 25

2.3.1 Advantages and Disadvantages 252.4 Dynamic Headspace Extraction 26

2.4.1 Advantages 272.4.2 Disadvantages 27

2.5 Solid Phase Microextraction (SPME) 272.5.1 Research 272.5.2 Practical 292.5.3 Advantages 322.5.4 Disadvantages 32

2.6 Stir Bar Sorptive Extraction 332.6.1 Research 332.6.2 Practical 342.6.3 Advantages 352.6.4 Disadvantages 35

2.7 PDMS Foam and Microvial 362.7.1 PDMS Foam 362.7.2 Microvial 36

2.8 Solvent Extraction 392.8.1 MIXXOR 392.8.2 Soxhlet Extraction 39

CONTENTS vii

2.8.3 Solvent Assisted Flavor Evaporation (SAFE) 422.9 Summary 42

References 42

3 Traditional Flavor and Fragrance Analysis of Raw Materialsand Finished Products 45Russell Rouseff and Kevin Goodner

3.1 Overview 453.2 Physical Attribute Evaluation 47

3.2.1 Color – Optical Methods 483.2.2 Turbidity 493.2.3 Water Activity 493.2.4 Moisture Content 50

3.2.4.1 Karl Fischer Method 503.2.4.2 Secondary Moisture Determination

Methods 513.2.5 Optical Rotation 513.2.6 Specific Gravity 523.2.7 Refractive Index 523.2.8 Sugars/Soluble Solids 533.2.9 Viscosity 54

3.3 Instrumental Analysis 543.3.1 Separation Techniques 55

3.3.1.1 Gas Chromatography (GC) 553.3.1.2 GC Retention Data 553.3.1.3 Standardized Retention Index Systems 553.3.1.4 GC Injection 563.3.1.5 GC Columns (Stationary Phases) 583.3.1.6 GC Detectors 60

3.3.2 Identification Techniques 633.3.2.1 Retention Index Approach 633.3.2.2 GC–MS 643.3.2.3 MS/MS 65

References 67

4 Gas Chromatography/Olfactometry (GC/O) 69Kanjana Mahattanatawee and Russell Rouseff

4.1 Introduction 694.2 Odor Assessors’ Selection and Training 704.3 Sensory Vocabulary 71

viii CONTENTS

4.4 GC/Olfactometers (Sniffers) 724.5 Practical Considerations 734.6 Types of GC/Olfactometry 73

4.6.1 Dilution Analysis 734.6.2 Time Intensity 764.6.3 Detection Frequency 794.6.4 Posterior Intensity Method 82

4.7 Sample Introduction 834.8 Identification of Aroma-active Peaks 84

4.8.1 Standardized Retention Index Values 844.8.2 Aroma Description Matching 854.8.3 MS Identifications 854.8.4 Use of Authentic Standards 86

4.9 Conclusion 86References 87

5 Multivariate Techniques 91Vanessa Kinton

5.1 Introduction 915.2 Hierarchical Cluster Analysis (HCA) 975.3 Principal Component Analysis (PCA) 985.4 Classification Models 99

5.4.1 k-Nearest Neighbors (k-NN) 1005.4.2 Soft Independent Modeling of Class Analogy

(SIMCA) 1005.5 Principal Component Regression 1015.6 Example of Data Analysis for Classification Models 102

5.6.1 Tabulating Data 1025.6.2 Examining Data 1035.6.3 Multivariate Exploratory Analysis 1035.6.4 Creation of a Classification Model with a

Training Set and Validation with a Testing Set 106References 109

6 Electronic Nose Technology and Applications 111Marion Bonnefille

6.1 Introduction 1116.2 Human Smell and Electronic Noses 1126.3 Techniques to Analyze Odors/Flavors 113

6.3.1 Sensory Panel 1136.3.2 GC and GC/MS 114

CONTENTS ix

6.3.3 GC/Olfactometry 1146.3.4 Electronic Nose 1156.3.5 Electronic Nose Technology

and Instrumentation 1156.3.5.1 Architecture 1156.3.5.2 Air Generator 1176.3.5.3 Sampling 1186.3.5.4 Detection Technologies 121

