ANA 1: Analytical Standards for Lipid Analysis
Chair(s): J. King, University of Arkansas, USA
Development of Standard Reference Materials for Fatty Acid Analyses. K. Sharpless, M. Schantz, L. Sander, S. Wise, NIST, Gaithersburg, MD, USA
The National Institute of Standards and Technology (NIST) has been providing food-matrix Standard Reference Materials (SRMs) with values assigned for fatty acids for a number of years. The early SRMs with values assigned for fatty acids consisted of foods with a number of different fat/protein/carbohydrate compositions such as infant formula, peanut butter, chocolate, and fish tissue. The proximate composition of the test food being analyzed can then be matched to that of the SRM being used as a quality control material. However, in the case of fatty acids, matching of the fatty acid profile may be just as important. More recently, NIST has initiated the development of SRMs for dietary supplements, including an oil-based carrot extract, four plant oils (evening primrose, perilla, borage, and flaxseed), and a vegetable oil mixture. In addition, biodiesel SRMs are being produced. All of these natural-matrix SRMs are intended for use in (1) validating the accuracy and precision of new analytical methods, and (2) providing quality control for analysis of similar materials.
Use of Lipid Compounds as Analytical Standards. J.D. Moore, W.V. Caufield, W.A. Shaw, Avanti Polar Lipids, Inc., Alabaster, AL, USA
Lipids function as essential and abundant building blocks of cells and their organelles, neurological second messengers, nutrients, hormones, protein modifiers and substrates of enzymes to name a few and are implicated in many disease processes. However, there are few sources of chemically pure and structurally characterized lipid reference standards. None are available for major lipid classes through certified sources such as the United States Pharmacopoeia, National Formulary, National Institute of Technology and Standards, nor from international agencies. Available lipid compounds chosen for preparation as standards are difficult to maintain as pure entities of stable concentration due to their physical and chemical interactions. Qualification, preparation and use of lipid compounds as analytical reference standards are daunting endeavors. The lipid chemist must understand these constraints for each chosen molecule to construct a standard material which provides accurate measurement for a practical length of time. Chemical stability and therefore constant concentration is a major concern for any standard material. Once chemically characterized, they should be packaged and stored into containers which achieve the best stability for extended use. And lastly, these packaged standards require testing over time to document their continued structural and concentration integrity.
The Need for Standards in the Execution of Single Lab Validations and Collaborative Studies of Lipid Methods. D.M. Sullivan1,2, 1Covance Laboratories, Madison, WI, USA, 2AOAC International, Gaithersburg, MD, USA
There has been a significant number of new lipid methods developed in the last several years that improving the way we measure total fatty acids, trans isomers of fatty acids, and omega 3 fatty acids. These new methods require validation and in some cases need to be subjected to collaborative study trials. In order to fully validate these methods analytical standards are needed for these lipid components in order to evaluate the selectivity, specifity and accuruacy of the analysis.In the collaborative study matrix standards are a very useful tool. They allow the study to evaluate the accuracy of the different laboratories participating in the studies.This presentation will provide detailed information about analytical method validation of lipid methods and the collaborative study process. The AOAC International and other collaborative study protocols will be presented. Several examples will be given on how the use of analytical standards and matrix standards are critical to the success of these studies.
Synthesis and Gas Chromatographic Separation of Monounsaturated Fatty Acids. P. Delmonte , Q. Hu, A.-R. Fardin Kia, J.I. Rader, US Food and Drug Administration, College Park, MD, USA
Following the finding of an association between consumption of foods containing trans fat and increased risk of coronary heart disease, several countries implemented new regulations regarding the labeling of trans fat content of foods. Trans fat quantitation is commonly achieved by GC using long polar capillary columns. Monounsaturated fatty acids (MUFA) are generally the primary constituents of trans fat in foods. This study is focused on the synthesis of mixtures containing MUFA not available as reference material, and therefore generally not quantitated during fatty acid analysis. A MUFA with the double bond in position n is brominated using hydrobromic acid in glacial acetic acid, yielding the n and n+1 monobrominated fatty acid. The products are then reacted with sodium t-butoxide in t-butanol yielding the positional isomers of the starting fatty acid with the double bond in position n-1, n and n+1. Mixtures containing potentially all of the positional isomers of the staring material were obtained by multiple repetitions of the process. The final products were then separated into cis and trans geometric isomers by silver ion HPLC. Positional isomers of each monounsaturated fatty acid were identified by GC coupled with covalent adduct chemical ionization mass spectroscopy (GC-CACI-MS) using acetonitrile as the reagent gas.
Reference Material Characterization and the Quality of Your Product. B. Schaneberg, ChromaDex, Inc., Irvine, CA, USA
The market for the use of natural products in one's diet and health regime is increasingly growing and changing all at the same time. Old products are re-formulated or new hip products take the market by storm and then fall by the way side after the current dietary fad. Now with the FDA cGMPs official for dietary supplements, guidelines are now in place to ensure consumers are purchasing a safe and quality product. The quality and safety of a product is highly dependent on the reference material used during the analysis process, whether qualitative or quantitative. Proper testing procedures begin with a proper reference material and/or standard. The level of characterization of the reference material can have a drastic effect on the quantitative values obtained. This can then in turn have a major effect on the biological and health benefits of the product. Depending on the testing protocols used for the characterization of a reference material, there can easily be a difference of 10% or higher in the analytical values. This presentation will discuss and define the proper characterization of a reference material and give examples on how different reference materials will have a direct effect on the quality and safety of a product.
Isotopically-Labelled Fatty Acids. R.O. Adlof, Retired, USDA, NCAUR, Peoria, IL, USA
Fatty acids (FAs) labelled with stable isotopes such as deuterium (2H) or carbon-13 (13C), or with radioactive isotopes such as tritium (3H), carbon-11 (11C) or carbon-14 (14C), have found prominent roles in the study (in vivo and in vitro) of the incorporation, metabolism, oxidation and interaction of lipids. Isotopically-labelled FAs have been prepared chemically by well-documented syntheses and biologically by incubating algal or bacterial cultures with labelled-FAs, -water, -carbon dioxide or -acetates. FAs labelled with stable and radioactive isomers may also be purchased from a variety of commercial sources, and a listing of suppliers (including Internet addresses) will be included. The presentation will conclude with general methods for analysis of labelled fatty acids, their storage, as well as specific applications in living organisms, from bacteria and viruses, to plants and animals and, as 2H or 13C-labelled isomers, in humans subjects.
Development of Commercial Analytical Standards for Oil Fat Methodologies. K. Kiefer, J. Walbridge, L. Sidisky, S. Cecil, Supelco / Sigma Aldrich, 595 North Harrison Road, Bellefonte, PA 16823-0048, USA
Every laboratory involved in oil/fat testing must maintain appropriate standards for the various methods generated by the American Oil Chemists' Society (AOCS). It is impractical for each laboratory to develop and test all the necessary standards. Therefore, commercial analytical standards are a necessity for any oil fat program. This presentation will cover the steps involved in developing high quality, readily available biodiesel analytical standards for consumers. A commercial laboratory is properly equipped with trained personnel, documentation (MPS and SOP), and calibrated equipment and instrumentation to manufacture and test accurate and reliable analytical standards. ISO certified manufacturers of commercial analytical standards have systems in place that document methods, procedures, and records that provide continued assurance of compliance through external audits.The quality assurance program must combine thorough testing of raw materials and solutions. Raw materials are tested by several independent methods; solutions are tested for component concentration, compatibility with solvent and other potential components in the solution, homogeneity and shelf life. The methods and technical requirements will be described for neat compounds and solutions in developing analytical standards.
HPLC Analysis of Triacylglycerol in CLA Enriched-Soy Oil: Role of Standards. R. Lall, V. Jain, A. Proctor, University of Arkansas, Fayetteville, AR, USA
A 20% CLA rich-soy oil has been produced by soy oil photoisomerization with an iodine catalyst. However, the isolation and characterization of the TAGS in CLA-rich oil has not been described. The objective of this research was to analyze CLA-rich oil TAGS by RP-HPLC. This was done by using analytical HPLC method with an acetonitrile / dichloromethane gradient (2 C18 5µ columns, 4.6mm x 500mm) with ELSD and UV detectors. New TAG peaks were observed that were probably derived from linoleic acid containing TAGS. The LnLL, LLL, LLO and LLP declined after isomerization with an increase in adjacent peaks that co-eluted with LnLnO, LnLO, LnOO and LnPP respectively. The newly formed peaks were wider than those of the original oil, possibly suggesting presence of various TAGS containing various CLA isomers. FAME analysis by GC was challenging due to limited sample size injections. Since no CLA TAG-standards were available, a semi-preparative HPLC system (5µ column, 10mm x 500mm) was used to enable injections of 25mg oil samples. This allowed subsequent FAME fraction analysis. Newly formed peaks were, once again, wider than those of the original oil. Therefore, new peaks may represent a collection of CLA containing TAGS. Quantification may have been simpler if CLA TAG-standards were available.
Using Theoretical Correction Factors (TCFs) for Quantitative Analysis of Sterols. C.D. Costin, S.L. Hansen, D.P. Chambers, Cargill Inc., Global Food Research - Scientific Resources Group, Wayzata, MN, USA
A common detector for the analysis of sterols by gas chromatography is the flame ionization detector (FID). The detector measures the response of ions from a molecule as it is pyrolyzed in a hydrogen flame. The response is relative to the number of hydrogen and carbon atoms in a molecule and it gives different responses for the same amounts of different analytes. Theoretical Correction Factors (TCF) can be used to account for these differences during data analysis and are based upon the number of active carbons in a molecule and can be calculated from know structures. TCF have been used to provide a more reproducible approach to the analysis of sterols in raw materials. Use of Empirical Correction Factors (ECF) requires determination of relative responses for each analyte and also requires a high purity standard (and determination of that purity), which can be expensive or difficult to obtain for the more uncommon sterols. Experiments have been conducted to determine ECF for Campesterol, Stigmasterol, and b-Sitosterol and compare them to the calculated TCF for the same sterols. The experiments evaluated differences in concentration, standard purity, and injection technique. The comparison between ECF and TCF is made by determining the percent difference between ECF/TCF.
AOCS Methods Development: Process, Verification, and International Participation. R. Cantrill, G. Clapper, AOCS, Urbana, IL, USA
Since the foundation of AOCS almost 100 years ago, the methods have been developed and used by the fats and oils industry for routine analysis of feedstock and product quality. The latest printing of The Official Methods and Recommended Practices of the AOCS (5th edition) still contains methods from those early days that remain industry standards today. There is a rigorous process applied to method acceptance. Methods Subcommittees and Expert Panels review the preliminary request for recognition, may request additional information and laboratory results prior to recommending the acceptance of a procedure as a Recommended Practice, Standard Procedure or Official Method. Final acceptance is in the hands of the Uniform Methods Committee. The use of AOCS methods in global trade has meant that our methods acceptance procedures conform to internationally agreed-upon standards such as the AOAC/IUPAC Harmonized protocol or ISO 5725:1994. Both of these standards set the requirements for collaborative study and the statistical approach for the determination of method precision data. When the method performance data conform to these standards, the analyst can determine if the method is suitable for their application (fit for purpose). Analysts can also determine their analytical ability in the AOCS Laboratory Proficiency Program and through the use of AOCS reference materials.To facilitate trade, for many years AOCS has maintained strong relations with other standards development organizations (SDO) to ensure that our methods are in line with those of other organizations. Therefore AOCS is both a liaison organization and the US representatives at the relevant ISO committees. AOCS is also represented at many other organizations including the International Union of Pure and Applied Chemistry, and as a non-governmental organization (NGO) with observer at Codex Alimentarius.
