SPRINGER BRIEFS IN FOOD, HEALTH, AND NUTRITION Hong-Sik Hwang Advances in NMR Spectroscopy for Lipid Oxidation Assessment SpringerBriefs in Food, Health, and Nutrition More information about this series at http://www.springer.com/series/10203 Hong-Sik Hwang Advances in NMR Spectroscopy for Lipid Oxidation Assessment Hong-Sik Hwang Agricultural Research Service United States Department of Agriculture Peoria, IL, USA ISSN 2197-571X ISSN 2197-5728 (electronic) SpringerBriefs in Food, Health, and Nutrition ISBN 978-3-319-54195-2 ISBN 978-3-319-54196-9 (eBook) DOI 10.1007/978-3-319-54196-9 Library of Congress Control Number: 2017933956 © The Author(s) 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Abstract Although there are many analytical methods developed for the assessment of lipid oxidation, different analytical methods often give different, sometimes even contra- dictory, results. The reason for this inconsistency is that although there are many different kinds of oxidation products, most methods measure only one kind of oxi- dation product. For the quality control of food products and for better understanding of the factors affecting lipid oxidation, it is necessary to improve the current meth- ods and to develop new analytical methods that provide more accurate assessment of lipid oxidation. NMR spectroscopy techniques including 1H, 13C, and 31P NMR are very powerful and reliable tools to determine the level of lipid oxidation, to identify oxidation products, and to elucidate oxidation mechanism. 1H NMR spectroscopy has demonstrated its reliability, accuracy, convenience, and advantages over conven- tional analytical methods in the determination of the level of oxidation of edible oils during frying and storage by monitoring changes in several proton signals of oil including olefinic, bisallylic and allylic protons. This modern analytical method has been used to identify oxidation products, including primary oxidation products such as hydroperoxides and conjugated dienes and secondary products such as aldehydes, ketones, epoxides, alcohols, dimers and polymers, and their derivatives. By identify- ing intermediates and final oxidation products, mechanisms for lipid oxidation were elucidated. Another type of NMR method, 13C NMR, has also been used to identify oxidation products. The relatively newer method, 31P NMR spectroscopy, can also provide additional information on the molecular structure of an oxidation product. Backgrounds, principles, advantages over conventional methods, most recent advances, and future prospects of these methods will be discussed. Keywords Lipid oxidation • NMR • Oxidation products • Edible oil • Vegetable oil • Oxidation mechanism Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. v Acknowledgements I thank Matthew I. Hwang and Esther Y. Hwang for improving the use of English in and organization of the text. vii Contents 1 Conventional Analytical Methods to Assess Lipid Oxidation ............... 1 1.1 Mechanisms of Lipid Oxidation ........................................................ 2 1.2 Methods to Determine Primary Oxidation Products and Their Problems ............................................................................ 3 1.3 Methods to Determine Secondary Oxidation Products and Their Problems ............................................................................ 6 2 Application of NMR Spectroscopy for Foods and Lipids ...................... 11 3 1H NMR Spectroscopy for Assessment of Lipid Oxidation ................... 15 3.1 Assessment of Lipid Oxidation During Oil Storage .......................... 15 3.2 Assessment of Oxidation During Frying ........................................... 21 3.2.1 Methods Monitoring the Changes of Major NMR Signals .... 22 3.2.2 Methods Using the NMR Proton Relaxation Time ................ 27 3.2.3 Methods Measuring Acyl Groups .......................................... 27 3.2.4 Methods Measuring Aldehydes and Other Oxidation Products ................................................................. 28 4 1H NMR Spectroscopy for Identification of Oxidation Products and for Elucidation of Reaction Mechanisms ........................ 33 4.1 1H NMR to Indentify Oxidation Products During Storage of Oil ...... 33 4.2 1H NMR to Indentify Oxidation Products at Frying Temperatures ... 37 5 Use of 13C NMR Spectroscopy for Determination of Lipid Oxidation ..................................................................................... 43 6 31P NMR Spectroscopy for Assessment of Lipid Oxidation .................. 47 7 Conclusions and Future Prospects .......................................................... 49 References ........................................................................................................ 51 ix Chapter 1 Conventional Analytical Methods to Assess Lipid Oxidation Lipid oxidation is a major cause of the deterioration of quality in food, and accu- rate qualitative and quantitative assessment of lipid oxidation is important for qual- ity assurance of food products (Shahidi and Zhong 2010; Pignitter and Somoza 2012). Lipid oxidation occurs during manufacturing, transportation, storage, cook- ing, and frying of edible oils and oil-containing foods. Although sensory analysis is the most reliable method to evaluate the extent of lipid oxidation, it is not practi- cal for routine analyses and generally lacks reproducibility (Gray 1978). For this reason, numerous chemical and physical analytical methods have been developed to assess lipid oxidation, including conjugated diene value, per oxide value, alco- hols, epoxides, p-anisidine assay, HBR titration, iodometric titration, xylenol orange, total polar compounds (TPC), high performance liquid chromatography (HPLC), fatty acid composition determined by gas chromatography-mass spec- trometry (GC-MS), Fourier transform infrared spectroscopy (FT-IR), volatile products using gas chromatography (e.g. solid phase microextraction), and dimers/ polymers by size exclusion chromatography (SEC) (Schaich 2013b). Again, it is inevitable to have such a huge number of analytical methods for the accurate assessment of the level of oxidation. This is because lipid oxidation is a very com- plicated process involving numerous oxidation products. Compounds formed dur- ing lipid oxidation may vary with different oils (different fatty acid compositions), antioxidants (inherent and/or added), oxidation temperatures, contents of water, acid and other minor ingredients since these factors can alter the mechanisms and routes of the oxidation reactions. Unfortunately, despite tremendous efforts on understanding mechanisms of lipid oxidation, lipid oxidation is not completely understood, and it is almost impossible to accurately predict oxidation products under different oxidation conditions. Among numerous oxidation products, there are two important questions: which oxidation product is the best one to represent the oxidation level of lipid? And which analytical method should be used? The answer depends on the goal of the analysis. Some people may be interested in volatile aldehydes that affect the smell of food © The Author(s) 2017 1 H.