6.3.6 Data Treatment Tools 1276.4 The Main Criticisms Directed at the Electronic Nose 1346.5 Market and Applications 136

6.5.1 Application Range 1366.5.2 Perfumery Compound Detection in a Fragrance 1386.5.3 Cosmetic Natural Raw Materials:

Characterization of Volatile Constituentsof Benzoin Gum 139

6.5.4 Home Care Products: Identificationand Quantification Using an Electronic Nosein the Perfumed Cleaner Industry 142

6.5.5 Pharmaceutical Products: Flavor Analysisin Liquid Oral Formulations 146

References 151

7 MS/Nose Instrumentation as a Rapid QC Analytical Tool 155Ray Marsili

7.1 Introduction 1557.2 Operating Principle 1577.3 Advantages of MS over Solid State Sensors 1607.4 Using Other Sample Preparation Modes 1607.5 Techniques for Improving Reliability and Long-term

Stability 1617.5.1 Calibration Transfer Algorithms 1617.5.2 Internal Standards 162

7.6 Two Instruments in One 1637.7 Application Examples 1637.8 Classification of Coffee Samples by Geographic Origin 1647.9 Classification of Whiskey Samples by Brand 1667.10 Future Directions: Partnering MS/Nose with GC/MS 1687.11 Conclusion 170

References 170

x CONTENTS

8 Sensory Analysis 173Carlos Margaria and Anne Plotto

8.1 Introduction 1738.2 The Purpose of Sensory Analysis 1748.3 Flavor Perception 1778.4 Sensory Analysis Techniques 178

8.4.1 Overall Difference Tests 1798.4.1.1 Triangle Test 1808.4.1.2 Duo–Trio Test 1828.4.1.3 Simple Difference Test 182

8.4.2 Single Attribute Difference Tests 1848.4.2.1 Difference from Control 1848.4.2.2 Paired Comparison Test 1848.4.2.3 Ranking Tests 185

8.4.3 Descriptive Tests 1868.4.4 Affective Tests 188

8.5 Preparation and Planning 1898.5.1 Experimental Design 1898.5.2 Environment 1918.5.3 Sample Preparation 191

8.6 Panel Selection 1928.6.1 Trained Panels 1938.6.2 Consumer Panels 194

8.7 Conducting a Panel 1958.8 Expression of Results 1968.9 Conclusions 197

References 198

9 Regulatory Issues and Flavors Analysis 201Robert A. Kryger

9.1 Introduction 2019.2 Regulatory Overview 202

9.2.1 History 2029.2.2 Safety Regulations 2049.2.3 Product Labelling Regulations 2069.2.4 Fair Trade/Conformity with Established

Standards 2089.2.5 Flavor Types 2099.2.6 Governing Authorities 2119.2.7 Role of Flavor Analysis in Regulatory

Conformance 212

CONTENTS xi

9.3 Specific Regulatory Issues 2139.3.1 Identifying the Presence of ‘Forbidden’

Substances 2139.3.1.1 Heavy Metals such as Pb, As, Hg,

and Cd 2149.3.1.2 Pesticides 2149.3.1.3 Environmental Toxins 2159.3.1.4 Allergen Testing 216

9.3.2 Testing Whether a Product is ‘Natural’or Meets a ‘Standard of Identity’ 216

9.3.3 Testing for Other Regulatory ComplianceRequirements 219

References 220

Index 223

Preface

PRACTICAL ANALYSIS OF FLAVORAND FRAGRANCE MATERIALS

Flavor is one of the most important factors in consumer purchases andlong term consumption. However, flavor is not easily quantified as thefactors that impact flavor are almost always trace components. So froma chemical point of view, flavor analysis is essentially trace organicanalysis. The human factor is essential to understanding flavor becausehumans have different genetic and cultural backgrounds which willalter their perception of flavor. Therefore all flavor analysis should beguided by human sensory panels. For too many years the study of flavorwas conducted by analytical chemists who measured what they couldmeasure using traditional analytical techniques rather than quantifyingthose trace impact compounds which should be measured. For manyyears the use of human assessors (sensory analysis) was conductedwithout interest in determining what was producing flavor changesin products being evaluated. Because sensory panels are impractical forroutine quality control purposes, most food and fragrance manufacturershave chosen a middle ground where sensory panel data is used to guidechemists as to which compounds should be monitored to maintainquality or a specific sensory profile.

This book is an attempt to demonstrate how to develop this hybridapproach to flavor analysis. The few books that exist for flavor analysishave exclusively detailed either chemical analysis with sensory input orexclusively sensory analysis without regard to chemical composition.

xiv PREFACE

This book is aimed at the practical side of analytical analyses. Weattempt to produce a book as a reference book or as a primer for ana-lytical chemists who are starting out in the flavor and fragrance industrywith useful chapters on some of the major topics that someone new tothe industry might encounter, including some of the basic tests one mightsee in the labs such as ◦Brix, water activity, turbidity, and similar tests.