The Combined Application of Empirical Correction Factors (ECF) and Theoretical Correction Factors (TCF) for AOCS Ce 1i-07. S. Hansen1, G. Perri 1, J. M. Curtis 2, S.L. Ehler3, S. Diltz4, W.M.N. Ratnayake5, 1Cargill Incorporated, Wayzata, MN, USA, 2University of Alberta, Edmonton, Alberta, Canada, 3Canadian Food Inspection Agency, Dartmouth, Nova Scotia, Canada, 4Martek Biosciences, Columbia, MD, USA, 5Food Directorate, Health Canada, Ottawa, ON, Canada
A pre-collaborative study was performed for AOCS Ce 1i-07 for the determination of saturated, cis-monounsaturated and cis-polyunsaturated fatty acids in marine and other oils containing C20 and C22 long chain polyunsaturated fatty acids (LC PUFA) by capillary gas-liquid chromatography (GLC). A similar AOCS official method Ce 1h-05 strictly employs theoretical correction factors (TCF) for the determination of cis-, trans-, saturated, monounsaturated and polyunsaturated fatty acids in vegetable oils, where the primary fatty acids are not C20 and C22 LC PUFA. It was determined that the use of TCF for marine and other oils which do contain high amounts of C20 and C22 LC PUFA does not ensure the best possible accuracy. Instead, the use of empirical correction factors (ECF) is required in order for the fatty acid content obtained using GLC to approach the known total fat content of the sample and to ensure the accurate determination of individual C20 and C22 LC PUFAs including arachidonic acid (ArA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). The ability to accurately determine EPA and DHA in oils and extracted fat is now highly desirable since food products containing these fatty acids in sufficient quantities can now be labeled with a qualified health claim for omega-3 fatty acids.
ANA 2.1/BIO 2.1: Analytical Methodology in Functional Genomics
Chair(s): R. Weselake, University of Alberta, Canada; and J.T. Lin, USDA, USA
A Method of Regiospecific Analysis of Triacylglycerols by ESI-MS3 and Its Use for Olive Oil Analysis. J.T. Lin, United States Department of Agriculture, Albany, CA, USA
A method of regiospecific analysis of triacylglycerols (TAG) in vegetable oils and animal fats is reported here using electrospray ionization MS3 of TAG lithiated adducts. The fragment ions of the MS3 from the loss of fatty acids at the sn-2 position as α,β-unsaturated fatty acids were used for the regiospecific identification and quantification. The ratio of the regiospecific TAG, ABA and AAB, in a oil sample usually fraction collected by HPLC can be determined by the ratio of the abundances of the fragment ions of [ABA + Li – ACOOH – BCH=CHCOOH]+ and [ABA + Li – ACOOH – ACH=CHCOOH]+. The method was used to analyze regiospecific TAG in extra virgin olive oil. The results showed that the saturated fatty acids, palmitic acid and stearic acid, were mostly located at the sn-1,3 positions and unsaturated fatty acids, oleic acid and linoleic acid, were mostly located at the sn-2 position.
Tilling in the Botanical Garden: a Reverse Genetic Technique Feasible for all Plant Species. G.W. Haughn, E. Gilchrist, University of British Columbia, Vancouver, BC, Canada
TILLING is a reverse genetics technique that can identify a series of alleles or SNPs in a target gene. It can be adapted for use in a high-throughput facility, and has been successfully applied to all organisms tested in our laboratory. To date we have established TILLING in Arabidopsis thaliana, Caenorhabditis elegans, and Brassica oleracea, and we are currently testing this technique in Brassica napus. We have also used the technique to look at natural variation in Populus trichocarpa (here known as Ecotilling).The Brassica project on which we are currently working, is part of an oilseed initiative funded by Genome Canada/Genome Alberta project entitled Designing Oilseeds for Tomorrow's Markets and the Alberta Value-Added Corporation-sponsored project, Bioactive Oils Program. The goals of the project are to develop Canola with optimal seed coat characteristics and minimal levels of anti-nutritional factors. It is anticipated that the results of this research will enhance the overall usefulness of canola seed leading to improved meal for new food and feed applications, and increased seed oil content. We are generating a population of 6000 EMS-mutagenised B. napus lines for TILLING, and have begun to use TILLING to identify mutations in several genes involved in oil biosynthesis and or modification. Our results to date indicate that TILLING will be a powerful addition to the reverse genetic techniques available to Brassica napus researchers.
Chemical and Flavor Profile of Genetically Modified Peanut Varieties. N.T. Dunford1, R. Jonnala1, E. Ng1, K. Chenault2, 1Oklahoma State University, Stillwater, OK, USA, 2USDA, ARS, Stillwater, OK, USA
Peanut (Arachis hypogaea L.) is an economically important crop throughout the world. It is susceptible to many types of fungal pathogens. Genetic engineering offers great potential for developing peanut cultivars resistant to a broad spectrum of pathogens that pose a recurring threat to peanut health. In an effort to improve the disease resistance of peanuts, transgenic peanut lines were developed. Somatic embryos of the peanut cultivar Okrun were transformed with a chitinase gene from rice and/or a -1-3- glucanase from alfalfa via microprojectile bombardment. Modified peanut lines have been tested for S. minor resistance under greenhouse and field conditions Three transgenic peanut lines, 188, 540 and 654 showed increased resistance to fungal diseases, as compared to the parent line.The main objective of this research project was to assess the “substantial equivalence” and nutritional safety of biotechnology-derived peanut lines. The three transgenic peanut lines were analyzed for their oil, protein, ash, moisture, sugar, total dietary fiber, mineral and fatty acid, tocopherol, phytosterol and phospholipid compositions. The flavor analysis was performed using a gas chromatograph/mass spectrometer (GC/MS) equipped with an olfactory detector. The compositions of transgenic lines were compared to those of the parent cultivar Okrun.
Improved Resolution of Reverse Isomers of 1, 2-Diacylglycerols as 3, 5-Dinitrophenylurethanes by Reversed-Phase HPLC on Polymeric Octadecyl Silica Columns. Y. Itabashi1, C. Aizawa1, A. Kuksis2, 1Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan, 2Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
The resolution of reverse isomers still remains a major unsolved problem in glycerolipid chromatography. We have investigated HPLC behavior of the reverse isomers of 1,2-diacylglycerols (DAG) on different types of octadecyl silica (ODS) columns. The reverse isomers of DAG having various pairs of acyl groups including highly unsaturated chains were chromatographed as 3,5-dinitrophenylurethanes (DNPU). Complete resolution of the reverse isomers possessing minor differences in chain lengths and degree of unsaturation, such as16:0-18:0 and 18:0-18:1, which had not been resolved previously by any chromatography techniques (Itabashi et al., J. Chromatogr. A 893, 261-279. 2000), was achieved within 60 min on a polymeric ODS column (150 x 4.6 mm i.d., 3 μm particles) using methanol as the mobile phase. Excellent resolution was also obtained for the reverse isomers of very different pairs of acyl groups, such as 16:0-22:6. The less polar isomers with the acyl chains of the higher degree of unsaturation or shorter chain in the sn-2 position were retained more strongly than the respective sn-1 isomers. The highly dense packing of octadecyl groups in polymeric ODS and the planar structure of the DNPU moiety in the derivatives may contribute to the separation.
Plant Hormone Profiling—A Targeted Metabolomics Technology for Oilseed Crop Improvement. S.R. Abrams, National Research Council of Canada, Saskatoon, SK Canada
Plant hormones regulate gene expression, control development, and regulate plants' responses to the environment through complex signaling networks. In particular, the plant hormone abscisic acid (ABA) regulates synthesis of genes involved in lipid synthesis and modification. For functional genomics research programs, we are developing and implementing technology for measuring multiple plant hormones in plant tissue samples (Chiwocha et al. 2005 Plant Journal 42: 35-48). We measure biologically active forms of the hormones and also inactive catabolites, which together afford an overview of hormone metabolism. Our approach is based on liquid chromatography coupled with tandem mass spectrometry, using specific deuterated internal standards to quantify more than twenty hormones and metabolite of interest in a single experiment, without derivatization and with minimal sample work up. Examples to be discussed include hormone profiling of developing seeds of Brassica napus, correlating with, oil content and gene expression information obtained through microarray analysis; determining the roles of ABA catabolites in regulating oil synthesis; hormone profiles of developing Brassica napus seeds and Arabidopsis seedlings subjected to freezing, drought, and biotic stresses.
Detection and Quantification of TAG Accumulation in Recombinant Yeast Systems. R.M.P. Siloto, M. Truksa, R.J. Weselake, Dept. of Agricultural, Food and Nutritional Science; University of Alberta, Edmonton, AB, Canada
Metabolomic Approach in Studying Biochemical Effects of Alfa-Tocopherol Using a Rat Model. A. Moazzami1, C. Sandström2, A. Kamal-Eldin1, 1Dept. of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2Dept. of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden
Metabolomics is a new approach in which profiles of metabolites in different tissues and/or biofluids are investigated to understand the changes, which is induced in the profile of metabolites, following an induced modulation. We have used this approach to investigate the biochemical activities of α-tocopherol in the liver using a rat model. 21-day Rats were either fed a sufficient control (n=10) or a deficient diet (n=12) for two month before sacrifice. Their livers were collected and extracted with chloroform-methanol-water. The extracts were analyzed using 1H-NMR and GC-MS to profile the metabolites and the data was analyzed using multivariate statistical method (PCA and PLS). The statistical analysis revealed that α-tocopherol modulates the contents of some amino acids, amino acid intermediate and lactate suggesting the possible impact of α-tocopherol on energy metabolism in rat liver.
ANA 2: Antioxidants and Natural Extracts Analysis
Chair(s): R. Della Porta, Frito-Lay Inc., USA; and A. Proctor, University of Arkansas, USA
Interaction of Terpenyl Esters and Polydimethyl Siloxane with Tocopherols in Frying Oil Degradation. J.A. Gerde, E.G. Hammond, P.J. White, Dept. of Food Science and Human Nutrition, Iowa State University, Ames. Iowa, USA
The protective effect of terpenyl esters and polydimethyl siloxane (PDS) in vegetable oil held at frying temperature has been previously demonstrated. In the current work, soybean oil containing 0.1 % of linalyl oleate (LO), menthyl oleate (MO), menthyl caprate, menthyl stearate or 5 ppm of PDS were heated at 180 °C for 32 hr, and the oil degradation monitored. Fatty acid compositions were determined at selected times and the linoleic to palmitic acid ratios were calculated. The slopes of the logs for this parameter decreased as a consequence of oil degradation. Oil with added PDS had a slope of -0.004, whereas all other treatments had slopes ranging from -0.0181 for oil with added MO to -0.0199 for oil with no additives. For LO, the rate of degradation increased with increasing surface-to-volume ratios. Further, tocopherol concentrations were determined and their degradation rates calculated to evaluate the impact of their disappearance on oil degradation as related to the additives.
