AOCS Annual Meeting Archives
102nd Annual Meeting | Abstracts | Hot Topics | Keynote Address | Schedule of Events | Short Courses | Sponsors
ANA 1 / BIO 1: Lipidomics and Metabolic Analysis
Chair(s): W.C. Byrdwell, USDA, ARS, USA; and R. Weselake, University of Alberta, Canada
Plant Lipidomics to Identify the Roles of Lipids in Plant Stress Responses. R. Welti1, H.S. Vu1, M. Roth1, P. Tamura1, S. Shiva1, S. Sarowar2, V. Nalam2, G. Klossner2, K. Lorenc Kukula2, M. Li3,4, G. Gadbury1, J. Shah2, X. Wang3,4, 1Kansas State University, Manhattan, KS, USA, 2University of North Texas, Denton, TX, USA, 3University of Missouri at St. Louis, St. Louis, MO, USA, 4Danforth Plant Science Center, St. Louis, MO, USA
Our group is investigating how membrane lipids change when plants are exposed to environmental stresses. Direct infusion electrospray ionization triple quadrupole mass spectrometry, quadrupole time-of-flight, collision induced dissociation time-of-flight, and Fourier transform ion cyclotron resonance mass spectrometry are being employed to profile lipids as a function of plant genotype and treatment. Currently we are analyzing a large-scale experiment examining the role of oxidized lipids in plant response to biotic and abiotic stresses, including freezing, salinity, bacterial infection, and fungal infection.
Acylated Monogalactosyldiacylglycerols: Their Detection and Possible Biological Roles in Plant Stress Responses. H.S. Vu1, R. Welti1, M. Roth1, P. Tamura1, S. Shiva1, S. Sarowar2, V. Nalam2, M. Li3,4, G. Gadbury1, J. Shah2, X. Wang3,4, 1Kansas State University, Manhattan, KS, USA, 2University of North Texas, Denton, TX, USA, 3University of Missouri at St. Louis, St. Louis, MO, USA, 4Danforth Plant Science Center, St. Louis, MO, USA
Although acylated monogalactosyldiacylglycerols were first discovered almost four decades ago, they have been ignored due to the lack of evidence to support their biological roles. We have developed various mass-spectrometry-based methods to profile monogalactosyldiacylglycerols acylated with both oxidized and non-oxidized fatty acids. Many acylated monogalactosyldiacylglycerols significantly increase in Arabidopsis leaves challenged by different stresses. In responses to biotic stresses, oxidized acylated monogalactosyldiacylglycerols are induced to a much greater extent by avirulent bacterial interactions than by virulent interactions. When challenged by abiotic stresses such as mechanical wounding, freezing or salinity, unique patterns of induction are also observed.
Carbon Flux Analysis in Oil Crops. I.A. Guschina1, M. Tang1, U.S. Ramli2, J.J. Salas3, P.A. Quant4, R.J. Weselake5, J.L. Harwood1, 1Cardiff University, Cardiff, Wales, UK, 2Malaysian Palm Oil Board, Kuala Lumpur, Malaysia, 3CSIC, Seville, Spain, 4Oxford University, Oxford, UK, 5University of Alberta, Edmonton, Canada
Oil-producing plants represent an extremely important group of agricultural crops. The demand for their products has been increased recently by their use (and potential use) for chemical feedstocks, speciality nutraceuticals and, possibly, for biofuels. This is in addition to providing basic edible oils. In order to optimise oil production by crops we need to know how it is regulated both qualitatively and quantitatively. For the latter, flux analysis provides much useful information. We have applied one particular method (top-down flux control analysis) to a variety of important oil crops. Our data show that different crops show distinct properties, making wide generalisations difficult. However, it is possible to use the information from flux control analyses to inform genetic manipulations. This emphasises the importance, as well as the utility, of defining the details of carbon flux to plant storage oils.
Core Aldehydes of PtdCho as Possible Activators of Hydrolysis of Plasma Lipoproteins by Group IIA sPLA2. A. Kuksis, A. Ravandi, W. Pruzanski, University of Toronto, Toronto, ON, Canada
Recent studies have demonstrated that bee venom PLA2 (group III PLA2), which possesses structural similarity to group IIA sPLA2, is activated in liposomes by 1-palmitoyl-2-(9’-oxo-nonanoyl) GroPCho (a core aldehyde) and has suggested that other lipid oxidation products might produce similar effects. Other work had shown that group IIA sPLA2 preferentially attacked the hydroxyl and hydroperoxy linoleates and other oxygenated fatty acids, which were released from PtdCho of plasma lipoproteins at early times of incubation. Later oxygenated arachidonates (isoprostanes) were also identified among the products of plasma lipoprotein hydrolysis by group IIA sPLA2. In the present work we have identified both 1-palmitoyl(stearoyl)-2-[(5-oxo- valeroyl( 9-oxo-nonanoyl)] GroPCho among the products of group IIA sPLA2 incubation of HDL and LDL of normal and acute phase plasma. The aldehydes were identified by LC/ESI-MS with reference to synthetic core aldehyde standards. The above liposomal studies and the present demonstration of formation of core aldehydes during a prolonged enzyme digestion provides a plausible explanation for the increased activity of group IIA sPLA2 observed occasionally with PtdCho, but which until now had remained unaccounted for.
Rapid Characterization of Lipids by MALDI MS. J.O. Lay, Jr., J. Gidden, R Liyanage, University of Arkansas, Fayetteville, AK, USA
Lipids can be analyzed after minimal processing in crude extracts without resort to hydrolysis and esterification. For glycerol lipids direct MALDI results in detection of sodium adduct ions of intact triacylglycerols (TAGs) and abundant diacylglycerol (DAG) like fragments. Unless protonated molecule formation is suppressed these fragments are abundant and preclude detection of DAGs in mixtures. DAGs like fragments can be eliminated by addition of base to preclude formation of unstable precursor protonated molecules during ion formation. With phospholipids the loss of the head group can be competitive with fatty acid loss. This can be useful for elucidation of phospholipids class. Indeed direct MS/MS of phospholipids in crude mixtures is very useful for the confirmation of the lipid class when obtaining phospholipids profiles because of the ability to establish parent-ion and product-ion relationships directly from mixtures. Lipid mixtures containing both glycerol- and phospho-lipids show only the phospholipids by direct MALDI because of suppression of the TAGs by the more polar phospholipids. We have developed rapid SPE separation approaches to produce separate TAGs and phospholipids fractions. We have applied these techniques to the analysis of lipids, lipids-decomposition, olive oil adulteration, microbial taxonomy, lipid metabolism/biochemistry, and lipid oxidation.
Triple Parallel Mass Spectrometry (LC1/MS3) Method for Lipidomic Analysis of Vitamin D and Plant Triacylglycerols in Dietary Supplement Capsules. W.C. Byrdwell, USDA, ARS, BHNRC, FCMDL, Beltsville, MD, USA
Three methods are demonstrated for a complete analysis of vitamin D and triacylglycerols (TAGs) in fortified dietary supplements that virtually eliminates all chemical pretreatment prior to analysis. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used simultaneously. The contents of gelcaps that contained 1000 IU (25 mcg) vitamin D3 in safflower oil and 2000 IU (50 mcg) vitamin D3 in rice bran oil were analyzed without the need for lengthy saponification and extraction. Three to five gelcaps were analyzed, each in triplicate or quintuplicate, using vitamin D2 as an internal standard. Vitamin D3 was analyzed using UV detection, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS4, the ELSD and the CAD. Triacylglycerols (TAG) containing fatty acids up to 28 carbons in length were identified. The gelcaps contained more than the label amount of vitamin D3 and differences were seen between synthetic vitamin D and that from fish oil.
LC-MS/MS as a Tool for Probing Industrial Oil Biosynthesis in Seeds. J.M. Dyer1, T.R. Larson2, L. Whitehead2, A. Gilday2, C.R. Dietrich3, P. Yang3, J.M. Shockey4, C. Lu5, E.B. Cahoon6, I.A. Graham2, 1USDA-ARS, US Arid-Land Agricultural Research Center, Maricopa, AZ, USA, 2Center for Novel Agricultural Products, University of York, York, UK , 3Donald Danforth Plant Science Center, Saint Louis, MO, USA, 4USDA, ARS, Southern Regional Research Center, New Orleans, LA, USA, 5Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA, 6Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE, USA
The seed oils of crop plants, composed primarily of triacylglycerols (TAGs), represent a major source of calories for human and animal nutrition and an excellent feedstock for the production of biofuels. In recent years, there has been increasing interest in engineering plants to accumulate high amounts of industrially important fatty acids in the oil, which can substitute for similar types of oleochemicals that are typically derived from non-renewable petroleum. The genes and enzymes required for producing high amounts of industrial oils in plants, however, are not fully understood, and host plants often respond in unexpected ways that limit the production of desired fatty acids. As such, sophisticated analytical techniques are needed to help identify the enzymes and metabolic processes that influence the production of industrial fatty acids in seeds. We have recently developed LC-MS/MS procedures for identifying and quantifying individual TAG molecular species. Application of these techniques to the study of plants producing either hydroxylated or conjugated fatty acids has shed light on both the mechanisms of fatty acid "channeling" into TAG, as well as several bottlenecks that limit the production of industrial fatty acids in developing seeds. Implications for producing industrial oils in transgenic plants will be discussed.
Applying Genomics and Biotechnology to Design Soybeans for 21st Century Markets. Richard F. Wilson, United Soybean Board, Raleigh, NC, USA
Future gains in U.S. soybean productivity will be a function of superior technology. Advances in soybean genetics and biotechnology will: 1) help ensure an adequate supply, 2) provide cost-effective ways to adapt to government regulations, 3) help sustain domestic livestock markets, and 4) provide health conscience consumers with high quality foods. The United Soybean Board has enabled genetic modifications of soybean composition and yield enhancement that are now filling seed-industry pipelines with a series of elite soybean cultivars distinguished by specific quality traits. For example, soybeans that produce high-oleic oils under private labels are now becoming commercially available. Looking forward, knowledge gained from the sequence of the soybean genome will accelerate the development and deployment of additional traits that will continue to reset the ingredient paradigm for food and feed products. Soybeans with high levels of omega-3 fatty acids, and soybean meal with greater amounts of digestible phosphorus are among the soybean quality trait innovations. This review envisions how these next generation technologies will influence soybean consumption and trade in oilseed markets that are increasingly driven by customer demand for quality, nutrition and value.
Sterol Glycosides in Various Plant Materials Reflect Unique Sterol Patterns. L. Nyström1, A. Schär1, A.-M. Lampi2, 1ETH Zurich, Zurich, Switzerland, 2University of Helsinki, Helsinki, Finland
Plant sterols from various plant materials have gained significant scientific interest in the past decades due to their use in functional foods. Recent studies have shown that also glycosylated sterols and natural intake levels of plant sterols can inhibit the cholesterol absorption in the gut. Very little information is available of the occurrence of these polar sterol conjugates in food materials, and even less information is available on the sterol composition found as sterol glycosides. We have demonstrated that the sterol composition as sterol glycosides (SG) and acylated sterol glycosides (ASG) can be significantly different from the total sterol composition found in various edible plant materials (e.g. beans, seeds, fruits, vegetables), and that there are unique patterns of sterol composition in SG from various plant families. Unlike commonly stated in the literature, sitosterol may not always be the most abundant sterol in the glycosylated sterols. Due to their more polar nature, incorporation of glycosylated sterols provides new dimensions to formulation of plant sterols in functional foods. Further, the sterol composition of SG mixtures may be modified by selection of SG source.