David Rowe summarized much of the descriptive information fromhis recent book on Chemistry and Technology of Flavour and Fragranceinto the first chapter. Sample preparation techniques are described byRussell Bazemore in the next chapter. It provides a detailed descriptionof classic and cutting edge sampling techniques that ultimately determinethe success of any flavor analysis. Traditional analytical techniques thathave been used to measure the quality of raw flavor materials and finishedproducts are presented next. Gas chromatography-mass spectrometry isincluded in this chapter as it is the most common technique employedby flavor chemists.

Gas chromatography-olfactometry, GC-O, is a hybrid techniqueemploying the separation power of high resolution gas chromatog-raphy with the particular selectivity and sensitivity of human olfaction.This chapter written by Kanjana Mahattanatawee and Russell Rouseff,covers the hardware, software, and various techniques used for GC-O,along with selected applications, and benefits.

Vanessa Kinton wrote the next chapter on multivariate techniqueswhich are commonly used for data analysis. This chapter describesthe mathematical background and theory behind these techniques. Thefocus is to provide a basic understanding of the theory behind thesemathematical approaches knowing that in practice the procedures arehandled as a ‘‘black box’’. These techniques are used extensively in manyareas of analysis (electronic nose, MS chemsensor, sensory analysis, etc.)and this chapter provides the basics while the other chapters provide theapplication examples.

Chapters 5 and 6, by Marion Bonnefille and Ray Marsili respectively,employ many of the multivariate data treatments for two very differentsensor types. Chapter 5 concerns the metal oxide based electronicnose while chapter 6 is on the MS-based chemical sensor. Althoughboth techniques employ pattern recognition software from instrumentalsensors to mimic human olfaction, they differ profoundly in the typesand number of sensors used to obtain the data arrays.

The chapter on sensory analysis by Carlos Margaria and Anne Plottois likely to be an area in which most chemists have little familiarity. Thischapter provides a wealth of practical information about conducting

PREFACE xv

sensory panels both trained and untrained with many anecdotes fromtheir own experience.

The last chapter describes the ever changing regulations that affectflavor analysis in the industry and is written by Robert Kryger. This is anextremely important issue that is rarely taught in schools or universities.He discusses many of the basic terms and regulations as well as someof the complications in interpreting these regulations which vary fromcountry to country.

The editors hope that this compilation will benefit those scientistsbeginning their careers in the area of flavor. Finally, and most impor-tantly, we wish to thank each contributor for their time and efforts theyput into their respective chapters. This book was a long time in themaking and we are most appreciative of individual authors for theirdedication and expertise in making this book possible.

About the Editors

Kevin L. Goodner received both a B.S.Ch. in Chemistry and a B.S.in Mathematics from the University of Memphis in 1992 and a Ph.d.in Analytical Chemistry from the University of Florida working withFourier Transform Mass Spectrometry. His focus changed to flavorchemistry after a 1.5 year post-doctorial position at the University ofFlorida with Russell Rouseff. Kevin then worked for 9 years at the USDACitrus and Subtropical Products Laboratory researching flavor andquality aspects of many products. In January of 2008, Kevin switchedto industry at Sensus, LLC working on tea, coffee, and other productswhere he currently is the Director of Research and Development. Kevinhas over 50 peer-reviewed publications.

Russell Rouseff is a professor at the University of Florida’s CitrusResearch and Education Center specializing in flavor and color chem-istry. He has 35 years experience in the Florida citrus industry, firstwith the Department of Citrus and then the University. He has writtenor edited five books, 37 book chapters and over 108 referred journalarticles. He has mentored scores of domestic and international students,8 post docs and numerous visiting scientists. He has worked with aromavolatiles in fruits, coffee, wine, flowers and foliage and bitter non-volatiles. He is a Fellow of the American Chemical Society’s Agriculturaland Food Chemistry Division, recipient of the IFT’s Citrus ProductsDivision Research and Development Award and received the AmericanChemical Society’s Award for the Advancement of Food and Agricul-tural chemistry in 2009. Hobbies include salt water reef aquariums,tennis and motor cycles.