Relationship Between Free Radical Scavenging Activity of Non-Polar Antioxidants and Their Ability to Inhibit Lipid Oxidation in Food Systems. J. Alamed, D.J. McClements, E.A. Decker, University of Massachusetts, Amherst, MA, USA
Antioxidants are useful tools to inhibit oxidation of lipids. The effectiveness of antioxidants varies as a function of the chemical and physical properties of both the antioxidants and the foods to which they are added. Free radical scavenging activity of non-polar antioxidants [butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), rosmarinic acid (RA) and α-tocopherol (TOC)] was measured using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method. It was found that RA had the highest free radical scavenging activity followed by BHT, TBHQ and TOC. To determine if free radical scavenging activity correlated to antioxidant activity in foods, the same group of antioxidants was added to bulk oil and oil-in-water emulsions. Oxidation was followed by measuring lipid hydroperoxides and headspace hexanal. In bulk oil the antioxidants were largely ineffective even though they were able to scavenge DPPH radicals. In emulsions, TBHQ was more effective than RA while BHT and TOC were prooxidative. These data indicate that determination of an antioxidant′s free radical scavenging activity with DPPH was not able to accurately predict the ability of the antioxidant to inhibit lipid oxidation in complex food systems.
Antioxidant Protocols for Food and Frying Applications. E. Frankel, University of California, Davis, CA, USA
A great multiplicity of methods has been used to evaluate the activity of synthetic and natural antioxidants for various food applications. Unfortunately, variable and confusing results have been obtained depending on the methods and conditions used to test activity. Many different substrates, system compositions and methods have been employed. Because antioxidants inhibit lipid oxidation in complex foods by a multi-step interfacial mechanism more than one specific evaluation methods are required. It is important to consider the food system composition, the type of food, the mode of accelerating oxidation, the methods to assess oxidation, and how to quantify activity. The use of vegetable oils for frying involves complex sequences of reactions affecting the quality of fried foods. With current trends towards avoiding hydrogenated fats containing undesirable trans isomers, the use of less polyunsaturated fats may result in greater losses of tocopherols in frying oils. Supplementation with synthetic or natural antioxidants and rapid turnover rates of higher oleic oils used for frying will be important parameters to improve the shelf life of fried foods.
Evaluation of Water-Soluble and Oil-Soluble Natural Antioxidants in Meat. K. Robbins, J. Antony, Kemin Food Ingredients, Des Moines, IA, USA
Quirks and Quandries in ORAC,TEAC, and DPPH Assays for Antioxidant Activity. K.M. Schaich1, X. Tian1, D. Havkin-Frenkel2, 1Rutgers University, Dept. of Food Science, New Brunswick, NJ, USA, 2Rutgers University, Agricultural Biotechnology Center, New Brunswick, NJ, USA
Documentation of antioxidant capacity by ORAC (oxygen radical absorbance capacity), TEAC (Trolox equivalent antioxidant capacity) and DPPH (diphenylpicrylhydrazyl) assays has become mandatory for characterizing natural product extracts and developing applications. While somewhat "standard" procedures have evolved by common practice, systematic consideration of fundamental factors affecting kinetics and pathways of reactions shows that oxygen, reagent and sample relative and absolute concentrations, pH, temperature, analysis timing, and nature of the molecule or extract all critically influence assay results, and use of incorrect conditions can lead to very misleading pictures of antioxidant actions. E.g, in ORAC assays heat generates nonreactive C•, yet also expels oxygen required to transform these to reactive ROO•. Fluorescein concentrations commonly used lead to self-quenching and apparent low reactivity; some components of extracts appear to complex with this reagent, extending its lifetime and erroneously indicating very high reactivity. Antioxidant reactions in TEAC and DPPH assays are complete within milliseconds; extended assays measure primarily molecular size and access rather than reactivity. This paper documents how various factors affect ORAC, TEAC, and DPPH antioxidant assays, and suggests modifications necessary to improve and standardize these methods.
ANA 3: Mass Spectrometry-Based Analysis
Chair(s): W. Byrdwell, USDA, ARS, BHNRC, FCL, USA; and X. Han, Washington University, USA
Quantification of Long-, Medium-, and Short-Chain Triacylglycerols in Butterfat by Normal-Phase HPLC with Electrospray Ionization Tandem Mass Spectrometry in the Presence of Ammonium Ions and Ammonia. P. Kalo1, A. Kemppinen2, V. Ollilainen1, 1Department of Applied Chemistry and Microbiology, Helsinki, Finland, 2Department of Food Technology, Helsinki, Finland
Here we report the identification and quantification of the molecular species of long-, medium-, and short-chain triacylglycerols (TAG) in butterfat (BF), including TAG with odd number of acyl carbons (ACN). We studied previously quantification of short-chain TAG by NP-HPLCâ€“ESIâ€“MS and the plausible fragmentation mechanisms of TAG and partial acylglycerols by flow injection MS2 and MS3. In the present study, a large set of methods, each recording MS2 for 1â€“4 ions, were used for identification of TAG species. For the quantification of long-chain, odd ACN, and unidentified TAG, molar correction factors (MCF) were calculated from the area data of each ACN:DB class and internal standard recorded for randomized butterfat (RBF) and the calculated random composition of TAG species. Similarly, acyl-chain- and regiospecific MCFs were calculated for butyrate and caproate TAG from their areas in RBF and mol% in the calculated random composition. These methods enabled identification of ca 450 TAG species in quantified 184 peaks of 88 ACN:DB classes.
Regioisomeric Structure Determination of α– and γ–Linolenoyldilinoleoylglycerol in Blackcurrant Seed Oil by Silver Ion High Performance Liquid Chromatography and Mass Spectrometry. H. Leskinen, J.-P. Suomela, H. Kallio, Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland
Mass spectrometry (MS) offers rapid and accurate analysis methods for the determination of the regioisomeric structure of triacylglycerols (TAG), but chromatographic separation of TAGs prior to MS is essential because e.g. TAGs containing double bond positional isomers such as α– and γ–linolenic acid cannot be distinguished by MS. In this study the regioisomeric structures (sn–ABA and sn–AAB+sn–BAA) of α– and γ–linolenoyldilinoleoylglycerol (18:3(n–3)/18:2/18:2 and 18:3(n–6)/18:2/18:2) in blackcurrant seed oil were determined by silver ion high performance liquid chromatography and different MS methods. The MS methods used were APCI–MS and ESI–MS/MS (APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; MS/MS, tandem mass spectrometry). The regioisomerism studies were based on the proportions of formed diacylglycerol (DAG) ions. The ratios of these DAG ions are different in different regioisomers because the cleavage of the fatty acid from the secondary sn–2 position is less favoured than from the primary positions sn–1/3. The regioisomerism of the sample TAGs were determined based on the calibration curves obtained from the reference TAG analyses.
Differential Profiling of Triacylglycerols: a New Approach for Mining LC/APCI-MS Data. M. Holbrook1,2, G. Seaborn1, J. Schwacke2, 1National Ocean Service, Charleston, SC, USA, 2Medical University of South Carolina, Charleston, SC, USA
A signal-based or chemometric approach to data analysis, commonly used in proteomics and metabolomics experiments, is applied for high-throughput processing of triacylglycerol profile data obtained using RP-HPLC/APCI-MS. Raw instrument data are converted to mzXML format and imported into the R statistical program where a suite of algorithms for data processing is applied. Data are treated as a two-dimensional image and methods from signal processing, statistics and machine learning are used to find patterns in the data that differentiate fats and oils from different biological sources. Peak identification is achieved via a translation-invariant wavelet transform and triacylglycerol peaks are aligned in the retention time dimension. Feature selection is achieved by selecting peaks which best differentiate sample classes and the random forest algorithm is used for classification. Data processing algorithms and the application of this methodology to classify edible oil samples will be discussed.
Alkaline Methanolysis of Lipid Extracts Extends Shotgun Lipidomics Analyses to the Low Abundance Regime of Cellular Sphingolipids. X. Han, X. Jiang, Washington University School of Medicine, St. Louis, MO. USA
Sphingolipids that contain an sphingoid base are comprised of hundreds of distinct compounds, many of which serve as lipid regulators of biological functions. The global analysis of the large number of low abundance sphingolipid species has been hampered in many cases by the fact that sphingolipid molecular species are overwhelmed by the quantity of other classes of lipids (e.g., phospholipid) present, thereby imposing severe restrictions of the dynamic range for their measurement using shotgun lipidomics. We developed a facile approach in which the sphingolipids of cellular extracts were dramatically enriched by direct alkaline methanolysis of lipid extracts followed by extraction to remove the large majority of other endogenous lipid classes. Through direct infusion of the resultant enriched solution, we identified and quantitated a variety of very low abundance sphingolipid classes (e.g., sphingosine, psychosine, and lysosphingomyelin) and molecular species by using electrospray ionization mass spectrometry. Accordingly, through utilization of these facile enrichment techniques, direct penetrance into the sphingolipidomes has been greatly extended facilitating new insights into their metabolism and signaling functions in biological systems.
Online Mass Spectrometry Analysis of Oxidized Lipids Generated in Situ via Ozonolysis. A. Brown, W. Holmes, T. Benson, D. Sparks, R. Hernandez, Mississippi State University, USA
Lipids comprised of unsaturated fatty acids are susceptible to oxidation. In the edible oil industry, this oxidation leads to rancidity in food products. However, in the oleochemical industry, useful products are generated from the oxidation of unsaturated lipids. In both applications, identification of the oxidation products is key to elucidating the reaction mechanisms. One of the most common and widely studied unsaturated lipid components is oleic acid. It is the primary unsaturated fatty acid in canola oil, tall oil, and palm oil and is oxidized industrially to produce azelaic and pelargonic acids. An automated, analytical system was developed for rapid on-line determination of oxidation products. This system was comprised of a corona ozone generator and a Varian 3600 GC equipped with a Saturn 2000 MS. The mass spectrometer allows for both electron impact and chemical ionization modes for product identification. Multi-port valves were added to the GC to allow manipulation of ozone flow into the built-in microreactor. The valves purged ozone prior to GC analysis, thus preventing damage to the column stationary phase. Oleic acid was used as a model compound to validate the analysis/reaction system. The goal of this work is to develop a rapid method for identification and quantification of lipid oxidation products.