Biotechnological Approaches to Remove Chlorophyll Components in Plant Oils. Rene Mikkelsen1, Janne Brunstedt1, Birgitte Wittschieben1, Heidi Pedersen1, Lis Byrsting Møller1, Charlotte Poulsen1, Masoud Zargahi1, Susan Madrid2, Ken Carlson3, 1Danisco, Brabrand, Denmark, 2Danisco USA, Palo Alto, CA, USA, 3Danisco USA, New Century, KS, USA
Plant oils contain a vast number of compounds which potentially interfere with processing, stability, organoleptic properties, health effects etc of the final oil. For example, vegetable oils derived from oilseeds such as soybean and rape seed (canola) typically contain some chlorophyll which has to be removed in the final oil. Modern biotechnology provides a new powerful toolbox to modulate and control the properties of various components found in plant oils. Part of this toolbox is recombinant DNA and protein engineering technologies that make it is possible to create new enzymes targeted specifically towards selected compounds found in plant oils. This combined with modern and improved enzyme production capabilities have made it possible to generate new enzyme solutions for the fats and oil industry. We have isolated enzyme candidates from the chlorophyllase family of enzymes and tested the ability to degrade different chlorophyll components in an oil matrix.
ANA 1.1 / S&D 1.2: Advances in Analytical Methods for Surfactants and Detergents
Chair(s): D. Scheuing, The Clorox Company, USA; and K. Ma, Cognis Corp., USA
Quantifying Adsorption of Surfactants and Polyelectrolyte Complexes at the Solid-Liquid Interface by Quartz Crystal Microgravimetry with Dissipation. Mona Marie Knock, David R. Scheuing, Michael I. Kinsinger, Clorox Technical Center, Pleasanton, CA, USA
The suitability of quartz crystal microgravimetry with dissipation (QCM-D) for measuring the adsorption of surfactants and polyelectrolyte complexes at the solid-liquid interface is examined. The effect of sensor hydrophobicity on surfactant adsorption is investigated for sodium dodecyl sulfate (SDS), ′dioctyl′ sulfosuccinate sodium salt (AOT), hexadecyltrimethylammonium bromide (CTAB), and dihexadecyldimethylammonium bromide (DHDAB). Surfactant solutions were studied above their respective critical micelle concentration (CMC) and Krafft point in the absence of added electrolyte. Surfactant adsorption at the hydrophobic octadecanethiolate sensor surface was compared with adsorption on hydrophilic gold and silicon dioxide sensor surfaces. The adsorption behavior of net anionic and cationic polyelectrolyte complexes on silicon dioxide was also characterized.
Consumer-preferred Rheology of Surfactant-thickened Cleaning Products. D. Fritter, The Clorox Company, Pleasanton, CA, USA
Surfactants are commonly used to thicken oxidant-containing cleaners through the formation of a viscoelastic network of cylindrical micelles. These systems can exhibit a range of flow properties that influence ease of use and consumer perception of the product. Relevant measures go beyond the traditional thin-to-thick scale of viscosity and include ease of dispensing, elasticity, and cling to vertical surfaces. Rheological characterization provides a way to quantitate flow behavior that allows definition of the consumer-preferred space for a particular application.Viscoelastic materials can be characterized by the zero-shear viscosity, relaxation time, and static shear modulus, as obtained from a Frequency Sweep. For surfactant-thickened formulations, the consumer-preferred rheology for a product that is squirted or sprayed onto a vertical surface is a relaxation time less than about 0.01 s and a zero-shear viscosity greater than about 100 cP. The low relaxation time ensures a smooth and fluid appearance as the product hits and covers the surface, while the zero-shear viscosity requirement ensures that the coating doesn't drain too quickly. This combination of parameters leads to a product with consumer-preferred cling to vertical surfaces.
Applications of Fourier Transform Infrared Spectroscopy to Studies of Surfactant Behavior. David R. Scheuing, Clorox Services Company, Pleasanton, CA, USA
Liquid water is an intense absorber of infrared light and is thus commonly considered to interfere significantly in the analysis of aqueous systems via FT-IR. However, when the pathlength through the sample is restricted to below about 20 micrometers, a variety of analyses of surfactants in aqueous systems can be readily accomplished with FT-IR. Modern spectrometers, coupled with newer designs of optical accessories, can be used routinely and efficiently to characterize the behavior of surfactants or formulations on clean surfaces or on controlled dirty surfaces through the use of internal reflection (attenuated total reflectance or ATR) techniques. Due to the short effective pathlengths achieved with ATR and the excellent reproducibility of signals in the frequency domain with modern instruments, the elimination of the normally intense absorption due to water can be readily achieved via spectral subtraction or ratio techniques. Spectra of a variety of water-solid interfaces can be obtained, and hence the chemical analysis of these interfaces, including time-resolved changes is possible. The combination of FT-IR data with results from other surface analytical techniques can be used to probe formulation performance or more fundamental properties of phase separation of aqueous systems of practical interest.
Application of LC-MS to Surfactant Analysis. David Dabney, Stepan Company, Northfield, IL, USA
Mass spectrometry has taken an increasing role in all segments of analytical analysis over the last 20 years. From quantitative analysis related to biologics to structural elucidation of novel compounds, mass spectrometry continues to improve our understanding of the materials we use everyday. The application of mass spectrometry to the study of surfactants while not new has generally been limited to the identification of surfactants related to biologic matrices. Here we apply mass spectrometry to the analysis of surfactants for the purpose of understanding the composition and properties of the surfactant as a stand alone entity. By understanding the total composition of our materials we provide for the information necessary for regulatory, manufacturing, performance and quality of the surfactants. Mass spectrometry is well suited for this endeavor due to its dynamic range, ability to be coupled to various separation techniques and array of ionization and mass selection configurations as well as rapidly improving data evaluation techniques. In this talk, the basic principles of mass spectrometry will be reviewed along with examples of mass spectrometry being applied to the evaluation of various surfactant chemistries.
Emerging Ambient Ionization Methods and Their Use to Characterize Substrate Modifications. Ismael Cotte-Rodriguez , The Procter & Gamble Co., Global Analytical, Cincinnati, OH 45252, USA
Tools that allow rapid in-situ analysis of ambient surfaces with minimal or no sample preparation are ideal to monitor substrate modifications. This presentation focuses on the use of desorption electrospray ionization (DESI) as an imaging tool to understand substrate modification. DESI is a powerful ambient ionization method that combines features of desorption ionization with those of spray ionization. DESI allows mass spectrometry to be performed on samples that are in ambient environments. The method is based on directing a pneumatically-assisted electrospray onto a given surface, from which small organics and large biomolecules are picked up, ionized and delivered as desolvated ions into the mass spectrometer. Increased selectivity is achieved both by MS/MS and by including additives in the spray solvent (Reactive-DESI). DESI has high sensitivity, is suitable for characterization of both large and small molecules, and allows the direct analysis of pure compounds and formulated mixtures deposited on surfaces or in tissue without sample preparation. Examples of applications in consumer product development will be covered including analyte-substrate interactions and quantitation approaches for selected analytes.
Analytical Toolbox to Unveil Complex Mixtures of Surfactant-Based Systems. Michele Mangels1, José Andrés Rojo1, Bob Strife2, Kevin Garber2, 1The Procter & Gamble Co., Miami Valley Innovation Center, Cincinnati, Ohio, USA, 2Mason Business Center - Analytical GCO, Cincinnati, Ohio, USA
The characterization of the complex mixtures found in many surfactant-based systems and detergent products poses unique analytical challenges. The surfactant raw materials themselves are often intricate mixtures of active molecules that generally need to be formulated into blends with other raw materials involving co-surfactants, functional polymers and many other organic and inorganic chemicals and solvents. A variety of analytical approaches are available for the characterization of these mixtures. Presented are a broad suite of analytical techniques currently in use for understanding various polar and non-polar surfactants with nonionic, cationic, or anionic functionalities. Chromatographic approaches discussed range from the use of gas and high pressure liquid chromatography, including the use of supercritical fluids and the so-called aqueous normal phase separation mode. Detection techniques discussed which prove useful in the characterization of these complex systems include flame ionization, evaporative light scattering, and both low and ultra-high resolution mass spectrometry with various types of ionization sources. Also discussed are some approaches used to help visualize the complex data sets generated by these techniques.
ANA 2: Advances in Spectroscopic Techniques
Chair(s): T. Mason-West, Bunge, USA; and TBD
E-Nose and TD GCMS on Oxidized Canola Oils. M.D. Evenson, J.A . Flook, T.G. Patterson, A. Syed, C.J . Kahl, D.H. Meyer, Dow AgroSciences, Indianapolis, IN, USA
Oxidative stability is a key factor in the selection of oils for food applications. Volatile compounds generated during the oxidation of oil will impact both the flavor and aroma characteristics of cooked food products. A comparative study was conducted to evaluate the composition and sensory attributes of volatiles emitted from two different cooking oils using Thermal Desorption Gas Chromatography Mass Spectroscopy (TD/GCMS) and Electronic Nose (E-Nose) technologies. Over one hundred volatile compounds were identified by TD/GCMS from used oils. Many were common to both oils and show a linear relationship to the accelerated aging, several had a plateau at a specific time point and others were specific to the individual oils. The same sample set was also evaluated on the E-Nose and aroma maps were generated. Aroma sensing by E-Nose offers a powerful new technology for determining oil quality and characteristics on the basis of an aroma profile from a complex mixture of volatile compounds. As detailed information is generated on the aged oils, specific compounds that produce undesired or desired aromas or flavors could be determined, altered or masked in the oil to improve the stability or sensory profiles.
Analysis of Color Bodies in Vegetable Oil. J.B. Soe, R. Mikkelsen, L. Lauridsen, T. Jorgensen, Danisco A/S, Brabrand, Denmark
Vegetable oils like soya oil and rapeseed oil produced form extraction of beans or seeds contain a number of oil soluble minor components including carotene, tocopherols, chlorophylls or degradation products of chlorophylls like pheophytins and pyropheophytins. Some of these components are crucial for the quality of oil.Over the years a number of analytical methods have been developed to analyse these minor components and methods can among others be found as AOCS standard methods.Many of these methods rely on photometric measurements, which can be used in the quality control during refining of vegetable oils.Analysis of chlorophyll, pheophytin and pyropheophytin is however a challenging task,at least in refined oil where these components are found in low ppb level.In order to understand the effect of enzymatic degradation of green color bodies it is crucial to be able to analyse these components.Development of a analytical method based on advanced HPLC/MS equipment, is shown to give the possibility for detection of chlorophyll, pheophytin and pyropheophytin components individually as well as their degradation products in the low ppb range.In the presentation examples of analysing these colour components in oil samples from crude to refined oil will be shown.