List of Contributors

Russell Bazemore, Volatile Analysis Corporation, USA

Marion Bonnefille, Alpha MOS, Toulouse, France

Kevin Goodner, Sensus, LLC, Hamilton, USA

Vanessa Kinton, Alcohol and Tobacco Tax and Trade Bureau (TTB),Ammendale, USA

Robert A. Kryger, Citrus Resources LLC, Lakeland, USA

Kanjana Mahattanatawee, Department of Food Technology, SiamUniversity, Thailand

Carlos Margaria, US Distilled Products, Princeton, USA

Ray Marsili, Marsili Consulting Group,Rockford, USA

Anne Plotto, USDA, ARS, Citrus and Subtropical Products Laboratory,Winterhaven, USA

Russell Rouseff, IFAS, Citrus Research and Education Center, Universityof Florida, USA

David Rowe, Riverside Aromatics Ltd , Poole, UK

1Overview of Flavorand Fragrance Materials

David RoweRiverside Aromatics Ltd, Poole, UK

The nature of this chapter must be that of an overview as the alternativewould be a multivolume series! The difficulty is not a shortage ofmaterial but rather a surfeit, and a second issue is how to give a rationalcoverage; should the materials be classified by chemistry, by odor orby application? The approach here is a combination of all three, and isbased in part on a precis of The Chemistry and Technology of Flavoursand Fragrances [1].

There is, of course, a massive overlap between flavor and fragrance;for example, cis-3-hexenol, discussed below, has a ‘green’, cut-grassodor, and hence contributes freshness to both flavors and fragrances.The division between the two Fs is itself not always a natural one!

1.1 FLAVOR AROMA CHEMICALS

1.1.1 Nature Identical

The vast majority of the aroma chemicals used in flavor are nature identi-cal (NI), that is, they have been identified as occurring in foodstuffs in the

Practical Analysis of Flavor and Fragrance Materials, First Edition.Edited by Kevin Goodner and Russell Rouseff. 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.

2 PRACTICAL ANALYSIS OF FLAVOR AND FRAGRANCE MATERIALS

human food chain. This is a key method of identifying the most impor-tant components which create a flavor, and until recently, there werealso regulatory implications. European Council Directive 88/388/EECdefined these as ‘‘flavouring substances identical to natural substances’’,with the alternative being ‘‘artificial flavouring substances’’, with the lat-ter leading to the stigma of ‘‘artificial flavors’’. The newest regulations,REGULATION (EC) No 1334/2008 OF THE EUROPEAN PARLIA-MENT AND OF THE COUNCIL, no longer differentiates betweenNature Identical and artificial, but the concept is still important – as aguide to flavorists, knowing a material is NI is important, and it canbe especially so the context of ‘‘from the named food’’ type of flavours.Regulation 1334/2008 now only differentiates between ‘‘flavouring sub-stances’’ and ‘‘natural flavouring substances’’, which harmonizes toan extent with the USA, where the NI classification has never beenused. Even there, though, the NI concept has value, as materials haveto be on the FEMA GRAS list, that is they are ‘‘Generally Recog-nized As Safe’’, and the vast majority of such substances are foundin Nature.

1.1.1.1 Alcohols

It should be noted that ethanol 1 itself is a flavor component of ‘alcoholicdrinks’ as anyone tasting alcohol-free drinks will report! In fact it mayconsidered as a solvent (especially in fragrances), as a flavour substance(FEMA 2419) or an additive (E1510)! cis-3-Hexenol 2, mentionedabove, is produced in nature as a ‘wound chemical’, that is, whenplant tissue is damaged, ingressing oxygen is ‘mopped up’ by reactionwith linoleic acid, which generates the unstable cis-3-hexenal, which isenzymatically reduced to the alcohol. Also formed are trans-2-hexenal 3,which has a harsher, more acrid greenness and trans-2-hexenol 4, whichis rather sweeter:

OH

1 2 3

OHOH O

H

4

1-Octen-3-ol, ‘mushroom alcohol’ 5, has the earthy note character-istic of mushrooms. The ‘terpenoid’ alcohols, C10 derivatives, includegeraniol 6 and its isomer nerol 7, citronellol 8 and linalool 9 [2]. Cyclicterpenoid alcohols include α-terpineol 10 and menthol 11:

OVERVIEW OF FLAVOR AND FRAGRANCE MATERIALS 3

6

OH

OH5

97 OH

OHOH

8

10

OH

OH

11

Benzyl alcohol 12 has relatively little odor and is more commonlyused as a solvent in flavors; phenethyl alcohol 13 is a component ofrose oil and has a pleasant rose-like aroma. Two important phenols arethymol 14 and eugenol 15, which are also major components of thymeand clove oils respectively:

OHOH

OH

OOH

12 13 14 15

1.1.1.2 Acids

Simple acids contribute sharp notes which often become fruity on dilu-tion. Butyric acid 16 is indisputably ‘baby vomit’ in high concentration;valeric acid 17 is cheesy, whereas 2-methylbutyric acid 18 is fruitier.Longer chain acids such as decanoic 19 are fatty and are important in