The Identification of Mono-, Di-, Tri-, and Tetragalactosyl-diacylglycerols and Their Natural Estolides in Oat Kernels. R.A. Moreau1, D.C. Doehlert2, R. Welti3, G. Isaac3, M. Roth3, P. Tamura3, A. Nunez1, 1Eastern Regional Research Center, USDA, ARS, Wyndmoor, PA, USA, 2ARS, USDA, Fargo, ND, USA, 3Kansas Lipidomics Research Center, Manhattan, KS, USA
Oat kernels were extracted with methanol and glycolipid-enriched fractions were prepared using silica solid phase extraction. Using direct infusion electrospray ionization (ESI) tandem mass spectrometry (MS), HPLC-ESI-MS, and HPLC-atmospheric pressure chemical ionization (APCI)-MS, we confirmed previous reports that digalactosyldiacylglycerol (DGDG) was the most abundant glycolipid in oat kernels and confirmed a previous report of the presence of a DGDG mono-estolide in oat kernels. In the current study we also identified several additional natural galactolipid estolides: two new DGDG estolides (di- and tri-estolides), two trigalactosyldiacylglycerol estolides (mono- and di- estolides), and one tetragalactosyldiacylglycerol estolide (mono-estolide). To our knowledge, this report is the first evidence of natural di- and tri-estolides of polar lipids.
The Highlights, Perils, and Pitfalls of Vitamin D Analysis by Mass Spectrometry. W.C. Byrdwell, USDA, ARS, BHNRC, FCMDL, Beltsville, MD, USA
Vitamin D has received a great deal of attention in recent years due to findings that deficiency may play a role in numerous types of cancer as well as heart disease, stroke, hypertension, autoimmune diseases, diabetes, depression, chronic pain, osteoarthritis, osteoporosis, muscle weakness, birth defects, periodontal disease and others. As the interest in vitamin D becomes more widespread, the need for high quality analytical data regarding the levels of vitamin D in foods increases. Although methods for vitamin D analysis are available in the literature, these have shortcomings and caveats that must be understood if accurate results are to be obtained. This report describes some of the special considerations that must be taken into account for different sample matrices. A comparison of results by ultraviolet (UV) detection to those obtained by selected ion monitoring (SIM) atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) is presented. The special case of processed cheese, which produced erroneous results by UV detection but good results by APCI-MS, is highlighted. Unusual results obtained during analysis of commercially available standards are also presented.
Regiospecific Identification of 2-(12-Ricinoleoylricinoleoyl)-1, 3-Diricinoleoyl-Glycerol in Castor Oil by Mass Spectrometry and Sn-1, 3 Specific Lipase. J.T. Lin, USDA, ARS, WRRC, Albany, CA, USA
(12-Ricinoleoylricinoleoyl)diricinoleoylglycerol (RRRR), a tetraacylglycerol, was identified earlier in castor oil. Using ESI-MS4, 95% of the 12-ricinoleoylricinoleoyl chain was identified at the sn-2 position of the glycerol backbone of RRRR. Regiospecific location of the 12-ricinoleoylricinoleoyl chain of RRRR on the glycerol backbone was identified and quantified by the ions from the losses of the acyl chains at the sn-2 position as α,β-unsaturated fatty acids from the lithium adduct of RRRR. The regiospecific location was confirmed by hydrolysis of RRRR using sn-1,3 specific lipase. By comparing to the mass spectrum of 1-O-palmityl-2,3-palmitoyl-rac-glycerol containing one ether bond, the 12-ricinoleoylricinoleoyl chain of RRRR is indeed the ester bond between the two ricinoleoyl chains, not the ether bond formed from the two hydroxyl groups of the two ricinoleoyl chains.
ANA 4: Biodiesel Analysis and Characterization
Chair(s): T. Alleman, National Renewable Energy Lab , USA ; I. Clark, Grace Davison Discovery Sciences , USA ; and S. Fenwick, Archer Daniels Midland, USA
Investigation of Biodiesel Cold Soak Filtration Time and Characterization of Trace Components. T.L. Alleman, R.L. McCormick, NREL, Golden, CO, USA
The Solubility of Partial Glycerides (PG's) as a Factor in the Blocking of Biodiesel Fuel Filters During Cold Weather. R.W. Heiden, R. W. Heiden Associates LLC, Lancaster, PA USA
We recently reported in Vienna that SPG's (saturated PG's) are implicated in an unpublicized breakout of fuel filter blockages associated with B20 use during the winter of 2005-6. Analyses of B20 fuels associated with these filter blockages indicate that the original B100's have PG's that are near or below total glycerin limits (0.24%). Gel flocs nonetheless form at 1.5-2C, and when isolated, the compositions of the flocs are consistent with those of substances found on the filters, namely SPG's. Although numerous types of substances might also cause fuel filter blockages, in these instances the role played by SPG's is dominant. Monoglycerides are frequently the most prevalent PG in commercial biodiesel, so we experimentally determined the solubility of the saturated mono-glycerides (SMG's) in various diesel fuels, blends and 100% soy methyl esters. Diagrams of the solubility of SMG's as a function of temperature were constructed and are presented here and discussed. Both the temperature and fuel blend composition exert a strong influence on the solubilities of SMG's. In B20 at a concentration of 0.2% wt MG total (as glycerin) and 1/5 of the MG's as SMG in the original B100, the diagrams show that the solubilities of the SMG's are exceeded at temperatures well above OC.
Comparison of Analytical Techniques for Glycerin Analysis in Biodiesel. T.L. Alleman1, A. Patel1, J. Morton1, I. Clark2, L. Tucker3, 1NREL, Golden, CO, USA, 2Grace Davidson, USA, 3Metrohm Peak, Houston, TX, USA
More Advances in the Use of Networked Fourier Transform Infrared Spectroscopy for Biodiesel Quality Analysis. B. Stefl, N. Wang, C. Tseng, Cognis Corporation, Cincinnati, OH, USA
Fourier Transform Infrared Spectroscopy is a useful tool for measuring a variety of components and properties of biodiesel quickly and easily. Previous work demonstrated the effectiveness of a networked FT-IR system correlated to ASTM methods for the biodiesel reduced specifications of moisture, acid number, cloud point, methanol, total and free glycerin. Recent work investigates correlating FT-IR spectra to oxidative stability, cold flow plugging point, and sterol glucoside content. Additionally, calibrations for biodiesel blends have been developed.
The Evaluation of Biodiesel Characteristics Utilizing Impedance Spectroscopy. C.J. Koehler1, R.W. Hirthe1, M.A. Seitz2, D.L. Wooton3, 1Paradigm Sensors, Milwaukee, WI, USA, 2Marquette University, Milwaukee, WI, USA, 3Wooton Consulting, Beaver Dam, VA, USA
The use of AC impedance measurements to characterize the electrical properties of materials systems provides an alternate means of measuring composition, as well as critical physio-chemical attributes. This methodology has been successfully applied to multiple parameter determinations of biodiesel fuel quality. The evaluation of biodiesel/petroleum diesel blend concentration, total glycerin content, methanol contamination, and acid number using impedance spectroscopy will be presented.
Utilizing Nuclear Magnetic Resonance as an Alternative Research Tool for Fatty Acid Methyl Ester Characterization. M. Ter Horst1, R. Burton2, S. Urbin1, C. MacMillan1, M. Brookhart1, 1University of North Carolina at Chapel Hill, Dept. of Chemistry, Chapel Hill, NC, USA, 2Piedmont Biofuels Industrial, Pittsboro, NC, USA
Nuclear magnetic resonance (NMR) is uniquely suited for the identification of components of mixtures since molecules have characteristic NMR spectra. Careful analysis can also determine relative amounts of identified components within a mixture such as biodiesel. Biodiesel is defined as the mono alkyl esters of long chain fatty acids of either vegetable oils or animal fats. Since biodiesel can be produced from a wide array of fatty acids, complementary characterization techniques can be useful for the discrimination of the various methyl esters (ME). Currently in the biodiesel industry, gas chromatography (GC) analysis is used to verify quantitative results for fuel certification. Quantifying biodiesel with alternative analytical tools will aid in GC analysis where overlapping peaks complicates analysis. In this research paper, we deconstruct NMR spectra of biodiesel using spectra of individual methyl esters. Using the unique fingerprints, we investigate the feasibility of using NMR spectroscopy to identify and quantify relative concentrations of ME within an array of commercial biodiesel samples.
Effect of Ambient Conditions on Moisture Content of Biodiesel. I. Lee, S.R. Fenwick, J.S. Lawyer, Archer Daniels Midland Co., Decatur, IL, USA
Water is known as a major source of fuel contamination. Water not only can cause corrosion and microbial growth problems but also can negatively affect the fuel filterability by interacting with other minor components in biodiesel. Greater affinity of biodiesel to water, than regular diesel, underscores the importance of proper handling of biodiesel. The objective of this study was to generate information on moisture absorption by biodiesel, from it ambience, with the intention of using such information to establish a guideline for proper biodiesel handling. Moisture absorption by biodiesel is a spontaneous and equilibrating process. The equilibrium could be reached relatively fast, and the moisture content in biodiesel at equilibrium depended on the temperature and relative humidity (RH) of the environment. Low RH and temperature was required to maintain the moisture level below 500 ppm. Surface area also affected the moisture absorption, and as much as 0.48g/ft2/hr water was picked up by soy methyl esters at 90°F/81% RH. Moisture in biodiesel could be desorbed in dry environment. However, final moisture content at the desorption equilibrium was several hundreds ppm greater than that at the absorption equilibrium because of the hysteresis of the sorption isotherm. Some recommendations are given for proper handling of biodiesel in this aspect.
Biodiesel Acidity: Method Review and Proposed New Method. K. Fichter, L. Tucker, Brinkmann Instruments, Inc., Westbury, NY, USA
Biodiesel acidity is specified at 0.5mg KOH/g maximum according to fuel specifications set in ASTM D6751. The current method to measure acidity is ASTM Method D664. This method is not optimal for the acidity analysis. The authors will present the changes necessary to improve accuracy and precision of the method and compare with ASTM Method D974 which has been proposed as an alternative method.
Soap Formation as Deposit Precursors in Diesel Fuel and Lubricant Systems. H. Fang, Cummins Inc., USA
The current use of lowered solvency of ULSD and extremely high pressure in today's fuel system promote the detrimental growth of soap formation in the fuel. The deposit precursor is believed to be an acid-base neutralization product, which exhibits low solubility in the fuel and high tendency to adhere to injector piston surfaces. For fuel systems without lubricant contamination, a three-way interaction can be used to monitor the precursor formation pathways. The basic components can be derived from tank debris or biodegradation prodcuts in the dirty water bottoms delivered into the fuel by dispersant or detergent. The acidic components can be lubricity additives or oxidation degradation products of the fuel. The emulsion components of dispersant or detergent serve as carriers for acid-base neutralization process. For systems with lubricant contamination, the interaction between acidic fuel additives and the overbased detergent serves as the obvious pathway. A similar detergency approach can be applied to oil filter plugging where carboxylate salts begin to form network and block the fiber pore.