Updating a Synchronous Fluorescence Spectroscopic Virgin Olive Oil Adulteration Calibration to a New Geographical Region. J.H. Kalivas1, M.R. Kunz1, J. Ottaway1, C.A. Georgiou2, G.A. Mousdis3, 1Idaho State University, Pocatello, ID, USA, 2Agricultural University of Athens, Athens, Greece, 3National Hellenic Research Foundation, Athens, Greece
Detecting and quantifying extra virgin olive adulteration is of great importance to the olive oil industry. Many spectroscopic methods in conjunction with multivariate analysis have been used to solve these issues. Successes to date are limited as analyses are specific to included geographical regions, growing seasons, cultivars, etc. (the composite primary condition). Samples from new geographical regions, growing seasons, etc. (secondary conditions) are not always correctly predicted by the primary data due to the secondary conditions not matching the primary conditions. Three Tikhonov regularization (TR) variants are used in this presentation to allow adulterant (sunflower oil) concentration predictions in samples from geographical regions not part of the original primary domain. Of the three TR variants, ridge regression with an additional 2-norm penalty provides the smallest validation sample prediction errors. While the presentation reports on using TR for model updating to predict adulterant oil concentration, the methods are applicable to updating multivariate analyses distinguishing adulterated samples from pure extra virgin olive oil. Additionally, the approaches are general and can be used with other spectroscopic methods and adulterants as well.
On-line Monitoring of the Transesterification Reaction Between Triglycerides and Ethanol Using Near Infrared (NIR) Spectroscopy. R. Richard1,2, B. Dubreuil1,2, S. Thiebaud-Roux1,2, L. Prat3, 1Université de Toulouse; INPT; LCA (Laboratoire de Chimie Agro-Industrielle); ENSIACET, F-31030 Toulouse, France, 2INRA; LCA (Laboratoire de Chimie Agro-Industrielle), F-31030 Toulouse, France, 3Université de Toulouse; INPT; CNRS; Laboratoire de Génie Chimique; UMR 5503, F-31030 Toulouse, France
To substitute fossil fuels, biodiesel can be produced from vegetable oils, animal fats, and waste cooking oils by transesterification with ethanol. Various factors such as free fatty acid content, water content, type/amount of catalyst, vegetable oil to alcohol molar ratio, or temperature can affect this process. Many analytical procedures using gas chromatography and high performance liquid chromatography have been developed to determine the composition of crude transesterification products but these techniques are long to handle, unreliable and expensive methods of on-line monitoring. In this work, an innovative method using NIR spectroscopy to on-line monitor the transesterification reaction of high oleic sunflower oil with ethanol in a one-liter-batch reactor was developed. Partial least squares regression was used to develop calibration models between NIR spectral data and analytical data obtained by a reference method: gas chromatography with flame ionization detection (GC-FID). The results indicated that the use of NIR spectroscopy is an appropriate technique to research optimal reaction parameters and obtain kinetic data for a range of temperature from 30°C to 80°C. It was also shown that the water content in ethanol or oil has a negative influence on ethyl ester production.
Using Fourier Transform Near Infrared (FTNIR) in Evaluation of Monoacylglycerides and Propyleneglycolmonoester in Edible Fats and Oils. Gabriela Sekosan, Tiffanie West, Bunge North America, Bradley, IL, USA
A rapid and new method for the quantitation of the amount of monoacylglycerides, α-monoacylglycerides and propyleneglycolmonoester in both emulsifier and oils and fats containing emulsifiers, was developed using NIR. For the calibration of the instrument more than 100 samples with ranges of 0-75% were used. The new method reduces the preparation and analysis time from 3 hours to less than 10 minutes and is eliminating the solvent use.
Analysis of Epoxidized Soybean Oil using Fourier Transform Near Infrared Spectroscopy (FT-NIR). H. Li1, M. Ochs2, M. Gulden2, 1Bruker Optics, Inc., USA, 2CHS, Inc., USA
Epoxidized soybean oil (ESBO) is widely used as a plasticizer and heat stabilizer for polyvinyl chloride (PVC) plastics. There are several parameters used to control the epoxidiation process and the quality of the finish product. Iodine value (IV) is used to help determine when all the carbon-to-carbon double bonds have been opened and when to stop the reaction. Moisture content is used to determine the moisture residule in finish product from the washing procedure, of which the value should be very low since water can cause degradation of epoxide group. The oxirane value (OV) is another measurement to determine if the epixidation reaction was completed during the reaction. While acid value (AV) needs to be checked to determine if any residual acid that is used in the reaction remains in the ESBO product.Fourier transform near infrared (FT-NIR) spectroscopy was used to in this study to analyze multiple measurement parameters in washed ESBO during process and finish ESBO production samples. For washed ESBO, partial least squares (PLS) calibration model was developed for IV and an independent test set validation of the calibration model yielded root mean square error of prediction (RMSEP) value of 0.79 (mg I2/g). A set of finish ESBO samples were used for developing PLS calibration models for measuring acid value, iodine value, moisture and oxirane value, and leave-one-out cross validations for each model gave values for RMSECV of 0.38 (mg KOH/g), 0.88 (mg I2/g), 0.05% and 0.14% respectively. Overall, the results of this study demonstrate the feasibility of FT-NIR spectroscopy for the routine analysis of ESBO finish product and process control of epoxidization of soybean oil.
ANA 3.1 / LOQ 3: Antioxidants and Oxidation Control: Analytical Methodologies and Efficacies
Chair(s): D. Luthria, USDA, ARS, USA; and F. Shahidi, Memorial University of Newfoundland, Canada
Efficacy and Measurement of Antioxidants. F. Shahidi, Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada
Antioxidants in food and biological systems have received increasing attention in recent years due to better appreciation of their role in food preservation as well as in health promotion and disease risk reduction. The efficacy of antioxidants from natural origin in foods depends on the source material, activity of the relevant phytochemicals related to their structural characteristics and lipophilicity/hydrophilicity. In addition, concentration of antioxidants, presence of other food constituents and storage conditions, among others, are important factors affecting antioxidant activity. The mechanism of action of antioxidants may be quite varied and testing of their efficacy should include several assays that are complementary to one another. In addition, sampling techniques as well as release of bound antioxidants from food matrix is essential when correlating in-vitro data with in-vivo results.
Comparison of Extraction Solvents on Assay of Phenolics Form Foods. Devanand Luthria, USDA, ARS, ERRC, USA
Increased interest in bioactive food components and phytochemicals has arisen from numerous epidemiological studies that suggest that certain phytochemicals can reduce risk of chronic diseases. This presentation will illustrates with examples the significance of optimization of extraction procedures in developing analytical methodologies for accurate estimation of bioactive compounds present in foods and dietary supplements. It will discuss the importance of different sample preparation parameters such as extraction solvent composition, solid-to-solvent ratio, temperature, and particle size for the accurate assay of phenolic compounds in different food matrices. Furthermore, effect of drying and grinding procedures on the assay of phenolic compounds from plant and food samples will also be presented. A comparison of current (pressurized-liquid, ultrasonic irradiation, and microwave-assisted) and classical (stirring, Soxhlet, shaker, vortex) extraction procedures on the assay of phenolic phytochemicals will also be discussed. A systematic protocol for optimizing sample preparation procedure for extraction and assay of phytochemicals from different plant matrices will also be presented. Accurate analysis of bioactive compounds is critical for their precise and reproducible quantification in different foods, establishing appropriate dietary intake and safety guidelines, and understanding their role in human health and nutrition.
Extraction and Analysis of Soluble and Bound Fruit Polyphenols. L. Howard, B. White, University of Arkansas, Dept. Food Science, Fayetteville, AR, USA
Polyphenols in fruits and their co-products are typically extracted with aqueous-organic solvent mixtures prior to HPLC analysis or determination of antioxidant capacity. Evidence is increasing that significant quantities of polyphenols in fruits, especially hydrolysable and condensed tannins are entrapped or bound to cell wall polysaccharides and resist extraction using conventional extraction techniques, suggesting levels of fruit polyphenols reported in the literature have been vastly underestimated. The release of bound polyphenols from fruits following conventional extraction can be performed using acid or alkaline hydrolysis methods, but both methods have limitations due to the lability of polyphenols and formation of side products under harsh chemical conditions, and lack of authentic standards for quantification. These methods along with their advantages and disadvantages will be discussed as well as the potential implication of bound polyphenols on gastrointestinal health.
Challenges with Antioxidant Analysis: Strengths and Weaknesses. W. Ellefson, D. Sullivan, Covance Laboratories, Madison, WI, USA
Today’s marketplace reflects consumers demand for healthy products. Products that are high in antioxidant content are one area of major interest. There are a variety of general methods (ORAC, DPPH, Folin-C, FRAP, etc.) to measure antioxidant potential. In addition there are many methods that assess the level of individual categories (e.g., catechins, isoflavones, etc.) or individual compounds. All methods have strengths and weaknesses. This presentation will focus primarily on these strengths and weaknesses. An overview of the application of a selected number of these methods will be presented along with estimates of what compounds each test is capable of measuring. Specific examples will be highlighted, including the stability indicating nature of some of these test methods. This discussion will provide an overview of many of the analytical techniques and highlight the need for harmonized methods of analysis. Some of these methods have more specific applications with selected matrices, while others have a broader application. When there are numerous methods for the same analyte there is significant room for disagreement. We will highlight a process to come together, discuss common methods, agree on an approach, and complete a study to develop `gold’ standard methods that everyone can accept. This AOAC process has been extremely successful for 125 years. Methods that successfully navigate this process are widely accepted by government agencies around the world and stand up in courts of law.
Can Antioxidant Activity Assays be Redirected to Guide Stabilization of Foods with Natural Compounds? K.M. Schaich, Rutgers University, New Brunswick, NJ, USA
A plethora of antioxidant activity assays have been developed to test putative antiradical action of natural compounds and extracts, and results are being widely used to predict protective effects in vivo. However, such application is being increasingly questioned due to very low absorption of phenols, rapid conjugation and elimination, and mounting evidence that phenolic compounds exert their effects through a variety of signal transduction pathways. Arguments will be presented for productively redirecting antioxidant assays to identify compounds with promise for replacing BHA and BHT in foods. Which assay(s) will most accurately predict phenol effects in food systems – H atom transfers or electron transfers? What new assays must be developed to supplement established assays? Predicting effects in foods will require modification of current antioxidant assays to provide kinetic and mechanistic information as well as stoichiometric measures of antioxidant reactions, increased attention to synergisms and antagonisms between polyphenols, consideration of phase partitioning, and determination of protein binding effects on phenol reactivity and availability in foods. Strategies for determining appropriate natural antioxidants and delivery modes for various food applications will be described.
Dietary Modulation of Oxidative Stress: Physiological Meaning of the Non Enzymatic Antioxidant Capacity (NEAC). Mauro Serafini, National Institute for Food and Nutrition Research (INRAN), Rome, Italy
Diet-derived antioxidants have been proposed to reduce oxidative stress-related disease development. However, contrasting results from clinical trials have raised strong concerns about the importance of antioxidants on human health. A vulnerable point of the research on antioxidants is the lack of information on the effect of the whole array of antioxidants in disease prevention, as so far mainly single galenic molecules have been investigated. Despite many food items rich in antioxidants have been accredited of an antioxidant action in vivo, the extent to which plant foods are able to tune oxidative stress in human is unclear and it represents a matter of debate. The first systematic review of dietary intervention studies with plant foods on markers of antioxidant function will be presented and the association between dietary and endogenous antioxidant defenses in vivo will be discussed in order to obtain a realistic portrait of the complex interactions at the basis of the postulated protective effect of antioxidants molecules. There is a strong need of increasing the existing knowledge on the real efficacy of antioxidants in vivo in order to clarify if redox molecules represent a scientific-based strategy for disease prevention or just ancillary ingredients of fruit and vegetables.
