A GUIDE TO CAROTENOIDA GUIDE TO ANAL This publication is made possible by support from Opportunities for Micronutri- ent Interventions (OMNI) Research, a project of the Office of Health and Nutrition, Bureau of Global Programs, Field Support and Re- search, the U.S. Agency for International Development (USAID) under cooperative A GUIDE TO agreement HRN-5122-A-00-3046-00 with the International Life Sciences Institute (ILSI) Research Foundation. CAROTENOID The information presented herein does YSIS IN FOODS not necessarily reflect the scientific recom- mendations or views of OMNI Research, ANALYSIS IN USAID, or ILSI. Additional copies of this and other OMNI Research publications are available FOODS free of charge to developing countries and for US$3.50 to developed countries. Copies can be ordered from OMNI Research at: RODRIGUEZ-AMAYA OMNI Research ILSI Human Nutrition Institute One Thomas Circle, NW Washington, DC 20005-5802, USA Email: [email protected] Internet: hni.ilsi.org/publications Delia B. Rodriguez-Amaya, Ph.D. A GUIDE TO CAROTENOID ANALYSIS IN FOODS Delia B. Rodriguez-Amaya, Ph.D. Departamento de Ciência de Alimentos Faculdade de Engenharia de Alimentos Universidade Estadual de Campinas C.P. 6121, 13083-970 Campinas, SP., Brasil The use of trade names and commercial sources in this document is for purposes of identification only, and does not imply endorse- ment by ILSI. In addition, the views expressed herein are those of the individual authors and/or their organizations, and do not necessarily reflect those of ILSI. Other publications from OMNI Research are available through the ILSI Human Nutrition Institute website: http://hni.ilsi.org/ publications. OMNI Research ILSI Human Nutrition Institute One Thomas Circle, N.W. Washington, D. C. 20005-5802 Email: [email protected] ILSI PRESS International Life Sciences Institute One Thomas Circle, N.W. Washington, D. C. 20005-5802 ISBN 1-57881-072-8 Printed in the United States of America—2001 CONTENTS Nature of Carotenoids in Foods ......................................................................................................................................... 1 Common Food Carotenoids ...................................................................................................................................... 2 Composition of Carotenoids in Foods ...................................................................................................................... 5 Factors Influencing Carotenoid Composition .........................................................................................................10 Some Physicochemical Properties of Carotenoids ..........................................................................................................14 Solubility ..................................................................................................................................................................14 Light Absorption......................................................................................................................................................14 Adsorption and Partition Properties ........................................................................................................................18 Isomerization and Oxidation ....................................................................................................................................20 Chemical Reactions of Functional Groups ...............................................................................................................22 General Procedure and Sources of Errors in Carotenoid Analysis ................................................................................23 Special Precautions in Carotenoid Work..................................................................................................................23 General Analytic Procedure .....................................................................................................................................24 Prechromatographic Steps .......................................................................................................................................25 Chromatographic Separation ...................................................................................................................................27 Identification and Quantification .............................................................................................................................31 Do’s and Don’ts in Carotenoid Analysis ...........................................................................................................................34 Sampling and Sample Preparation ...................................................................................................................................37 Sampling ...................................................................................................................................................................38 Sample Preparation...................................................................................................................................................38 Sampling and Sample Preparation in Food Carotenoid Analysis ............................................................................39 Open-column Method ........................................................................................................................................................41 Precautions ..............................................................................................................................................................41 Reagents and Apparatus..........................................................................................................................................41 Extraction .................................................................................................................................................................42 Partitioning to Petroleum Ether ................................................................................................................................42 Saponification ..........................................................................................................................................................42 Concentration ..........................................................................................................................................................42 Chromatographic Separation ...................................................................................................................................42 Thin-layer Chromatography ....................................................................................................................................43 Chemical Tests .........................................................................................................................................................44 Identification ............................................................................................................................................................45 Calculation of the Concentration .............................................................................................................................45 iv A Guide to Carotenoid Analysis in Foods High-performance Liquid Chromatographic Methods ....................................................................................................46 Method of Bushway et al. ........................................................................................................................................46 Method of Heinonen et al. .......................................................................................................................................47 Method of Hart and Scott ........................................................................................................................................47 Method of Khachik et al...........................................................................................................................................49 Conclusive Identification ..................................................................................................................................................51 Method Validation and Quality Assurance .......................................................................................................................54 Validation of Methods .............................................................................................................................................54 Quality Assurance ...................................................................................................................................................56 Calculation of Retention in Processed Foods ..................................................................................................................58 References ........................................................................................................................................................................60 v PREFACE There is a worldwide consensus — in different fields of studies and in programs to control micronutrient deficiency and promote better human health — that more extensive and accurate data on the carotenoid composition of foods are urgently needed. Carotenoid analysis, however, is inherently complicated. Nevertheless, the difficulty can be eased