ANA 5.1: General Analytical
Chair(s): M. Collison, Archer Daniels Midland Co., USA; and F. Eller, USDA, ARS, NCAUR, USA
A Rheo-NMR System for the Determination of SFC Under Controlled Shear Flow. E.M. Mudge, E.Y. Anom, G. Mazzanti, Dalhousie University, Halifax, NS, Canada
The mechanical, sensorial, thermal and diffusional properties of multicomponentedible lipid systems depend dramatically on their solid fat content (SFC) and theirnanostructures, which are affected by the shear rate applied. A mini-Couette cell wasdeveloped to shear fat samples, and was tested with blends of canola stearin in canola oil in a 20MHz pulsed NMR spectrometer. The blends were placed in the mini-Couette cell,melted at 80 °C, and then crystallized under different shear rates (50 s-1 to 400 s-1) at 40 °Cinside the spectrometer. Time averaged NMR free induction decay curves were captured at20 s intervals, and the SFC values calculated using parameters determined by a calibrationprocedure.The SFC values determined by the direct method with and without the shaft of the Couettedevice, and with and without shear in the Couette device were reasonably close. The FID curvesdid not show a significant difference either. Therefore we conclude that this system is accuratefor in-situ time-resolved determination of SFC under shear flow. A combination of the direct andindirect method was successfully used to determine viscous heating.The system developed will help in understanding the effects of shear flow on the volumefractions of nanostructured lipid multicomponent systems. This will permit to optimizemanufacturing processes.
Ananlysis of FFA: Method Comparison. V. Barthet1, L. Tucker2, F. Fiddler3, K. Waszkoviak1, 1Canadian Grain Commission, Canadian Grain Commission, Winnipeg, MB, Canada, 2Brinkmann Instruments Inc., Mississauga, ON, Canada, 3Brinkmann Instruments Inc., Austin, TX, USA
Free fatty acids (FFAs) are produced from triacylglycerides (TAG) through chemical or enzymatic hydrolysis. They are usually associated with seed damage and undesirable flavor and textural changes when they are present in fats and oils. FFAs have to be removed during the oil processing. Reference methods to have been developed by AOCS (Ca 5a-40, 1997), AOAC International (940.28, 2003) and ISO (1996) to measure FFA in oils. These methods are titration methods; the samples are dissolved in an organic solvent and the acids are titrated with a base (KOH or NaOH) and the end point of the reaction could be measured using indicators (phenolphtalein, thymolphthalein or Alkali blue 6B) or an electrode (potentiometric method). In sound seeds, the FFA levels are usually lower than 1%; the FFA ten year average for Western canola is 0.24%. All these methods require large amount of sample to analyze FFA lower than 1% (20to 56.4 g of oil). This large sample requirement makes difficult the analysis of FFA in seed using an official method. The purpose of this study was to compare the results obtained by the three different official methods and a modified Ke & Woyewoda (1978) method (requesting only 1g of oil) to measure FFA in oil containing less than 1.5% FFA using potentiometric measurement.
Evaluation of a Compact Variable Filter Array (VFA) IR Spectrometer for the Analysis of FFA in Biodiesel Feedstocks. D.Y. Lee, D.L. Garcia-Gonzalez, F.R. van de Voort, McGill IR Group, Dept. of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada
The transterification of triacylglycerol-based oils for biodiesel production is greatly affected by the initial free fatty acid (FFA) content of feedstocks. A portable low-resolution variable filter array (VFA)-IR spectrometer equipped with a transmission flow cell was evaluated for its ability to quantitatively analyze FFAs in biodiesel feedstocks or edible oils, and its performance was compared with that of a conventional FTIR spectrometer. FFA analysis was based on methodology originally developed for FTIR spectrometers whereby FFAs are extracted from the oil sample into a solution of sodium hydrogen cyanamide (NaHNCN) in methanol to convert them into their sodium salts, producing a strong, readily measurable COO- band at ~1573cm-1. The energy-limitation of the relatively weak pulsed IR source was compensated for by replacing methanol by the less absorbing ethanol and moving the source closer to the detector. A calibration over an FFA range of 0-5% yielded an overall SD of ±0.07% FFA (R>0.97), very similar to that obtained with more sophisticated FTIR spectrometers. Thus, the VFA-IR spectrometer combined with the modified NaHNCN method is suitable for rapidly determining the FFA content of biodiesel feedstocks and provides portability, versatility and high sample throughput (~20-30 samples/h) relative to conventional titration.
A Novel FTIR Sampling Approach for Accelerated Oxidation Studies Under Moderate Temperature Conditions. D.L. Garcia-Gonzalez, D.Y. Lee, F.R. van de Voort, McGill IR Group, Dept. of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada
Autoxidation is a natural deteriorative process that results in a loss of sensory quality and nutritional value of edible oils. Most of the methods developed to assess the oxidative stability of oils measure the formation of indicator products under accelerated oxidation conditions that do not generally reflect those under which oxidative products actually accrue under normal storage conditions. A novel approach has been developed to monitor the oxidation process and products under more moderate conditions by FTIR spectroscopy. A stainless steel mesh IR "cell" (SSMC) was designed, in which oil deposited on a fine mesh is trapped by its inherent surface tension, resulting in a uniform sample with an effective thickness of ~150 μm. Owing to its high surface area, the mesh-entrapped oil is very susceptible to autoxidation at ambient or slightly elevated temperatures. The acquired spectra provide detailed information on oxidative changes and their time course, such as the formation of hydroperoxides, their breakdown products and cis/trans double bond conversion. Spectral oxidation time-profiles are presented and compared for some vegetable oils relative to pure triolein, trilinolein and trilinolenin to illustrate the performance of the SSMC/FTIR oxidative monitoring system.
Development of a Novel Ionic Liquid Mesoporous Complexation Adsorbent for the Extraction of Polyunsaturated Fatty Acid Methyl Esters (PUFAME) Triacylglycerols (PUTAG) and Free Fatty Acids (PUFA). P.J. Pham, M. Li, T. Li, Department of Chemistry, Mississippi State University, Mississippi State, MS, USA
Mesoporous silica (SBA-15) synthesized by using neutral organic triblock copolymers as structure-directing template was functionalized by immidazolium based ionic liquids.Silver salts were then immobilized unto the supported ionic liquids (SIL's) using the incipient wetness impregnation technique.This research work aimed to look at the effects of applying this novel adsorbent to polyunsaturated fatty acid methyl esters (PUFAME), feedstock for biodiesel production with emphasis on polyunsaturated triacylglycerols (PUTAG) as well as free fatty acids (PUFA).NARP-HPLC with Evaporative Light Scattering Detection(ELSD) methodology for quantification was also developed for triacylglycerols and RP-HPLC for free fatty acids. Approximately 14 mg of AgBF4/SBA15•IL•PF6 showed an adsorption capacity for trilinolenin (18:3) to be around 217.4 mg adsorbed/gram of sorbent and 192.6 mg/g for free fatty acids.Applying this adsorbent showed good selectivity and capacity for the more unsaturated species. Consequently,a stepwise methodology was also developed to increase the recovery of the adsorbed components.In addition, the synthesized adsorbent was also characterized by SAXS,Multi-point BET Analysis,SEM/TEM, TGA,XPS,ICP-AES,and Solid State NMR.
Investigation of the Selectivity of Room Temperature Ionic Liquids Stationary Phases for Capillary Gas Chromatorgaphy. L.M. Sidisky, G.A. Baney, K.K. Stenerson, J.L. Desorcie, Supelco, Bellefonte, PA, USA
Ionic liquids are a class of nonmolecular ionic solvents with low melting points. These liquids are unique combination of cations and anions and can provide a variety of different selectivities when used as stationary phases in capillary gas chromatography. We have recently begun evaluating the selectivity and thermal stability of a number of different ionic liquid cation and anion combinations. Selectivity was determined and compared using various isothermal and temperature programmed test mixes. Thermal stability was determined by examining column performance during incremental (destructive) and long-term stability testing at various upper temperatures. Particular cation and anion combinations appear to provide the selectivity similar to polar polyethylene glycol and highly polar cyanosilicone phases while possibly providing higher maximum temperature stability.
Soybean Fatty Acid Prediction by Near Infrared Spectroscopy and Model Standardization. B. Igne, G. Rippke, C. Hurburgh, Iowa State University, Ames, IA, USA
With the development of varieties such ultra-low linolenic soybean, the various actors of the field are looking for methods to manage their productions or ensure the quality of the raw material they use. NIRS has been used for more than ten years to determine the content in palmitic, stearic, oleic, linoleic and linolenic acids of whole and ground soybean. The goal of this study was to provide an update on the prediction of fatty acids by NIRS reflecting improvement in precision of wet chemistry analysis and advancement in chemometric techniques. New instrument standardization techniques were also applied. Samples (n=900) collected form 2003 to 2006 were scanned on two Foss Infratec units and partial least squares, artificial neural network and least squares support vector machines were used to develop prediction models. Fatty acids were predicted as %oil as well as in %weight. Results show a standard error of prediction of 0.64%oil, 0.3%oil, 1.62%oil, 1.51%oil and 0.64%oil for respectively palmitic, stearic, oleic, linoleic and linolenic acids, a significant improvement over previous reports. Results expressed as %weight provided similar precision. Linolenic results showed a possible detection limit at the 1% level. The linolenic PLS model was standardized; the difficulty of transferring more complex models than proximate analysis factors was determined.
What Are The Main Physicochemical Factors Involved in Antioxidant Action of Phenolics to Counteract Lipid Oxidation? A New Approach Through the Conjugated Triene Assay. M. Laguerre, L.J. Lopez Giraldo, J. Lecomte, P. Villeneuve, CIRAD PERSYST, UMR IATE, Montpellier, France
Growing evidence suggests that, in addition to organism antioxidant defences, dietary antioxidants may protect biological molecules from oxidative damages. Among these, phenolics are promising, owing to their ubiquity in plant kingdom and their strong antioxidant properties. Despite the interest of these compounds, many questions remain: first, what are the real antioxidant power of these compounds, then, how to evaluate them, and finally what are the physicochemical factors governing them? To answer these questions, a new spectrophotometric method called conjugated triene assay, CTA was firstly developed in microplate reader to measure antioxidant capacity using Tung oil TAGs, as UV probe. This oil contain 86% eleostearic acid with conjugated triene, exhibiting an absorption at 273 nm. In emulsion and under oxidation, the degradation of conjugated triene leads an absorbance decrease at 273 nm. Addition of phenolics results in a delay of oxidation and allows to quantify the antioxidant capacity. Afterwards, our study consisted in comparing the antioxidant capacity of phenolics via the CTA. The data obtained were then treated through a QSAR study to determine the main physicochemical factors involved in the antioxidant action of phenolics to counteract lipid oxidation.
ANA 5: Process Optimization / Quality Control
Chair(s): F. Galhardo, Bunge North America, USA; and Peter Domaille, Verenium Corporation, USA
Process Optimization and Quality Control—A Review. F. Galhardo, Bunge North America, Bradley, IL, USA
Analytical techniques traditionally used in R&D have now widespread use as tools for Quality Control in oil refining plants (e.g. FTIR, ICP, HPLC). Other instruments are now offered for in-line use (e.g. spectrophotometers). Allied with affordable process automation, these instruments are becoming essential for the ever searched optimal process efficiency.A review of concepts for quality and process control will be made in preparation for other presentations in this Session that illustrate the topic.