Methods for Assaying Antioxidants in Lipids and Emulsion Systems. D. Huang, Department of Chemistry, National University of Singapore, Singapore
High throughput assays have been developed for rapid measurements of antioxidant capacity in bulk oil and microemulsion (oil-in-water) with methyl linoleate as the substrate in both cases. A oxygen sensitive fluorescent probe coated 96-well microplate, Oxygen Biosensor SystemTM, were applied to conveniently monitor the oxygen concentration change kinetics during the autoxidation. From the area under the kinetic curves, the antioxidant capacity of food lipids and polyphenolic compounds were calculated using Trolox as the standard. In the microemulsion system, the synergistic effect of hydrophilic antioxidants and α-tocopherol was measured for representative flavonoids. Chlorogenic acid has the best synergistic effect of 44.8%. The assays, coined as ORACoil and ORACE (for microemulsion) provides a model system in evaluating antioxidant capacity of phenolic compounds in a heterogeneous system relevant to food and cosmetic applications.
ANA 3: Emerging Analytical Issues in Process Contaminants - The Search for Truth
Chair(s): J.D. Pinkston, The Procter & Gamble Company, USA; and M.W. Collison, Archer Daniels Midland Co., USA
Indirect Determination of 3-MCPD Esters and Glycidyl Esters in Oils and Fats. Correct or Wrong? K. Hrncirik, A. Ermacora, Unilever R&D, Vlaardingen, The Netherlands
Fatty acid esters of 3-monochloropropan-1,2-diol (3 MCPD) and glycidol are food-borne contaminants predominantly formed during high-temperature processing of fat-based matrices, namely oil refining. Although several analytical methods are available, no standard method has been established yet. Indirect methods for the determination of 3-MCPD esters are based on the acid- or alkaline-catalyzed cleavage of the esters to give rise to free 3-MCPD, which is quantified by GC-MS after derivatization. Main drawback of such an approach is the complexity of sample preparation, which involves a series of chemical reactions that are suspected to affect the reliability of the results. A recent proficiency test with participation of 34 European laboratories showed significant inconsistency in the results obtained by some of the indirect methods used. More recently, few direct methods for the analysis of the individual esters by LC-MS has been presented as an alternative. It is becoming obvious that the current situation requires more clarity and that a consolidated approach towards the analytical methodology should be taken. This speech is aimed to provide a critical evaluation of the reliability of the most common indirect methods for the determination of 3-MCPD and glycidyl esters and to identify their major critical points.
Advances in LC-MS Analysis of Glycidyl Esters. M. Blumhorst, M. Collison, Archer Daniels Midland Company, USA
Glycidyl esters have been found in all types of refined vegetable oils. The method presented is a robust LCMS analysis for GE in vegetable oils of all types including hard and lauric oils. It requires no extraction procedures prior to analysis.
Quantitation of 3-MCPD Esters and Glycidyl Esters via Stable Isotope Dilution Analysis. M. Granvogl1, P. Schieberle1,2, 1Technical University of Munich, Chair for Food Chemistry, Freising, Bavaria, Germany, 2German Research Center for Food Chemistry, Freising, Bavaria, Germany
Free 3-MCPD is a well-known food constituent, which is supposed to have carcinogenic potential. Recently, 3-MCPD esters were reported in food, e.g. in refined edible oils. Due to the fact, that after consumption a cleavage of the esters to free 3-MCPD is thinkable, efforts have been undertaken to minimize their concentrations. Very recently, the presence of glycidyl esters in oils was also suggested. Up to now, all these compounds have been quantified with methods only providing the sum of all 3-MCPD and glycidyl esters.Application of the newly developed stable isotope dilution analysis (SIDA) on edible oils revealed considerable differences in the concentrations of the esters. For the first time, definite contents of different 3-MCPD esters and of glycidyl esters could be analyzed using various labeled standards in contrast to previous analytical approaches, which only offered the sum of the respective esters. The new method provided reliable results with a high sensitivity and selectivity as well as low detection limits. The obtained results will be discussed and compared to existing methods, which are on the basis of a derivatization step of free 3-MCPD after methanolysis of the esters.Further, the lecture will give deeper insights into the formation pathway, which was investigated by labeled precursors in model systems.
Validation of Quantitative Method for Glycidol Fatty Acid Esters in Edible Oils. Hiroki Shiro, Naoki Kondo, Yoshinori Masukawa, Kao Corporation, Haga, Tochigi, Japan
Glycidol fatty acid esters (GEs) in edible oils are supposed to present as food processing contaminants and have become a serious concern over the world. In response to this, we developed a method to quantify those GEs in edible oils, which was improved into a generalized method using common analytical instruments that is applicable not only liquid oils but also solid oils. The further revisions made it more rapid and more eco-friendly. In this study, we evaluated the method in terms of limit of detection/quantification (LOD/LOQ), accuracy and how to quantify. LOD and LOQ of GEs in edible oils were 0.025-0.033 and 0.082-0.11 μg/g-oil, respectively, demonstrating the high sensitivity of the method under practical use situations. The recovery test using GE-free oil with spiked standard GEs revealed that the method is sufficiently accurate (recovery%: 94.5-109.9%) under practical use situations. The quantified levels and its deviations using an internal standard method were substantially the same as those using an external standard method, indicating the availability of the external standard method. The present study demonstrates that the method using external standards is sufficiently accurate and simple under practical use situations, so it can be used as a globally standardized testing method.
Update on the Development of a Sensitive, Accurate, and User-friendly Method for the Direct Determination of 3-MCPD Esters. J.D. Pinkston, P.J. Stoffolano, P.Y. Lin, The Procter & Gamble Company, Cincinnati, OH, USA
Occurrence of 3-Monochloropropanediol (3-MCPD) and its fatty acid esters (3-MCPD esters, containing one or two fatty acids at the sn-1 and sn-2 position of the glycerol backbone) has been reported for refined fats and oils by several groups. A number of groups have been working to strengthen analytical methods to determine these compounds. The first and most widely-used analytical method involves cleavage of esters with sodium methoxide, derivatization, and GC/MS. Later evidence indicates that this "indirect" method can overestimate the total level of 3-MCPD. Recently, a direct method for the 3-MCPD esters using LC/TOFMS of the sodium adduct ions has been described. This method addresses many of the issues surrounding the indirect GC/MS method, but requires frequent instrument maintenance. We have developed a direct LC/MS/MS method of the ammonium adducts of the esters. This method provides improved limits of detection and is very user friendly. The development and details of the method will be described in this presentation.
LC-MS Detection of Glycidyl Esters and 3-MCPD Esters in Edible Oils. S. MacMahon, T.H. Begley, G.W. Diachenko, FDA Center for Food Safety and Applied Nutrition, College Park, MD, USA
3-Monochloro-1,2-propanediol (3-MCPD) esters and glycidyl esters are potentially, through hydrolysis of the esters, carcinogenic chemical contaminants formed during the processing of edible oils. The compounds have proven to be difficult to detect and quantitate using conventional analytical methods. Currently the most widely used methodology is the Deutsche Gesellschaft für Fettwissenschaft (DGF) standard method C-III 18 (09). In the DGF method, both glycidyl and 3-MCPD esters are transesterified to release 3-MCPD and glycidol, the 3-MCPD is derivatized and analyzed by GC-MS, and the glycidol is chemically converted to 3-MCPD before derivatization and analysis. This method requires significant chemical manipulation of the contaminants before detection. It does not directly measure the fatty acid esters of 3-MCPD or glycidol which are actually formed during edible oil processing and likely differ in toxicity from free 3-MCPD and glycidol. The LC-MS method described in this presentation detects and quantitates intact 3-MCPD and glycidyl esters in edible oils. The method is rugged, sensitive and specific and allows for the direct determination of fatty acid esters of 3-MCPD and glycidol in a method suitable for regulatory analysis.
Indirect Determination of Bound Glycidol and MCPD in Refined Oils. Jan Kuhlmann, SGS Germany GmbH, Hamburg, Germany
Since bound 3-MCPD and glycidol have been determined as process contaminants occurring commonly in refined edible oils, there has been some concern due to the possible toxic effects of 3-MCPD and glycidol. In order to monitor these compounds there is an obvious need for reliable and practical analytical methods. Whereas the indirect determination of bound 3-MCPD using GC-MS techniques is more commonly established, analytical methods for the quantificatin of bound glycidol are somewhat new. In general two different analytical approaches are available presently. One is the direct determination of the original 3-MCPD and glycidyl fatty acid esters. The second, rather indirect approach allows the determination of the released agents, free 3-MCPD and glycidol, after applying an ester cleavage, derivatisation of the free analytes and determination by GC-MS. Meanwhile several direct and indirect methods are in development or have already been established but differences in the obtained results have raised issues which approach is more reliable. Furthermore, recent findings of bound 2-MCPD in refined oils seem to require analytical methods covering this kind of analyte as well.The main topic of this presentation is a novel indirect approach that allows the parallel determination of bound glycidol and 2- & 3-MCPD within one analytical method.
Direct Analysis of MCPD Esters and Glycidyl Esters in Various Edible Oils: Current Gaps and Future Challenges. Mathieu Dubois1, Alfred Donaubauer2, Walburga Seefelder1, 1Nestle Research Center, Lausanne,Switzerland, 2NQAC Weiding/Nestlé Deutschland AG, Frankfurt am Main, Germany
Fatty acid esters of 3-mono-chloro-1,2-propanediol (ME) and 2,3-epoxy-1-propanol (GE) have been detected in refined fats and oils. Although no evidence is available indicating any adverse health effects from ME and GE in food, these substances have raised safety concerns, considered as a source of 3-MCPD and glycidol human exposure. Today, ME and GE are mainly analyzed indirectly by GC techniques, after dedicated but uncontrolled hydrolysis and derivatisation procedures. The inaccuracy of these methods is a major hurdle to the establishment of a commonly recognized method. Furthermore studies conducted in model systems and data in actual food matrices confirm that structural diversity of these compounds may become important to understand their safety significance. Hence a strong need for the development of reliable and sensitive direct methods for GE and ME. Whereas their detection can easily be achieved under conventional LC-MS conditions, their isolation from edible oils for sensitivity reasons is far more challenging. The presentation gives an overview on different approaches for extraction (SPE, GPC, ASE, MPLC) and quantitation (standard addition, isotopically labeled standards) that have been investigated, resulting in a validated method for the simultaneous quantitation of ME and GE by LC-MS (TOF and QqQ).
ANA 4: General Analytical I
Chair(s): V. Jain, Mars Chocolate North America, USA; and A. Proctor, University of Arkansas, USA
Faster Techniques for Total Fat Determination in Various Food Products. P. Kopecká, F. Dabo, E. Gouèzec, S. Marmesat Rodas, P.-A. Golay , Nestlé Research Center, Lausanne, Switzerland
Individual food producers have to provide the consumers with true information on the composition of their products. Fat content and its composition is one of the main interests. Therefore, determination of total fat content and, in some cases, qualitative analysis of the fat are frequently performed in many laboratories.The current methods for fat content determination are based on official gravimetric methodologies. These methods, however, have been found time consuming, difficult to automate, and requiring high amounts of solvents. In our study, we have been investigating and optimizing several alternative approaches to total fats extraction from a variety of foods. In addition to normal fat content foods, the aim of this work is to find alternative techniques suitable to low fat food products. Techniques such as accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) have been investigated. These techniques offer lower solvent consumption, are well suited for automation and hence can be performed unattended. A comparison of these techniques with the official methods will be presented both in terms of extraction yield as well as sample pre-treatment requirements. The challenges encountered for some complex food materials will also be discussed.