-S. Hwang, Advances in NMR Spectroscopy for Lipid Oxidation Assessment, SpringerBriefs in Food, Health, and Nutrition, DOI 10.1007/978-3-319-54196-9_1 2 1 Conventional Analytical Methods to Assess Lipid Oxidation while some may be interested in non-volatile oxidation products, which remain in food and affect taste and health. It should also be noted that, in practical uses, con- venience and quickness of analysis are very important factors in addition to accu- racy and reliability. Since most standardized assays typically measure one kind of oxidation product satisfying one of these goals, one should take precaution when selecting standardized assays. Inconsistency in results from different assays is a serious issue of the current analytical methods for lipid oxidation, and there is no ideal method that correlates well with changes in organoleptic properties of oxidized lipids throughout the entire course of oxidation. For this reason, although it is trou- blesome, it is very common to measure two to four different indications of oxidation at the same time to have more reliable data on the deterioration of oils. To overcome the inconvenience, it has been urged to develop new analytical methods that can combine the concomitant detection of many different oxidation products for a more consistent assessment of lipid oxidation (Pignitter and Somoza 2012). 1.1 Mechanisms of Lipid Oxidation In the 1940s, there were intensive efforts to understand mechanisms of lipid oxi- dation and a free-radical mechanism, and the chain reactions of radicals were proposed (Bolland and Koch 1945; Gunstone and Hilditch 1945; Holman and Elmer 1947). The current understanding of lipid oxidation is based on the pro- posed radical reactions involving initiation, propagation, and termination as shown in Fig. 1.1 (Frankel 2012b). Unsaturated lipids such as oleic, linoleic, and linolenic acids are the most susceptible species to oxidation in food. During lipid oxidation, an unsaturated lipid (LH) loses a hydrogen to form a lipid free radical (L•), which rapidly reacts with oxygen to form peroxyl radicals (LOO•). Peroxyl radicals react with other lipid molecules to produce more lipid radicals in the Fig. 1.1 Free radical reaction mechanism of lipid oxidation (Frankel 2012b) 1.2 Methods to Determine Primary Oxidation Products and Their Problems 3 propagation step. Hydroperoxides (LOOH) undergo a variety of reactions, including thermal or metal-catalyzed homolysis, to produce peroxyl (LOO•), alkoxyl radicals (LO•), alkyl (L•), and other radicals. When these highly reactive radicals accumulate, they react with each other to form many kinds of non-radical products in the termination step. While being widely accepted as the mechanism of lipid oxidation, this free radical chain reaction mechanism cannot explain all the oxidation products. There are on-going efforts to further understand lipid oxidation reactions and courses of reactions other than the hydrogen abstraction in propagation, including β-scission of oxygen, internal rearrangement to epodioxides, addition, disproportionation of lipid peroxyl radicals and many other reactions to alter mechanisms and oxidation products were also proposed (Schaich 2005, 2012, 2013a). 1.2 Methods to Determine Primary Oxidation Products and Their Problems As shown in Fig. 1.1, hydroperoxides (LOOH) are the major primary reaction products of fatty acids. Hydroperoxides of lipid, which are generally referred to as peroxides, are the compounds responsible for further reactions to produce secondary oxidation products (Gray 1978). It is known that the peroxides themselves do not contribute to the off-aromas causing rancidity but that secondary oxidation products, especially car- bonyl compounds, do (Reindl and Stan 1982). Although the peroxides are readily decomposed to other secondary oxidation products in the presence of metals or at high temperatures such as frying temperatures, they are relatively stable at room tempera- ture and in the absence of metals and thus the concentration is built up during the oxi- dation process (Choe and Min 2006). The concentration of peroxides can be determined by several methods and the peroxide value has become one of the most widely used analytical methods for lipid oxidation (Dobarganes and Velasco 2002). Numerous methods were reported to determine the peroxide value, and among them iodometric methods are very widely used. The iodometric method determines the concentration of iodine produced from the reaction between hydroperoxides and hydrogen iodide, which was produced from potassium iodide and acetic acid (Wheeler 1932; IUPAC 1992). The concentration of iodine can be determined by titration with NaSO 2 2 3 (Gray 1978). Figure 1.2 shows the iodometric method in which iodine produced by the reaction of peroxides with hydrogen iodide is determined by titration with NaSO. 2 2 3 The peroxide value is reported in milliequivalents of O per kilogram of sample. 2 The iodometric method can be followed by some other detection methods such as the end-point potentiometric determination (Kanner and Rosenthal 1992; Hara and Totani 1988) and the colorimetric detection of I − at 290 or 360 nm (Frankel 3 2012a; Hicks and Gebicki 1979). However, the iodometric method has the intrinsic problem that iodine can react with double bonds of unsaturated fatty acids and that iodine can be formed by the reaction of potassium iodide with oxygen, which is present in the solution to be titrated (Lea 1952).