Oil Recovery from Gums—Analytical Aspects. W.D. Cowan1, H.C. Holm2, H.S. Yee3, J. Hemann2, 1Novozymes UK, Chesham, Bucks, UK, 2Novozymes DK, Bagsvaerd, Denmark, 3Novozymes MY, Kuala Lumpur, Malaysia
Enzymatic processes may be applied to the recovery of oil from gums produced by water degumming of oils. The oil is present entrained within the gums but is not the only lipid material present. This presentation will examine the recovery and quantification of lipids within those gums and their influence on overall yield
Analytical Monitoring of Phospholipase C-Mediated Vegetable Oil Degumming. P. Domaille, H. Huang, S. Marks, E. O'Donoghue, C. Deciu, D. Walsh, N. Barton, Verenium Corporation, San Diego, CA, USA
We have used 31P NMR to monitor phospholipid (PL) components in vegetable oil degumming in a high throughput mode. The large quantity of data from 200-400 samples/week with up to 20 peaks per spectrum to be identified required automated analysis which was done offline with peak fitting and alignment algorithms, which reduced the subjectivity of quantification. HPLC-ELSD for quantification of diacylglycerides (DAG's) was done to follow 1 gm scale degumming reactions. Small scale reactions were used to optimize application conditions of reaction temperature, water content, pH, and enzyme loading, during development of a phospholipase C enzyme that improves the yield of vegetable oil processing. Mass balance of the increased oil yield was predicted on the basis of the PL identifications, and small scale reactions were predictive of larger scale reactions, allowing a broad range of process conditions to be explored for optimization. We will describe the details of analytical methods used to monitor the reactions.
Determination of Oil in Soapstock. C. Dayton, Bunge North America, Bradley, IL, USA
The removal of phospholipids from vegetable oils have traditionally been accomplished by any number of different “refining” techniques including water degumming, acid degumming, enzymatic degumming, and / or caustic refining. The technique utilized typically will depend on the oil source, the ability to “value add” any co-products, and the existing physical unit operations. The ability to analytically measure the oil losses from the various “refining” processes have been difficult to determine utilizing the current AOCS Official Method G 5-40 due to the amount of phospholipids and other emulsifiers present in the heavy phase discharged from the centrifuge. A modified method for the determination of the neutral oil lost in the “refining” process will be reviewed.
Improved Analysis of Sterols in Vegetable Oils for Control of Cross-Contamination. E. Powers, J. Hammer, T. Haines, L. Pfalzgraf, M. Collison, Archer Daniels Midland Co., Decatur, IL, USA
Some regulatory agencies are now using the brassicasterol content of soybean oil as an indicator of the presence of contaminating canola oil. The existing official methods for determination of sterol content (ISO 12228 and similar methods) require sample preparation steps of transesterification in ethanolic KOH, chromatography on an alumina column to separate non-saponifiable matter, and thin layer chromatography for isolation of sterols prior to analysis by gas chromatography. Modern GC columns provide sufficient resolution that these labor intensive preparation steps are not necessary in a screening assay. The current procedure provides a rapid method for extraction of sterols from vegetable oils for routine screening resulting in detection limits equal to the compendial tests and better reproducibility.
FT-IR Analysis for Process Control. C. Dayton, Bunge North America, Bradley, IL, USA
Analytical measurements of lipids utilizing physical or instrumental techniques did not become truly viable until the advent of the personal computer (PC). The PC has allowed the use of strong mathematical tools such as Fourier Transform (FT) and Partial Least Squares (PLS) to be coupled with traditional analytical instrumentation tools. The utilization of these tools has given rise to a new field of analytical chemistry, chemometrics. Utilizing chemometrics allows analysis of samples with complex matrices and potential interferences without the need of "purifying" or concentrating the unknown analyte(s). The amount of useful information as well as the speed of the instrumental analysis is much faster than the traditional wet methods. My presentation will cover the utilization of FT-IR for measuring Iodine Value (IV), Trans, Saponification Value, Solid Fat Content (SFC), and Fatty Acid Composition (FAC) of vegetable fats and oils.
Chair(s): J.T. Lin, USDA, USA
Biodiesel Analysis Using HPLC with an Evaporative Light Scattering Detector.
D. Young, M. Wilcox, Grace Discovery Science, Deerfield, IL, USA
Global biodiesel production has quadrupled in the last five years and the standard methods for controlling the quality of biodiesel continue to improve. The current ASTM and EN methods for quantifying mono-, di-, and triglycerides in biodiesel are time consuming and suffer from poor reproducibility. A new HPLC method has been developed using an Evaporative Light Scattering Detector (ELSD) that provides numerous benefits over existing glyceride analyses of biodiesel. The new HPLC method has a short run time and is reproducible. The ELSD eliminates the need to derivatize and is highly sensitive. This new HPLC method with ELSD answers a need for better test methods for total glycerin in biodiesel during a time when the industry is experiencing explosive growth. Product quality is essential and this method may play a key role in biodiesel testing in the future.
Determination of Coenzyme Q10 and Q9 in Vegetable Oils.
F. Lacoste1, R. Rodriguez-Acuna2, E. Brenne1, 1ITERG, Pessac, France, 2IAT, Seville, Spain
A new sensitive and selective method was developed for the quantification of the total coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) concentration in vegetable oil samples. The coenzyme Q fraction is isolated by solid phase extraction (SPE) on amino-phase eluting with a mixture of heptane:ethyl ether. The fraction is evaporated under nitrogen, the residue is dissolved in a mixture of acetonitrile:tetrahydrofuran, and finally is analyzed by reverse phase high performance liquid chromatography (HPLC) with mass detector (MS). The sensitivity of the method is based on the high efficient formation of the radical anions [Mâ€“] of CoQ9 and CoQ10 by negative atmospheric pressure ionization, APCI(â€“). Interferences are minimized by using mass detection of the [Mâ€“] ions (m/z= 797.5 for CoQ9 and m/z= 862.5 for CoQ10) in selective reaction monitoring (SRM) mode (m/z= 797.5 m/z= 779.5 and m/z= 862.5 m/z= 847.5) using a triple-quadrupole mass spectrometer. The method was successfully applied to sunflower, soybean and rapeseed oils.
Comparison of Different Direct Saponification Methods for Analysis of Fat (triglycerides) and Fatty Acids in Selected Foods.
S.D. Bhandari, Silliker Inc., Chemistry R&D, Chicago Heights, IL 60411, USA
Recently O'Fallon et.al. (J. Anim. Sci. 2007, 85:1511-1521) reported a simplified direct method (JAS method) of fatty acid methyl ester (FAMEs)synthesis and its application in quantitation of fatty acids in wet meat tissues, oils and feeds. In the current study we tried to apply the JAS method without any further optimization to analyze fat (as triglycerides) and fatty acids in different food matrices. The samples were also analyzed using a direct saponification method using BF3 for fatty acid methylation (BF3 method). The FAMEs were analyzed by GC as descibed in the AOAC 996.06 method. The results of fat (triglycerides) and fatty acids obtained by both of the methods were compared for various food matrices. The values for meat and cheese by the JAS method were about 15% lower than those generated by the BF3 method. The results for fluid whole milk obtained by two methods also followed a similar trend but with more significant differences. Studies are in progress to further optimize the JAS method for certain food matrices
Automation of Fatty Acid Methyl Ester Derivatization for the Analysis of Omega-3 Oils.
S. Diltz, D. Vinjamoori, Martek Biosciences, Columbia, MD, USA
Determination of the fatty acid composition of fats and oils requires preparation of fatty acid methyl ester derivatives prior to analysis by gas chromatography which is time consuming as well as labor intensive. The most frequently used procedure saponifies the fat present using sodium hydroxide in methanol followed by acid catalyzed conversion to the methyl ester using boron trifluoride in methanol. We investigated the automation of this procedure using the Gerstel MPS 2XL system on algal oils, marine oils and vegetable oils. The Gerstel system capabilities include liquid handling via gas-tight syringe, block sample heating and vortexing. After introduction of the sample into a suitable vial, the instrument adds internal standard, the required reagents and heats the sample for the required time in the sequence it is programmed. After derivatization is complete, the extraction solvent is added, the samples are vortexed after which the hexane extract is transferred to a GC vial. 25 samples can be processed in approximately 5 hours. The results obtained compared favorably with those analyzed using manual procedure. This presentation will include optimization of sample preparation steps, precision & accuracy of results obtained and a brief discussion of future sample applications with suggestions for improvement.
Off-Odor Detection and Aging Monitoring of Omega-3 with an Electronic Nose.
J.C. Mifsud, M. Manach, V. Schmitt, Alpha MOS, Hanover, MD, USA
PurposeOmega-3s are increasingly used in the food & beverage industry. However, this ingredient can bring an undesirable odor / aroma to the final product. Consequently, it is of utmost importance for both omega-3 manufacturers and users to monitor the organoleptic features of this ingredient. Usually, food ingredients are evaluated by human sensory panels, which can be unpleasant and time-consuming. MethodRecently an electronic nose based on ultra fast Gas Chromatography, the HERACLES, has been used to assess Omega-3 sensory quality and to perform aging studies.Various samples of Omega-3 powder were both assessed by sensory panel and HERACLES Electronic Nose at 2 different times: t = 0 (freshly produced) and t = 2 months under various storage conditions (air or nitrogen). The scoring scale ranked between values 1 and 2.ResultsAfter building a calibration curve with the HERACLES, the model showed a good correlation with sensory panel scores (correlation coefficient > 99%) and predicted unknown powder's scores.ConclusionThe HERACLES electronic nose can bring the ability to screen a large number of samples for quality analysis. Thus, companies can reduce the time and costs of the analysis chain process, and significantly improve the product quality consistency.
Headspace Solid Phase Microextraction of Methanol in Biodiesel.
M. Paraschivescu1, E. Alley1, R. Hernandez1, T. French1, K. Armbrust2, 1Dave C. Swalm School of Chemical Engineering, Mississippi State University, Mississippi State, MS 39762, USA, 2Mississippi State Chemical Lab, Mississippi State, MS 39762, USA
The European Biodiesel Standards (EN-14214) impose a methanol content of less than 0.2% (m/m). The flashpoint method is used nowadays to restrict the amount of alcohol in biodiesel. The headspace solid phase microextraction method developed is a new, direct and economical way to quantify the alcohol in biodiesel. While the flashpoint method uses at least 70.00 mL of biodiesel, the HS-SPME needs only 1.00 mL. The analysis was performed by exposing a 75-µm Carboxen-Polydimethylsiloxane SPME fiber assembly to the headspace of the biodiesel sample. The gas chromatography used a HP-5 capillary column and flame ionization detection. A polynomial relationship was observed between the methanol concentration and its peak area. This method showed good reproducibility (average relative standard deviation 7.06%) and recovery (average recovery 100.2%).Optimization was performed in order to achieve the highest adsorption in the shortest time. Among the fibers that were tested, the CAR/PDMS gave the desired results at 50 ºC and 20 minutes adsorption time.
Degradation of Plasma Lipoprotein Cardiolipin and Phosphatidylglycerol by Human Group IIA, V and X Secretory Phospholipases A2.