Purification of Stearidonic Acid (SDA) from Modified Soybean Oil by Argentation Silica Gel Open Column Chromatography. L. Kleiner-Shuhler, L. Vázquez, C. Akoh, The University of Georgia, Athens, GA, USA
The objective of this study was to purify stearidonic acid (SDA, 18:4 ω-3) from modified soybean oil containing a mixture of over 20 fatty acids (23% SDA). Interest in obtaining purified fractions of SDA arises from reported health benefits associated with polyunsaturated fatty acids (PUFAs), such as cardiovascular disease prevention. In addition, SDA may also provide improved stability characteristics since its unsaturation index is less than longer PUFAs. First, a chemical ethanolysis of modified soybean oil was performed to transform the triacylglycerols into fatty acid ethyl esters (FAEE). Then, the FAEE were fractionated and SDA-EE was purified by argentation silica gel (10% AgNO3) open column chromatography, which allows selectivity based on degree of unsaturation. Different FAEE sample loads and mobile phases were explored until the best purification of SDA-EE was achieved. The solvents used were hexane and hexane:acetone mixtures (99% and 95%). At the optimal conditions, a fraction with high SDA-EE purity was obtained (98%) with 77% yield. Besides, it was possible to obtain another fraction enriched in α-linolenic acid-EE (37% purity and 68% yield) and γ-linolenic acid-EE (22% purity and 61% yield). A scaled-up process resulted in 840 mg of final product composed of 97% SDA-EE with 71% yield.
Determination of the Heptadecyl Fatty Acids of Thespesia populnea Seed Oil. M.K. Dowd, USDA, ARS, SRRC, New Orleans, LA, USA
As part of a survey of the fatty acid composition of wild Gossypium species and related plant seeds, the seed oil of Thespesia populnea was found to have relatively high levels of 17:1 and 17:2 fatty acids. Heptadecyl monoenes and dienes had been previously reported as minor components of cottonseed oil, but the location of the double bonds in these acids has not been determined. By forming dimethylsulfide FAME adducts and picolinyl esters from the oils, these components were separated by gas chromatography and identified by mass spectrometry to be the 8-17:1, 9-17:1, 10-17:1 and 8,11-17:2 fatty acids. The positioning of the double bonds suggests that many of these acids were formed by α-oxidation from the corresponding octadecyl fatty acids. The occurrence of α-oxidation in these oils is not entirely unexpected as the oils also contains malvalic acid, which is generally presumed to be formed from sterculic acid by the same process. However, that α-oxidation occurs to such a degree in Thespesia sp. among other acid classes might make the plant useful for studying this degradative pathway. The same processes occur in cottonseed oil to a much more modest degree, the oil differing by having reduced levels of the 8-17:1 and 8,11-17:2 acids and no detectable level of the 10-17:1 acid.
Rapid and Direct Quantitative Analysis of Positional Fatty Acids in Triacylglycerols on Natural Occurring Oils and Fats. S.W. Gouk, S.F. Cheng, A.S.H. Ong, C.H. Chuah, University of Malaya, Kuala Lumpur, Malaysia
There is increasing evidence that the positional fatty acid composition is more crucial than total fatty acid composition in the aspect of nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of triacylglycerols using 13C NMR was developed to overcome the tedious conventional methods which involve enzymatic/Grignard chemical hydrolysis and chromatography analysis. The applicability of current method had been demonstrated on various types of oils and fats at systematic error of 1.0 mol %. The carbonyl carbons of saturated, monounsaturated, and polyunsaturated acyl chains were quantified by using inverse gated decoupling pulse sequences and appropriate data processing methods. A repetition delay of 7 times the T1 of carbonyl carbons is required to achieve a 99.9 % recovery of z-magnetization after 90° pulse excitation. Moreover, a specific set of NMR acquisition parameters has been optimized for current work. As the current 13C NMR method is a direct measurement and no hydrolysis of the sample is needed, problematic acyl migration has been fully omitted and higher accuracy is achieved. In the absence of laborious chemical derivatization, this method is simple, accurate and user-friendly to obtain useful positional fatty acid compositions of edible oils and fats.
A Simple, One-step, Quantitative Analytical Method for the Analysis of Triglycerides in Edible Oils and Other Natural Products. R. Freeman1, T. Yuzawa2, C. Watanabe2, 1Frontier Laboratories, Antioch, CA, USA, 2Frontier Laboratories, Koriyama, Japan
Precise fatty acid profiling is an essential step in the characterization of natural products including many food stuffs, a variety of biodiesel processes and clinical diagnostic tests. Because gas chromatography is almost universally used to separate and quantitate fatty acids, it is necessary to isolate and derivatize the acids prior to analysis. This improves the thermal stability of the acids and increases their vapor pressures. The most prevalent method for converting the acids to methyl esters utilizes a methanolic solution of BF3. However, BF3 is toxic, is noted for its instability and is not universally available. This work describes a simple method for profiling fatty acids in a complex matrix. The method utilizes reactive pyrolysis (RxPy)-GC which is, actually, thermally assisted hydrolysis and methylation. Microgram quantities of the "untreated" sample and a few µL of a methanolic solution of an organic alkali are placed in a small sample cup. When heated, FAMEs are formed and separated by GC.This work will show the FAME profiles of several sample types: sardine oil. Soybean oil, oats, and algae. The impact of using various organic alkalis (THAH, TMSH & m-TFPTAH) on the degree of isomerization will be presented. The derivatization of C22:6 , C20:4 and C6:0 will be used to compare the BF3 and PxPy methods.
Gas Chromatographic Quantification of Acetic Acid and Furfural in Lignocellulose Hydrolyzate Fermentations for Microbial Lipids Production. A. Mondala1, R. Hernandez1, T. French1, D. Sparks2, W. Holmes1, M. Haque1, P. Pham1, W. Sweet3, 1Dave C. Swalm School of Chemical Engineering, Mississippi State University, Mississippi State, MS, USA, 2Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS, USA, 3General Atomics, San Diego, CA, USA
Currently, much research is being conducted for the utilization of lignocellulose biomass for biofuels production. Acid hydrolysis of lignocellulose releases fermentable sugars, which can be used by oleaginous microorganisms for lipid accumulation. These lipids can be extracted and converted into biodiesel or renewable diesel. However, the hydrolysis process also produces degradation by-products such as acetic acid and furfural, which could inhibit microbial growth and metabolism. This paper will present a rapid gas chromatographic technique for the simultaneous quantification of acetic acid and furfural from aqueous hydrolyzate mixtures and fermentation media. The target compounds were extracted from sample matrices using chloroform with hexanoic acid as internal standard. Separation of the compounds was achieved on a Restek Stabilwax DA column and flame ionization detection method was evaluated in terms of detection limits, linearity, and reproducibility. Synthetic and actual sample matrices (hydrolyzate and fermentation media) were also processed and analyzed using the proposed method to evaluate its real-time applicability in lipid accumulation cultures of activated sludge or oleaginous microbial consortium for the correlation of inhibitor levels with lipid productivity.
Saturated Monoglycerides' Impact on Low-temperature Performance. G.M. Chupka1, R.L. McCormick1, G. Chiu2, 1National Renewable Energy Laboratory, USA, 2Phase Technology, USA
In recent work we have shown that above a threshold or eutectic concentration, saturated monoglycerides (SMGs) can significantly raise the cloud point (CP) of distilled soy and animal fat-derived B100. SMGs have an even greater impact on the final melting temperature (FMT, as measured when the sample is heated) at concentrations above the eutectic point. It was demonstrated that a lower melting point crystalline form of the SMG forms upon cooling, and then transforms into a more stable, higher melting point crystalline form when slowly heated or held at constant temperature using a controlled temperature stage microscope. This could be an explanation for the observation that some fuels can develop precipitates above the measured CP of the fuel. Current work is focused on the effect of trace impurities, such as water and steryl glucosides, on the CP and FMT in combination with the SMGs. The effect of the trace impurities when they are blended with conventional diesel fuels ranging from B5 to B20 was also investigated.
Separation of Fatty Acid Methyl Esters Utilizing the Novel Ionic Liquid SLB-IL111 GLC Column. Pierluigi Delmonte1, Ali Reza Fardin Kia1, John K.G. Kramer2, Magdi Mossoba1, Len Sidiski3, Jeanne I. Rader1, 1US FDA, College Park, MD, USA, 2Retired from Agri-Food Canada, Guelph, ON, CA, 3Suplelco Sigma Aldrich, Bellefonte, PA, USA
The Supelco SLB-IL111 column is a novel ionic liquid fused capillary GLC column capable of providing enhanced separations of fatty acid methyl esters (FAMEs) compared to the highly polar cyanopropyl siloxane coated columns currently employed for the analysis of complex mixtures of isomeric FAMEs. Based on the McReynolds constants, the SLB-IL111 shows a polarity number of 111 compared to 81 for the Supelco SP-2560 column, normalized to the value of the SLB-IL100. The SLB-IL111 provides an enhanced separation of geometrical and positional isomers of mono- and poly- unsaturated FAMEs. Operated isothermally at 168ºC, with hydrogen as carrier gas at 1.0 ml/min, the SLB-IL111 tested (100m x 0.25 mm I.D., 0.20 μm coating) provided a separation of c9,t11-CLA from t7,c9-CLA, of t11-18:1 from t10-18:1, and of t15-18:1 from c9-18:1; these pairs previously required complimentary silver ion separations prior to their chromatographic separation on cyanopropyl siloxane columns. The SLB-IL111 column also provided partial resolution of t13/t14-18:1, c8-18:1 from c6/c7-18:1, and several t,t-CLA isomeric pairs. The column evaluated also provided an improved separation of the geometric and positional isomers of 16:1, 20:1 and 18:3 FAMEs. The elution order was sufficiently different from that of cyanopropyl siloxane columns to consider them as complementary techniques.
Effect of Different Silicates Clay Minerals on Decreasing the Mycotoxins in Food and Animal Feeds. S.M. Cham1, I. Gokmen1, F. Bozoglu1, O. Tokusoglu1, 1Middle East Technical University, Ankara, Turkey, 2University of The Gambia, Serrekunda,The Gambia
The contamination of animal feed and human food stuff by highly poisonous low-molecular-weight poisonous natural products of secondary metabolite produced by filamentous fungi, especially the species of Aspergillus, Fusarium Penicillium, Claviceps and Alternarian called Mycotoxins represents a worldwide problem for farmers and food industries. Mycotoxins-containing feed can cause serious diseases to farm animals resulting in suffering and even death, causing serious economic losses. This thesis focuses on the effect of different silicate clay minerals (SCM) on decreasing the bioavailability of common Mycotoxins. The SCM in general are effective adsorbents, and can efficiently bind the Mycotoxins in the gastro-intestinal tract of animals and decrease their bioavailability. In this study the SCM will be gathered from different sources and assayed in vitro on different Mycotoxins. Parameters like pH, temperature , time of contact , concentration, shaking and the size of the clay materials will determined the binding efficiency of the different SCMs. Measurement of the residual Mycotoxins will be performed by high performance liquid chromatography HPLC coupled with a diode array UV detector (UV/DAD) or fluorescence detector (FD).
Impact of Processing on Oil Quality Evaluated by GCxGC-ToF-MS. André Düsterloh, Karin Volz, Neil Macfarlane, DSM Nutritional Products, Kaiseraugst, Switzerland
Stability and sensorical properties are crucial parameters for the quality of oils. Oil refining consists of several steps to remove lipid oxidation products and to stabilize the resulting oils. Due to the high number of doubles bond fish oils are very susceptible to lipid oxidation. The formed lipid oxidation products like 2,6-nonadienal, 4-heptenal, 2,4-heptadienal can cause severe fishy and painty/chemical off-odors which are generally not accepted by consumers. Typical lipid oxidation products like 2,4-decadienal or 3-octene-2-one can cause a rancid or metallic off-odor in n-6 oils. In order to monitor lipid oxidation reactions during oil refining and further processing of oils, sensitive analytical methods are required to detect odor-active compounds below human threshold levels. A SPME-GCxGC-ToF-MS was developed which is capable of analyzing major lipid oxidation products ranging from the very volatile 2-propenal to 2,4-decadienal at low ppb levels. The method is routinely used to evaluate raw materials, refined oils and powder formulations thereof as well as applications in various food matrices. The obtained analytical results generally correlate well with data from sensory panel trials and can be used for optimization of production and formulation processes.
ANA 5: General Analytical II
Chair(s): S. Bhandari, Silliker Inc., USA; and R. Della Porta, FritoLay, USA
A Comparison of Ionic Liquid and Polymer Based Capillary Columns for the Analysis of FAME Isomers. L.M. Sidisky, Y. Ni, G.A. Baney, J.L. Desorcie, K.K. Stenerson, Supelco, Bellefonte, PA, USA
Analyses of fatty acid methyl esters (FAMEs) are continuing to gain importance as more research is focusing on their biomedical impacts. This includes the analysis of saturated and polyunsaturated FAMEs along with the cis & trans isomers. Traditionally, FAME analyses have been performed using silicone or polyethylene (PEG) based phases. Analysts performing the task of analyzing the fatty acid composition of food have a wide choice of capillary column selectivities available for resolving the fatty acids as FAMEs depending upon the information they require from their analyses. Nonpolar methyl silicone columns provide a boiling point separation of the FAME isomers with limited resolution of polyunsaturated isomers. Polar polyethylene glycol columns resolve the isomers by degree of unsaturation with minimal overlap of the carbon chain lengths. The highly polar cyanosilicone columns will resolve cis and trans isomers along with possibly providing positional geometric isomer separations depending upon the column type. A new class of phases based on Ionic Liquids have now been developed and have been demonstrated to provide unique elution patterns for FAMEs compared to the traditional silicone or PEG based polymer phases. We will compare and contrast the selectivity of the ionic liquid phases with the polymeric based phases for a variety of FAME samples.
Identification of Volatile Oxidation Products Responsible for Off-flavours in Oxidized Fish Oil. J.C. Sullivan, S.M. Budge, Dalhousie University, Halifax, NS, Canada
The long chain polyunsaturated fatty acids (PUFA) found in fish oil, specifically the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have many health benefits. Unfortunately PUFA is very unstable and prone to oxidation so fish oil supplements are often avoided due to fishy off-flavors formed during the oxidation process. Here we investigate a method to monitor oxidation in fish oils that correlates well with sensory parameters of the oil. SPME coupled with GCMS was used to monitor volatile oxidation products in fish oil that was incubated at 40 °C. Sensory assessment of oxidized oils was also conducted with an untrained panel. Discriminant function analysis was used to classify samples into acceptable and unacceptable groups. By monitoring these volatile compounds, a simple method to assess the extent of fish oil oxidation that correlates with sensory properties was developed.
Effective Separation and Analysis of Different Isomeric Forms of Oxidized Triacylglycerols Using Ultra-High-performance Liquid Chromatography and Tandem Mass Spectrometry. J.-P. Suomela, M. Tarvainen, H. Kallio, Department of Biochemistry and Food Chemistry, University of Turku, Finland
An analytical approach based on efficient ultra-high-performance liquid chromatographic (uHPLC) separation of different isomers of oxidized TAG species with two columns and their (tandem) mass spectrometric analysis is presented. In the study, a novel method utilizing positive ion ESI using ammonia supplemented in the nebulizer gas was used to produce ammonium adduct ions. The novel uHPLC method proved to be an efficient approach in resolving different regioisomers of various oxidized TAG species, although some overlapping was still present. The tandem mass spectrometric approach utilizing modified nebulizer gas composition allowed regiospecific analysis of oxidized TAG isomers and demonstrated interesting differences in the fragmentation patterns of these isomers. The chromatographic separation and regiospecific characteristics can be utilized in both qualitative and quantitative analysis of oxidized TAG molecules.
Monitoring the Epoxidation of Vegetable Oils by LC/MS for Process Optimization and Control. S. Tuan Anuar, Y.-Y. Zhao, G. Liu, J.M. Curtis, Lipid Chemistry Group, Department of Agricultural Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada
Epoxidised oils are useful starting materials for producing a wide range of renewable materials. In order to study the formation of reaction intermediates during the epoxidation of canola oil, a method using non- aqueous reversed phase liquid chromatography coupled to electrospray mass spectrometry was developed. By monitoring the formation and disappearance of partially epoxidised components, this rapid method has been used to elucidate reaction kinetics and to aid in process development. Furthermore, it was found that the end point of the reaction can be readily found by monitoring the ratio of fully to partially oxidized products in the mass spectra. Thus, by comparing various epoxidation reactions that occur in a natural oil mixture we have been able to establish simple and reliable criteria to aid in producing epoxidised oils with consistent profiles
Variations of Secoiridoid Derivatives, Lignans, Fatty Acids and Some Qualitative Characteristics of Sariulak Olive Oil as Induced by Growing Area. D. Arslan1, Y. Karabekir2, M. Schreiner2, 1Department of Food Engineering, Faculty of Agriculture, Selcuk University, Konya, Turkey, 2Division of Food Chemistry, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria
Sariulak olive variety comprises 6% of olive tree number cultivated in Mediterranean region of Turkey. The extra virgin olive oils of this monovariety were produced from olives cultivated in three different locations (Antalya, Karaman, Mersin) in the south region of Turkey and were analysed to select varieties and conditions that produce oil with favorable quality regarding oxidative stability, taste and nutritional properties. Peroxide value, free acidity, chlorophyll and carotene content, color values, fatty acid compositions were measured and the contents of secoiridoid aglycones and lignans were determined with a qualitative and quantitative analysis performed by GC-MS to characterize the different subclasses. In particular following sub-classes of phenols were identified and calibrated: simple phenols such as tyrosol and hydroxytyrosol (3,4-DHPEA and p-HPEA), their respective acetates (3,4-DHPEA-AC and p-HPEA-AC), secoiridoid derivatives such as the dialdehydic form of elenolic acid linked to 3,4-DHPEA or p-HPEA (3,4-DHPEA-EDA, p-HPEA-EDA, where EDA is elenolic acid dialdehyde) and isomers of oleuropein and ligstroside aglycones (3,4-DHPEA-EA and p-HPEA-EA, where EA is elenolic acid aldehyde), lignans such as (+)-1-acetoxypinoresinol, (+)-1-pinoresinol and hydroxy-pinoresinol.
Actual Ratios of Triacylglycerol Positional Isomers Consisting of Saturated and Highly Unsaturated Fatty Acids in Fishes and Marine Mammals. N. Gotoh1, Y. Matsumoto1, H. Yuji1, D. Zhao1, Y. Jiang1, T. Nagai2, H. Mizobe2, I. Otake2, K. Kojima2, I. Kuroda3, H. Watanabe4, N. Noguchi5, S. Wada1, 1Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo, Japan, 2Tsukishima Foods Industry Co. Ltd, Edogawa-ku, Tokyo, Japan, 3GL Sciences Inc., Iruma, Saitama, Japan, 4Department of Health Science, Kochi Women's University, Kochi, Kochi, Japan, 5Doshisha University, Kyotanabe, Kyoto, Japan
The actual ratios of triacylglycerol (TAG) positional isomer pairs, consisting of palmitic acid and highly unsaturated fatty acid (HUFA) such as DHA or EPA, in fish and marine mammals were investigated using a high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) system equipped with tandem jointed non-endcapped polymeric ODS columns. The results show that for combinations of DHA or EPA with two palmitic acids in the TAG of marine mammals, binding was almost all at the α (sn-1,3) position. In contrast, binding of DHA or EPA was mainly at the β (sn-2) position in fish. The preferred DHA and EPA positions in TAG were the same in the same marine mammal or fish. The binding position tendency of HUFA in TAG positional isomers consisting of two HUFAs and one palmitic acid was the same as that for combinations of one HUFA and two palmitic acids.
Omega Fatty Acid Analysis in Selected Matrices by Direct Transesterification Methods with and without an Acid Treatment. Sneh D. Bhandari, Jerry Leahy, Silliker Inc., Chicago Heights, IL, USA
A variety of matrices including fish, pasta and breaded shrimp samples were analyzed for omega-3 fatty acids by the AOAC 996.11 as well as different direct transesterification methods which do not require prior fat extraction. Performance of direct methods with and without inclusion of an acid treatment step for omega-3 fatty acid analysis was evaluated. Different acid steps were used during the evaluation. Comparison of the results of omega-3 fatty acid analysis obtained by different treatments will be presented.
Identification of Diacylglycerol and Triacylglycerol Containing 11,12,13-trihydroxy-9,14-octadecadienoic Acid in Castor Oil by Mass Spectrometry. Jiann-Tsyh Lin, U.S. Department of Agriculture, Albany, CA, USA
Castor oil has many industrial uses. Molecular species of acylglycerols containing monohydroxy, dihydroxy and trihydroxy fatty acids in castor oil have been reported. The identification of acylglycerols containing a triOH18:2 fatty acid in castor oil is reported here. The structure of this novel fatty acid was proposed as 11,12,13-trihydroxy-9,14-octadecadienoic acid by the electrospray ionization - mass spectrometry of the lithiated adducts of acylglycerols in the HPLC fractions of castor oil. The fragmentation pathways of the lithiated adduct of 11,12,13-trihydroxy-9,14-octadecadienoic acid were proposed. This fatty acid is predominately located at the sn-1,3 position of the glycerol backbone of triacylglycerols, triOH18:2-OH18:1-OH18:1. The regiospecific location was identified by the fregment ion from the loss of fatty acid as α,β-unsaturated fatty acid specific at the sn-2 position. The proposed biosynthetic pathway of polyhydroxy fatty acids in castor is also reported.
A Universal LC-MS/MS Method for the Quantification of Choline Containing Compounds and Other Phospholipids in Foods and Animal Tissue. Y. Xiong, Y.-Y. Zhao, C. Field, S. Goruk, R. Jacobs, K. Oilund, J.M. Curtis, Department of Agricultural Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada
Measurements are needed to understand the current sources and forms of choline present in diets of various populations. Here, we report on the development and validation of an LC-MS/MS method to quantify all of the major choline-containing compounds found in a wide variety of foods. Tissues and food samples were spiked with isotopically labeled extraction recovery standards, synthesized in-house by establised procedures, and by commercially available deuterated internal standards. HILIC chromatography was used coupled to positive ion electrospray mass spectrometry. The MS/MS method was optimized for each compound class so that the final method used a combination of precursor ion, fragment ion and neutral loss scans. In this way, 14 compounds or compound classes including 8 phospholipids and 6 choline containing compounds were quantified in a single LC-MS/MS method. Validation demonstrated that the method is sufficiently sensitive, and is accurate, precise and linear over two orders of magnitude. Recoveries in the range of 90% to 115% were obtained by spiking a range of sample matrices with authentic standards containing all of the target analytes. The total choline content of a large number of foods and tissues has been obtained using the validated method.