A. Kuksis, M. Bergqvist, W. Pruzanski, University of Toronto, Toronto, ON, Canada
We have recently reported on the hydrolysis of the minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2, but did not include the effects of these phospholipases on cardiolipin and PtdGro, each of which has been estimated to represent about 2% of total plasma phospholipids These values are in the range of the other minor plasma glycerophospholipids already analyzed by LC/ESI-MS. Using the knowledge of LC/ESI-MS properties of synthetic standards of cardiolipin and PtdGro established earlier, we monitored the appearance and disappearance of the parent ion peaks in the total ion profile of control HDL and LDL phospholipids and of their residues following 1-24 hour digestion with the three secretory phospholipases A2. The results showed a rapid loss of PtdGro but not cardiolipin due to group IIA enzyme, with the opposite result recorded for the group V and X enzymes. There was no accumulation of cardiolipin or PtdGro in the digestion residues following prolonged incubation with or without the enzymes suggesting an eventual peroxidative degradation of the polyunsaturated phospholipids, which was confirmed by the appearance of ions corresponding to the hydroperoxides of both cardiolipin and PtdGro in the digestion residues.
HCl Treatment Followed By Bligh and Dyer Extraction Extract More Fatty Acids than Stoldt Fat Extraction in Feed and Fecal Samples.
S.K. Jensen, C. Lauridsen, Aarhus University, Faculty of Agricultural Sciences, Tjele, Denmark
The official EU method for fatty acid analysis in feed is based on boiling of the feed sample in 3 M HCl followed by a filtration, drying, Soxhelet extraction of the fat with petrol ether, conversion of the fatty acids to their corresponding methyl esters and followed by GC separation. Besides being very time consuming, this method has several disadvantages, e.g. the filtration step may allow some shorter chain free fatty acids and some phospholipids to be washed through the filter and escape the analysis. Further, during the drying step of filter paper, oxidation of polyunsaturated fatty acids (PUFA) is a risk. Because of the mentioned disadvantages, a new method (â€œHCl-Bligh and Dyer methodâ€) has been developed, and compared with the traditional Stoldt fat extraction. The new method combines the HCl treatment of the sample with a Bligh and Dyer extraction (water-methanol-chloroform) of the lipid. Depending on the matrix (feed ?), the HCl-Bligh and Dyer extraction lead to 10-15% more total fatty acids and a 30-50% increase in some of the PUFA compared with the official EU method. Thus, besides being much more efficient for extraction of fatty acids, the HCl-Bligh and Dyer method is much faster, and allows inclusion of an internal standard from the beginning of the sample treatment.
Facilitating Export of Oilseeds to Meet Global Biofuel Production Demands—A Rapid, Economical GMO Testing Strategy to Assure EU Compliance.
J. Fagan, B. Schoel, P. Chhalliyil, Genetic–ID, Fairfield, IA 52556, USA
Global trade patterns for oilseeds are rapidly changing in response to expanding demand for biofuels feedstocks. In particular, reduced European demand for genetically modified canola and soy for food purposes is being balanced by increased demand for these commodities for fuel purposes. Products exported to fulfill this demand must still comply with EU GMO regulations. For example, shipments of Canadian canola can be imported into Europe, only if testing verifies that 4 of the 6 transgenic events commercialized in Canada are absent, since those 4 events are not approved in Europe. One testing strategy would be to conduct 4 separate, event specific tests, each detecting one of the four unapproved varieties. We have developed a much more economical test, using two target sequences. Together, these selectively detect all 4 unapproved canola varieties, yet ignore the approved varieties. This testing method offers more rapid and economical screening that provides definitive, yet economical and rapid, evidence of regulatory compliance.
Development of an Optical Inspection Technique for the Quality Separation of Olive Fruit.
A. Kiritsakis, S. Efstradtiadis, F. Stergiopoulos, K. Kiritsakis, A. Bizopoulos, K. Kokonas, Alexander Technological Education Institute of Thessaloniki, Sindos Thessaloniki, Greece
An optical inspection method for the automatic quality separation of the processed olive fruit is proposed. It is designed to improve the quality of the produced table olives and virgin olive oil in an olive factory. The method is an additional step in the existing processing procedure in order to reject bad or unsuitable olives. More specific, it is proposed to accomplish an effective removal of defective olive fruit as well as any foreign materials, based on the digital image of the olive fruit. The olives are classified during the image processing stage according to the level of the defection caused by insects and fungi. The classification is based on predetermined features, such as statistical or geometric features, of the fruit.Results on the application of the proposed method to various types of defected olive fruit are presented.
Determination of Free and Total Fatty Acids in Surfactants Using Conventional and Fast GC Methods.
S. Ellis, S. Rumbelow, Croda Inc., New Castle, DE, USA
Various approaches to the determination of free and total fatty acids in surfactants by capillary gas chromatography are discussed. For example, free fatty acids can be analyzed directly with an appropriate polar column, or a non-polar column following either silylation or methylation. The relative merits of these approaches are discussed using particular examples to illustrate their relative reproducibility and robustness. Total fatty acids in surfactants are typically determined by in-situ saponification followed by methylation (in the GC vial) followed by analysis by either conventional or fast GC. In the latter approach, it has been possible to successfully reduce the analysis time significantly, by using a combination of a short, narrow bore column, hydrogen carrier gas and fast oven program rate. This was achieved with a standard, commercially available GC that had been pre-configured (by the manufacturer) to operate at 240v, as opposed to 115v. The impact of these changes on the total analysis time are illustrated with several examples and the relative merits of achieving even faster analysis times with more dedicated systems, is discussed.
Analysis of Triglyceride Positional Isomers with LC/APCI-MS, 13C-NMR and HPLC-AgNO3-UV.
H. Yuji1, N. Gotoh1, N. Yoshimura1, T. Nagai2, S. Tokairin2, K. Ichioka2, N. Noguchi3, S. Wada1, 1Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, Japan, 2Tshukishima Foods Industry Co., Ltd., Edogawa-ku, Tokyo, Japan, 3Tokyo University RCAST, Muguro-ku, Tokyo, Japan
DHA and EPA are the fatty acids specifically contained in marine organisms and their location on the glyceryl backbone in triglyceride (TG) is very important for the expression of their functionality. Recently, the analyses on the binding position of fatty acid on the TG glycerol backbone, namely analysis of TG positional isomer, have been well examined for plant oil and animal fat with LC/APCI-MS. Furthermore, we have reported the analyses results of TG positional isomers in fish oils with the same manner so far. However, our results were obtained from only the results of LC/APCI-MS and have not been confirmed by the other methods such as 13C-NMR and HPLC-AgNO3-UV. Therefore, to verify the accuracy of the LC/APCI-MS to quantify the existing ratio of TG positional isomers in fish oil, β-DDP (AAB type) and β-DPD (ABA type) in yellowfin tuna body oil was analyzed with three analytical methods and the results were compared. As the results, the results of three methods were the same and all the positional isomer was β-DDP. These results strongly indicate that the analysis of positional isomers in fish oil with LC/APCI-MS is reliable method. (D: DHA, P: palmitic acid)
Polymorphic Behavior of Mixed-Acid Triacylglycerols Including Omega-3 Polyunsaturated Fatty Acids.
K. Sato1, S. Ueno1, H. Mizobe2, K. Ichioka2, N. Goto3, S. Wada3, 1Hiroshima University, Higashi-Hiroshima, Japan, 2Tsukishima Foods Industry Co., Edogawa, Tokyo, Japan, 3Tokyo University of Marine Science and Technology, Minato, Tokyo, Japan
We have examined physical properties of mixed-acid triacylglycerols (TAGs) composed of omega-3 polyunsaturated and long saturated fatty acid moieties. By applying chemical modification techniques, 1,3-distearoyl-2-alpha-linolenoyl-glycerol (S-ALA-S) and 1,3-distearoyl-2-docosahexanoyl glycerol (S-DHA-S) with purity of >98 % for both TAGs were synthesized. The following main results were obtained.1. gamma and beta of S-ALA-S and gamma of S-DHA-S were observed.2. Melting points were 37.3°C (gamma) and 43.1°C (beta) of S-ALA-S, and 38.0 °C (gamma) of S-DHA-S.3. Long spacing (LS) values were 7 nm (gamma) and 6.2 nm (beta) of S-ALA-S, and 7.2 nm (gamma) of S-DHA-S, all of which revealed triple chain length structure. In comparison, the melting points of alpha-linolenic acid and DHA are -13°C and -44.5°C, respectively. These results indicate that the leaflets (chains) of the polyunsaturated fatty acid moieties are different from stearic acid moieties to form the triple chain length structure and that strong van der Waals interactions within the stearic acid leaflets cause extended chain conformation of ALA and DHA. As a result, the melting points of S-ALA-A and S-DHA-S were remarkably higher than the corresponding free fatty acids.
Polymorphism of Mixed-Acid Triacylglycerols with Long-Chain and Medium-Chain Fatty Acid Moieties: 8S8, 10S10 and 8P8.
S. Kawakami3, H. Uehara1, S. Arimoto1, S. Negishi1, Y. Uda2, Y. Furukawa2, S. Ueno3, K. Satou3, 1Central Research Laboratory of Nisshin OilliO Group, Ltd, Yokosuka, Japan, 2The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan, 3Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan
Metabolic properties of medium-chain fatty acids (MCFA) are different from those of long-chain fatty acids (LCFA), particularly in terms of conversion into body fats. MCFAs are little accumulated as the body fats, since they are rapidly digested and used as energy. Therefore, clarification of the physical properties of mixed-acid TAGs including MCFA and LCFA moieties are quite interesting for various food applications. In this study, we have studied the polymorphic properties of structured-fat crystals in which MCFAs with carbon numbers of 8 and 10 are placed at the sn-1 and sn-3 positions, whereas LCFAs (stearic acid and palmitic acid) are placed at sn-2 position (referred to 8S8, 10S10 and 8P8), which were synthesized by enzymatic methods. DSC, X-ray diffraction, Micro-probe FT-IR and optical microscopy were employed to study polymorphic occurrence and structures, melting and crystallization behavior and crystal morphology.
Application of Temperature and Silver-I on HPLC to the Screening of Structured Lipids.
R.O. Adlof1, G.R. List1, 1Retired, USDA, NCAUR, Peoria, IL, USA, 2Retired, USDA, NCAUR, Peoria, IL, USA
Silver ion chromatography (Ag-HPLC), utilizing columns packed with silver ions bonded to a silica or similar substrate and a variety of different solvent systems [1,2- dichloroethane, dichloromethane, or hydrocarbons (hexane, heptane) with acetonitrile or alcohols (methanol, isopropanol) as polar modifier(s)] has, over the last several decades, proven to be a tremendously powerful technique for the analysis of a wide variety of geometric (cis vs. trans) and positional (location of olefinic/ acetylenic bonds on the fatty acid backbone) fatty acid (FA), fatty acid methyl ester (FAME), mono-/diacylglycerol (MAG/ DAG) and triacylglycerol (TAG) isomers. While temperature control and programming have become important and integral parts of gas chromatography, the technology has found only limited application(s) in HPLC. Recently, the combination of silver-ion HPLC, acetonitrile in hexane solvent and temperature (isothermal) has been applied to the analysis and/ or isolation of variety of FAME and TAG isomers. The impact of sample size, column temperature and solvent composition on resolution, peak shape and elution time(s) will be discussed, as will the application of temperature programming, "ultra" (temperature/ pressure) and capillary (sub-micron substrates) to Ag-HPLC.