Chair(s): F.J. Eller, USDA, ARS, NCAUR, USA
Chromatographic Analysis of Biodiesel from Several Oils with Potentiality for Production Diversification in South of Brazil.
Rosana de Cassia de Souza Schneider , Guilherme José Weiss , Maiara Priscilla de Souza , Thiago Rodrigues Bjerk , William Leonardo Teixeira da Silva , Pablo Diego Gressler, Rodrigo Augusto Klamt, Santa Cruz do Sul University, Santa Cruz do Sul, Rio Grande do Sul, Brazil
Recognition of oils composition is important because these raw materials are versatile, but there is a large regional variability and oxidation susceptibility. Because this, we attempted to identify the main fatty acids present in biodiesel obtained from oils and fats with potentiality for production in Rio Pardo Valley, southern Brazil. The fats and oils samples were transesterified in a pilot plant for biodiesel production developed at the University of Santa Cruz do Sul. In the reaction, the sodium methylate and methanol at a temperature of 65°C and with strong agitation were used. For derivatization of standard oils and fats it was used BF3/metanol according to Christie, 1992. The analysis was performed on GC-MS QP 2010 plus equipment. The seeds used were: canola, sunflower, tung, castor, tobacco and peanuts, all of them presenting potentiality for biodiesel production in Rio Pardo Valley. We also analyzed the chromatographic profile of the low-quality tallow produced in a local tannery. Among the samples analyzed, we highlight the sunflower biodiesel obtained from Atlantic seeds that presents 78% of C18: 1 and the tobacco and tallow biodiesel, because they present an adequate chromatographic profile for biodiesel production and they are waste of low economic value. CNPq, PUIC-FAP-UNISC, FINEP, SCT-RS, CAPES.
Identification and Quantitation of Intact Glucosinolates in Canola Meal by UPLC-MS/MS.
B. McNew, J. Flook, T. Patterson, D. Schwedler, C. Kahl, D. Meyer, A. Syed, Dow AgroSciences, Indianapolis, IN, USA
The purpose of this study was to improve the current method used to determine glucosinolate concentrations in canola and rapeseed. The current method is labor intensive and depends on standard calculated absorbance conversion factors to determine the concentration of individual desulfonated glucosinolates. An improved method is described using liquid chromatography (UPLC) coupled with ESI mass spectrometry in negative ion mode. Complete chromatographic separation is achieved in 30 minutes using an Aquity BEH Shield C18 (2.1x150mm, 1.7 µm) column. Following a simplified sample extraction, elimination of the desulfonation step, and UPLC separation, the intact glucosinolates are evaluated by monitoring specific transitions in MRM mode. These transitions are derived from the loss of the sulfate group [SO4H] - which produces the fragment ion at m/z 97. The proposed method provides the ability to characterize variations in glucosinolate composition and concentration due to the effects of genotypic and environmental influences.
High Resolution Mass Spectrometry Profiling of Triacylglycerols for Determining the Biofuel Potential of Microalgae Strains.
K.M. Glenn, J. McNichol, P.J. McGinn, S.J.B. O'Leary, J.E. Melanson, National Research Council of Canada, Institute for Marine Biosciences, Halifax, Nova Scotia, Canada
Biofuels from microalgae are gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves converting intact lipids into fatty acid methyl esters prior to analysis by gas chromatography. However, analysis of the intact lipid profile is vital for selecting the most appropriate algal strains, as more polar lipids such as phospholipids are less desirable for biofuel production than triacylglycerols (TAGs). Herein, lipid extracts of microalgae are directly analyzed by ultra-high pressure liquid chromatography (UHPLC) coupled to a high-resolution mass spectrometer (MS). Using a ternary solvent system, a complete chromatographic profile of intact polar lipids and triacylglycerols was achieved in less than 15 minutes. MS data was collected in a non-targeted fashion and TAGs were identified based on accurate mass and fragmentation patterns. For instance, over 50 TAGs were identified in the algae Botryococcus braunii, the most abundant being 18:1/18:1/18:1, 18:1/18:1/18:3, 28:1/18:1/18:1 and 28:2/18:1/18:1. Heat maps allowed the relative abundance of TAGs to be compared among six strains of microalgae to gauge their biofuel potential. The effects of various growth conditions and extraction procedures on the lipid profiles will also be described.
Quantitation of Underivatized Omega-3, Omega-6, and Omega-9 Fatty Acids in Foods by HPLC and Charged Aerosol Detection.
M.A. Plante, B. Bailey, C. Crafts, I. Acworth, J. Waraska, ESA - a Dionex Company, Chelmsford, MA, USA
The omega (ω) fatty acids are a group of compounds that include both essential ω-3 and ω-6, and non-essential ω-9 fatty acids. Recent studies have investigated the roles of these fatty acids in human physiology and health, with results suggesting that the ratio of ω-3 and ω-6 is important. To determine the amounts of these compounds in foods and supplements, a simple analytical method was developed using high performance liquid chromatography (HPLC) and charged aerosol detection.Omega fatty acids are measured using gas chromatography (GC). For foods, lipid analytes are extracted from the samples, are hydrolyzed to release the fatty acids from their triglycerides, and then esterified for analysis by GC. Regardless, this approach is tedious, time-consuming and the high temperatures can affect polyunsaturated fatty acid stability. Using this method, samples can be extracted, hydrolyzed and directly analyzed to low levels for the determination of ω-3, -6 and -9 fatty acids in supplements and in traditional and commercially-produced foods. Examples of several oils and foods are presented.
Short Peptides in Complex Mixtures: Hydrophilic Interaction Chromatography (HILIC) Contribution to the Identification Strategy.
C. Harscoat-Schiavo, E. Ronat-Heit, F. Fournier, R. Kapel, I. Marc, Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, 13 rue du bois de la Champelle, F-54500 Vandœuvre-lès-Nancy, France
The industrial oil extraction process from rapeseed leads to rapeseed meal, a protein-rich co-product which value could be enhanced after enzymatic hydrolysis. Indeed, due to their numerous bioactivities, short peptides (<1000 g/mol) from vegetable proteins are of great interest for the food, cosmetics and pharmaceutical industries. But hydrolysis leads to a complex mixture of peptides with various sizes and properties. Industrial production of peculiar peptides is then subjected to great fractionation, purification and characterisation issues. Rational separation strategies are needed to adjust downstream processing to focus on peptides or fractions of interest. Various analytical techniques have been combined to fractionate peptides according to their charge, hydrophobic and/or hydrophilic properties, and to determine their amino acid compositions. These chromatographic separations have been modelled, thus predicting physicochemical properties of peptides from their elution. Complementarity between HILIC and reversed phase chromatography (RPLC) was demonstrated: HILIC model gives knowledge about hydrophilic intensity that is not redundant with the hydrophobic one.Relating peptides retentions to their properties, this strategy enables to direct fractionation towards mixtures enriched in targeted bioactivities.
High-resolution NMR Spectroscopy: An Alternative Fast Tool for Qualitative and Quantitative Analysis of Diacylglycerol (DAG) Oil.
Emmanuel Hatzakis1, Alexia Agiomyrgianaki2, Sarantos Kostidis3, Photis Dais2, 1University of North Carolina Wilmington, Wilmington, NC, USA, 2University of Crete, Heraklion, Crete, Greece, 3National and Kapodistrian University of Athens, Athens, Greece
Multinuclear (1H, 13C, 31P) and multidimensional NMR spectroscopy was employed for the analysis of diacylglycerol (DAG) oil and the quantification of its composition. A number of gradient selected two dimensional NMR techniques (TOCSY, HSQC-DEPT, HSQC-TOCSY, and HMBC) facilitated the assignment of the complex one dimensional 1H and 13C NMR spectra. In several cases, the aforementioned 2D NMR techniques offered solid proof regarding earlier assignments based on the empirical substituent effects. Integration of the appropriate signals in the NMR spectra of the three nuclei allowed the determination of DAG oil composition, which was found to be within the limits accepted for this oil, namely 1-monoglycerols 0.40-0.60%; 2-monoacylglycerols 0.40-0.50%; 1,3-diacylglycerols 57-62%; 1,2-diacylglycerols 28-32%; triacylglycerols 9-11%; saturated fatty acids 3-5%; oleic acid 37-45%; linoleic acid 49-53%; and linolenic acid 5-6.5%; tocopherols 0.24-0.27%. The compositional results obtained from the NMR spectra of the three nuclei were compared and discussed in terms of accuracy, precision and performance.
Rapid Characterization of Edible Oils for Purity and Composition using Open Air Direct Analysis in Real Time (DART) Mass Spectrometry.
Brian Musselman, Elizabeth Crawford, Jordan Krechmer, IonSense, Inc., Saugus, MA, USA
Rapid analysis of edible oils for composition and presence of potential adulterants is facilitated by using direct analysis in real time (DART) ionization coupled with mass spectrometry. Characterization of the major components of oil including triglyceride, diglyceride and fatty acids without sample preparation is completed in a few seconds per sample. Recently, determination of the distribution of these components in different olive oils has been utilized to differentiate the quality grade of these oils (Vaclavik et al, Anal. Chim Acta., 10: 645 (1-2); 56-63, 2009,). We have investigated the use of this technology operated in a high throughput configuration to detect adulterants with little or no sample preparation. Demonstration of the utility of this method for detection of common adulterants in edible oils including; less expensive oils used for economic adulteration, contaminants such as 3-MCPD, and non-oil based products that might be mixed with oils in production errors will be described.
Sensitive Analysis of Fat-Soluble Vitamins and Antioxidants by RP-HPLC.
M.A. Plante, B. Bailey, C. Crafts, I. Acworth, J. Waraska, ESA - a Dionex Company, Chelmsford, MA, USA
Fat-soluble vitamins (FSVs) and antioxidants (FSAs) play essential roles in a wide spectrum of biochemical and physiological processes. Vitamin E (tocopherol) along with other FSAs (e.g., carotenoids, CoQ10) are purported to help mitigate the effects of oxidative stress that have been linked to numerous diseases including cancer, neurodegeneration, and atherosclerosis. These compounds are thought to exert their beneficial effects by acting as chain-breaking antioxidants, inhibiting lipid peroxidation of PUFAs contained within biological membranes, thereby preventing the formation of potentially cytotoxic and highly reactive aldehydes.We developed a simple, RP-HPLC-charged aerosol detection method for the measurement of 11 FSVs and FSAs in commercially available supplements including: vitamins A (trans-retinol, retinyl acetate and palmitate), E (α-,δ-,γ-tocopherols and succinate), D, and K1 (phylloquinone), lycopene, lutein, and CoQ10. The analysis was completed in 20 min. Correlation coefficients were > 0.999. LOQ values were < 30 ng on column. This method is an enhancement over current analytical approaches (e.g., UV and ELSD) and offers a simple means of quantifying FSVs and FSAs in supplements and foods. Recovery values from commercial products show the quantitative capacity of this method.
**Cancelled** Determination of a Benzo[a]pyrene in Palm-derived Tocotrienols.
Shary Ong, Davos Life Science, Singapore
Identification and Quantitation of Biodiesel and other Reaction Intermediates in Lipase-catalyzed Transesterified Oils and an Oil-fat blend by HPLC.