Determination of Deltamethrin in Oil Matrix.
H. Muhamad1, T. Yew Ai1, Y. Chee Beng1, I. Sahid2, 1Malaysian Palm Oil Board, Bandar Baru Bangi, Selangor, Malaysia, 2University Kebangsaan Malaysia, Bandar Baru Bangi, Malaysia
The efficiency extraction method and the clean-up steps for deltamethrin in crude palm oil (CPO) was evaluated. Quantification of the deltamethrin was by gas chromatography using an electron capture detector (GC-ECD). The deltamethrin in CPO was extracted into acetonitrile and the oil discarded after cooling at -20oC. The acetonitrile containing the deltametrin was passed through a solid phase extraction cartridge for further clean-up before injection into the GC. The GC detector efficiency was tested using standard solutions containing 0.05 to 2 µg/mL of deltamethrin. The retention time for deltamethrin was 23.05 min and the minimum detection limit was 0.05 µg/mL. The recovery of deltamethrin from CPO samples spiked with the pesticide at levels of 0.01 µg/g to 0.4 µg/g ranged from 75-98% with relative standard deviations of 2.4-9.9%. This method for determination of deltamethrin using liquid-liquid extraction followed by low temperature separation of oil and solid phase extraction for cleaning-up of extract was found to be satisfactory and acceptable for monitoring the presence of deltamethrin in oil matrix.
Aroma Quality Control of Oil Products with an Electronic Nose.
J.C. Mifsud, V. Schmitt, M. Manach, Alpha MOS, Hanover, MD, USA
PurposeFats and oils play a key role in food preparation and are an essential ingredient of a healthy diet. In order to guarantee an appropriate flavor and a conform quality, it is crucial for both manufacturers and users to test the organoleptic features of these oils or detect possible contamination. Currently, food products are assessed by human sensory panels or classical analytical techniques such as GC or GC/MS. However, these methods are time-consuming and sensory tests can be unpleasant for panelists. MethodA metal oxide sensor based Electronic Nose was used to evaluate oil samples with different known qualities. The objective of the study was to compare and differentiate the aroma of the various samples, then to identify the quality of blind samples. The industrial goal was to set-up a quality control model for a rapid assessment of production batches.ResultsInstrumental analysis showed a clear differentiation of the oil qualities, in conformity with known data. A quality control chart was set-up and allowed to determine if the blind samples were conform or out of specifications. ConclusionThe electronic nose can bring the ability to rapidly determine the quality of oil products, while assuring the consistency of testing methods. The E-Nose is a convenient tool to help companies optimize time and costs of analysis chain process.
Determination of Peroxide Value in Infant Formula Using a Spectrophotometric Method.
J.W.R. Liu1, C.I. Hastilow1, M.E. Larson1, V.L. Pound1, J.A. Alexander1, G.E. Katz1, J.M. Boff1, C.L. Clinger1, J.E. Dugle1, A.J. Angulo2, J.M. Romera2, J.D. Holton1, M.S. Bergana1, 1Abbott Nutrition, Abbott Laboratories, Columbus, OH, USA, 2Abbott Nutrition International, Abbott Laboratories, Granada, Spain
A spectrophotometric method was developed to determine the oxidation stability of nutritional powdered product. Lipid oxidation is a major cause of food quality loss potentially affecting several key properties such as nutritional content, organoleptic properties, emulsion stability, shelf life and safety and its understanding and avoidance is therefore of key importance to food manufacturers. Peroxide values for twenty nutritional formulations are compared to complementary analytical measurements of oxidation, including, HNMR, GC-MS (hexanal) and Sensory oxidation score (SOS). The Pearson correlations among the three analytical methods (NMROSI, PV and GC-MS (hexanal)) were positive and statistically significant. The three variables were input into a Principal component analysis, which yielded a first component explaining 86% percent of the variability, with nearly equal contributions by the three analytical methods. The high correlations and nearly equal component contributions indicated, individually and collectively, specific oxidation stabilizing formulation features.
Recent Advances in Lipid Analysis using Accelerated Solvent Extraction.
S.M.R. Ullah1, K. Srinivasan1, C. Pohl1, B. Murphy2, B. Dorich2, B. Richter2, 1Dionex Corporation, Sunnyvale, CA, USA, 2Dionex Corporation, Salt Lake City, UT, USA
Lipids are water insoluble organic compounds but soluble in nonpolar organic solvents such as hydrocarbons or ethers. Determination of lipid in food samples follow different methods based on the sample matrix. Several extraction methods (Mojonnier, Rose-Gottlieb and Werner-Schmidt) are based on acidic and base hydrolysis of sample followed by liquid-liquid solvent extraction (LLE) for lipids but all these methods are labor intensive and time consuming. The lipids in food are often bound to proteins or carbohydrates and require an alkaline or acidic pretreatment to liberate the lipids and allow efficient solvent extraction. The extraction of lipids is typically followed by either analysis using GC or gravimetry. Presently, accelerated solvent extraction (ASE) is used for rapid extraction of component of interest with organic solvents from food samples. It is difficult to pursue ASE extraction with acid or base hydrolyzed samples due to the operating conditions and material limitations. In this presentation we describe a new method for pursuing extraction of acid hydrolyzed samples by ASE. We present here performance of the new method for lipid analysis for a variety of food samples. We will also show performance data comparing the new ASE method with the Mojonnier method. Overall the new method would make ASE a more robust and versatile analytical tool for extraction.
Regulatory Science: Rapid Determination of Total trans Fatty Acids by Infrared Spectroscopy.
M.M. Mossoba1, V. Milosevic2, M. Milosevic2, J.K.G. Kramer3, H. Azizian4, 1Food and Drug Administration, College Park, MD, USA, 2MeV Photonics, Westport, CT, USA, 3Agriculture and Agri-Food Canada, Guelph, ON, Canada, 4NIR Technologies, Oakville, ON, Canada
In 2007 we reported the presence of interferences from saturated fats in ATR-FTIR spectra observed for trans fats by using the recently proposed negative second-derivative infrared procedure for the rapid determination of total trans fats and oils. In this publication we determined that best two non-trans reference standard fats to be used as substitutes for the commercially impure cis fat triolein (TO) (which contains 0.5% trans fat impurity as determined by GC) were the saturated tripalmitin (TP) and triarachidin (TA). This is because they were the two saturated fats that exhibited the least interference/overlap with the trans band at 966 cm-1 and had the lowest infrared absorptivities. Therefore, with respect to the present study on the quantitative determination of total trans fats by ATR-FTIR, TP (or TA) were used to prepare calibration standard mixtures that consist of the trans standard trielaidin (TE) in TP (or TE in TA) at trans levels as low as ~0.5% of total fat. Various calibration linear regression equations were used to measure by IR a number of unknown test samples that cover the quantitation range of interest, and were found to be in good agreement with GC data. This IR procedure will be used to accurately determine trans fats and oils test samples in a planned international collaborative validation study.
Prediction of Trans- and Cis-Fatty Acids Directly in Ground Cereal Products by NIR Reflectance Spectroscopy.
S.E. Kays, Y. Kim, W.H. Morrison, USDA, ARS, RRC, Athens, GA, USA
The potential of near infrared (NIR) reflectance spectroscopy for prediction of cis- and trans-fatty acids directly in ground cereal products was investigated. NIR spectra (400-2498 nm) of ground cereal products were obtained with a dispersive spectrometer and trans-fatty acids, cis-fatty acids and total fat determined by a modification of AOAC Method 996.01. Partial least squares 1 (PLS1) and partial least squares 2 (PLS2) regression were applied to calculate models for prediction of trans- and cis-fatty acids using spectral regions related to lipid absorption. PLS2 models were found to be suitable for quality control for cis-fatty acids and screening for trans-fatty acids. PLS2 improved prediction of trans-fat at all wavelength regions tested. PLS1 regression coefficients indicated that optimum wavelengths for prediction of trans-fatty acids were in the overtone regions for lipid absorption and for prediction of cis-fatty acids were in the overtone and combination regions.
Critical Lipid Oxidation Products in Artificial Digestion Model.
M. Tarvainen1, J.P. Suomela1, H. Kallio1, M. Ahotupa2, A. Kuksis3, 1Dept. of Biochemistry and Food Chemistry, University of Turku, Turku, Finland, 2Dept. of Physiology, University of Turku, Turku, Finland, 3Banting and Best Dept. of Medical Research, University of Toronto, Toronto, Canada
Lipid oxidation is an unwanted process that takes place during the manufacturing and storage of foods. Human digestive system modifies these preformed oxidized lipids as well as causes de novo oxidation of dietary unsaturated fatty acids. Low pH in stomach promotes the oxidation of double bonds in unsaturated fatty acids. Antioxidants can suppress the formation of oxidized lipids but the antioxidant activity in stomach needs to be verified. Our artificial digestion model consists of three simulated processes: oral, gastric and small intestinal. Each part has specific fluids and digestive enzymes which modify a standard meal in a sequential manner. Rapeseed oil was oxidized artificially (heat, UV-light) and added to the artificial digestion model. Chyme was extracted with solvents to isolate the oxidized lipids and studied with RP-HPLC-ELSD. RP-HPLC-MS/MS was used to identify individual oxidized lipid molecules formed in the artificial digestive model. Also different food preparation methods were evaluated in order to minimize the formation of lipid oxidation products, and the antioxidant activity of tocopherol acetate in stomach was studied. Preliminary data indicate that the artificial model of digestion and absorption of oxidized lipids provides a valid approximation of the natural process. (This work is funded by the Academy of Finland)
Improved Solid Phase Extraction of Ferulate Phytosterol Esters from Corn Distillers Dried Grain Extract.
K.A. Rennick, J.K. Winkler, R. Holloway, S.F. Vaughn, NCAUR, ARS, USDA, Peoria, IL, USA
We recently reported on the retention of valuable phytochemicals such as ferulate phytosterol esters (FPE), tocopherols (T), and tocotrienols (T3) in solvent and supercritical CO2 extracts of distillers dried grains (DDG). However, the extraction and purification of these phytochemicals, especially FPE, from the oil is time consuming, and requires the use of hazardous solvents. Silica cartridges were used to compare two different solid phase extraction (SPE) methods for the purification of FPE. Method 1 involved elution of neutral lipids with 98/2heptane/ethyl acetate followed by elution of FPE with 90/10heptane/ethyl acetate. In method 2, a two-step elution of neutral lipids followed by elution of FPE with 90/10heptane/ethyl acetate was used. Fractions collected using methods 1 and 2 were analyzed qualitatively by TLC and quantitatively by HPLC for T, T3, FPE, and contamination by neutral lipids. Analysis by HPLC showed that method 1 was capable of isolating T, T3, and FPE from neutral lipids, while method 2 was superior at purifying PFE away from neutral lipids, T and T3. This data indicates that two-step SPE using 90/10heptane/ethyl acetate allowed for efficient and quick separation and purification of FPE from neutral lipids and T and T3, with less hazardous solvents.