Alberta N.A. Aryee, Benjamin K. Simpson, Department of Food Science and Agricultural Chemistry, Faculty of Agricultural and Environmental Sciences, McGill University, Ste. Anne de Bellevue, QC, Canada
A high performance liquid chromatography (HPLC) unit equipped with size exclusion column and a refractive index (RI) detector was used for simultaneous monitoring, identification and quantitation of the reaction components from lipase-catalyzed transesterification of three oils and fat. The procedure simultaneously separated and detected the unreacted triacylglycerols (TAG), diacyl- and monoacyl- glycerol (DAG and MAG) co-products, residual alcohol as well as free fatty acid (FFA) based on retention times. The chromatograms showed well separated and resolved peaks. The elution of the components from the transesterification reaction in increasing order was: TAG < DAG < FFA < MAG. Generally, higher alcohol ratios decreased the conversion of TAG in all the lipids studied with between 14 and 94% of TAG remaining at all the treatment combinations. Higher amount of SSO TAG was generally converted to DAG than Rothsay composite (RC) and olive oil (OO) TAG. Relatively higher amount of OO DAG was converted to MAG than SSO and RC with only 5-14% DAG remaining in OO. RC and OO generally accumulated less MAG and this was reflected as lower MAG levels in RC (< 6%) and OO (< 14%) compared with SSO (< 27%). For the various treatment combinations and the three lipids used in this study, the least amount of FFA was recorded in transesterified OO with a maximum of approximately 4%. This HPLC method can be used as a simple and fast technique to analyze the reaction components and products of transesterification reactions without the need for additional derivation steps.
Selective Fatty Acid Methyl Ester Binding in Starches of Different Botanical Origin.
B. Manion, K. Seetharaman, University of Guelph, Guelph, Ontario, Canada
Amylose, linear glucose chains in starch, forms helices with hydrophobic interiors that encapsulate non-polar molecules such as fatty acids. Amylose content analysis involves adding iodine to form blue polyiodide complexes and measuring absorbance at 620 nm, since branched amylopectin binds smaller triiodide complexes that absorb at lower wavelengths. Anhydrous milkfat, with fatty acids ranging from C4-C18, was methylated and added in excess to starch:water mixtures heated at 95C. Hardened starch paste balls formed and were methanol washed then bound fatty acid methyl esters (FAMEs) were extracted with dichloromethane and analyzed using gas chromatography. Starch:water/1:1.25 ratio was optimal. Less water did not hydrate starch pastes while more water caused increased swelling of starch balls that dissolved during methanol washing and bound less FAMEs. Waxy starches preferentially bound C4 FAMEs while starches with amylose preferentially bound longer-chain FAMEs (C14-C18). Interestingly, an R2 of 0.78 was observed when plotting 11 starches for amylose content and bound FAMEs with the exception of corn, potato and wheat starch binding more FAMEs and tapioca starch bound iodine preferentially over FAMEs. Results show different starches bind varying amounts of FAMEs based on amylose content but also based on starch granule architecture differences.
High-throughput Time Domain NMR Technology for Fats/oils Analysis.
Xenia Tombokan, Supriyo Ghosh, Bruker Corporation, The Woodlands, TX, USA
Time domain nuclear magnetic resonance (TD-NMR) can be implemented very easily using a small bench-top or hand-held instrument for fats and oils quality control and R&D. The technique is applied for determining oil sources quality (oil and moisture content in seed batches), extraction process efficiency (oil and moisture content in residues), and extracted oil characteristics (solid fat content, melting profile, iodine value). Subsequently, these oils go through further refining/processing for applications in many other areas including food, polymer, biofuel, etc, and TD-NMR can be utilized as a noninvasive analytical tool for formulations research (melting/solidification hysteresis), processing conditions optimization and control (oil migration, water mobility), and product stability examination (o/w or w/o emulsion stability, product spoilage). TD-NMR measurement is based on extracting the signal from hydrogen nuclear spins (e.g. water/moisture, oil/fat) in the sample and mathematical calculation on the signal to rapidly obtain physicochemical information on the sample at a molecular level. In this presentation, TD-NMR principles and applications in fats/oils industry will be elaborated. Future research direction and other TD-NMR applications will be touched upon.
Simple and Direct Analysis of Phytosterols by Reversed-Phase HPLC and Charged Aerosol Detection.
M.A. Plante, B. Bailey, C. Crafts, I. Acworth, J. Waraska, ESA - a Dionex Company, Chelmsford, MA, USA
Phytosterols are a group of naturally occurring steroid alcohols found in plants. They are key structural components of plant cell membranes, assuming the role that cholesterol plays in mammalian cells. There is considerable interest in phytosterols as dietary supplements as they are reported to lower cholesterol levels and also have a positive impact on cardiovascular diseases, but may also aggravate atherosclerosis and lead to aortic valve stenosis. Phytosterols are typically measured by gas chromatography (GC). However, this approach is time-consuming since it requires complex sample preparation. Here, a straightforward method is presented using reversed-phase HPLC and charged aerosol detection, for the direct measurement of several phytosterols. Five standards–campesterol, cholesterol, stigmasterol, β-sitosterol, and stigmastanol–were resolved in < 35 min. Calibration curves (5-2500 ng on column) were linear and the LOD was ≤ 5 ng on column for all analytes. The method was used to directly determine the phytosterol content of diluted red palm oil, matching literature values. Furthermore, as the mobile phase is also compatible with the LC-MS, this technique can be used to further identify analytes in the sample. This approach can be used to examine product purity and authenticity, supplement content, and possible adulteration.
Novel Fatty Acid, 12,17-dimethyloctadecanoic Acid, from Thermogemmatispora Strain T81.
M. Vyssotski1, J. Ryan1, K. Lagutin1, H. Wong1, X. Morgan2, M. Stott2, 1Industrial Research Limited, Lower Hutt, New Zealand, 2Institute of Geological and Nuclear Sciences, Wairakei, New Zealand
The major fatty acids of a novel species of Thermogemmatispora (strain T81) from the phylum Chloroflexi were identified as i18:0 (42.8% of total fatty acids), i19:0 (9.7%), and i17:0 (5.9%). Also observed was an unidentified fatty acid (16.3%) with ECL of 19.04 (BP-1), and 18.76 (TG-WAXMS A). GCMS revealed that this compound is a saturated 20-carbon atom branched fatty acid. The acid was isolated by preparative HPLC. 1H- and 13C-NMR, with 1H-1H-COSY and 1H-13C-HSQC experiments suggested the structure of dimethyl octadecanoic acid with iso-branching, and an extra middle-chain methyl group. A pyrrolidide derivative demonstrated the characteristic gaps in GCMS indicating methyl branching at C12 and C17, the fact eventually confirmed by 1H-13C-HSQCTOCSY experiment. A search of www.reaxys.com database for 12,17-dimethyloctadecanoic acid resulted in no hits. However, a recent description of a phylogenetically related species of Thermogemmatispora (Yabe et al. 2010, doi:10.1099/ijs.0.024877-0), noted an unidentified 20:0 fatty acid with matching GC behavior and GCMS data. These data suggest that Thermogemmatispora share an ability to synthesize the same fatty acid.
Analysis of Lipid Classes by HPLC-CAD: A Viable Alternative to Common GC-based Approaches.
M.A. Plante, B. Bailey, C. Crafts, I. Acworth, J. Waraska, ESA - a Dionex Company, Chelmsford, MA, USA
Lipids are a structurally diverse group of compounds that can be challenging to measure. Typically, the sample is first extracted, prior to derivatization to either render the lipid more volatile for gas chromatography (GC) or to introduce a chromophore for UV detection. Sometimes a combination of techniques are used to more fully characterize a sample, including GC, high performance liquid chromatography (HPLC) with evaporative light scattering detection, and LC-mass spectrometry. The use of charged aerosol detection (CAD) allows for direct and low-level quantitation of these non-chromophoric analytes without derivatization and with most LODs below 10 ng on column.Several analytical methods will be presented to illustrate both global and targeted approaches. Chemistries discussed include the use of a universal, reversed-phase method to resolve steroids, free fatty acids, free fatty alcohols, phytosterols, acylglycerides (mono-, di-, and tri-), phospholipids, and paraffins in a single analysis. A normal-phase (NP) LC method is also presented for single-peak phospholipid quantification. Practical examples are also presented, including total glycerides in biodiesel, phytosterols in natural oils, and fat soluble vitamins found in commercially available supplements.
Comparison of AOAC Official Method 922.06 (Fat in Flour) to AOCS Official Method Ce 1k-09 (Determination of Total Fat by Gas Chromatography).
Kathryn Reihel, Tiffanie West, Bunge North America, USA
A new method for determining the amount of fat in food was compared to AOAC Official Method 922.06, referred to as the Mojonnier method. For the comparison, 18 samples were prepared using both methods. A paired t-test was used to statistically compare the two sets of samples. The new method reduces the preparation time and eliminates the use of ether as a solvent.
Supercritical Fluid Extraction for the Removal of Lipid and Interfering Compounds Prior to Radiocarbon Dating of Archeological Artifacts.
J. King1, J. Phomokay1, K. Steelman2, M. Rowe3, 1University of Arkansas, Fayetteville, AR, USA, 2University of Central Arkansas, Conway, AR, USA, 3Texas A&M University-Qatar, College Station, TX, USA
Supercritical extraction (SFE) utilizing carbon dioxide and select cosolvents has been applied for the removal of chemical compounds such as caffeine from coffee or tea, pesticides from ginseng, fungicides from cork, as well as reducing the lipid content of food products. In this study, a SC-CO2 using methanol has been used to treat valuable archeological samples or artifacts prior to using low-temperature plasma coupled with accelerator mass spectrometry radiocarbon dating. SC-CO2 with methanol applied at 3000 psi and 40 C was applied to fiber, macroflora, charcoal, and wood to reduce the soil humic acid content which contaminate the sample and contribute to inaccurate radiocarbon dating of the artifact samples. Similarly, burial gauzes from Egyptian mummies containing lipid constituents were treated with SC-CO2-cosolvent mixtures. The SFE pretreatment can replace harsh acid-base pretreatment methods, and when coupled with a non-destructive argon or oxygen plasma for microgram carbon removal for accelerator MS isotope ratio determinations. This combination of techniques requires as little as 0.05 milligrams of carbon-equivalent weight for accurate determination of the age of the artifact without visible change to the artifact.
Rapid Analysis of EPA, DHA, and Other Critical Parameters for Fish Oil.
Kangming Ma, Kirsten Kramer, Ching-hui Tseng, Barbara Stefl, Cognis, Cincinnati, OH, USA
EPA/DHA are critical quality parameters for fish oil. Chromatrographic methods are commonly used to accurately measure EPA/DHA contents during refining process and on refined fish oils. This method is time consuming and not practical when rapid results are needed. New methods based on NIR and Mid-IR are developed using patented technology
A Non-invasive Optical Method for Quality Control of Maturing Wine Grapes.
P. Pinelli1, E. Fierini1, A. Romani1, G. Agati2, S. Miele3, 1Department of Pharmaceutical Sciences, University of Firenze, Firenze, Italy, 2IFAC CNR, Sesto F.no, Firenze, Italy, 3Department of Agronomy & Agroecosystem Management, University of Pisa, Pisa, Italy
Accumulation of anthocyanins (Anth) in whole winegrape (Vitis vinifera L.) bunches still attached to vine plants was followed non-destructively using a fluorescence-based sensor. The method is based on the screening of fruit chlorophyll fluorescence and allows the Anth contents of intact berry skin to be measured. With increasing Anth content, less excitation light is transmitted to the deeper chlorophyll layers, and thus the chlorophyll fluorescence signal decreases proportionally. Tests were carried out in 2009-2010 in vineyards of Central Italy. Measurements were performed weekly from véraison to harvest on Sangiovese, Cabernet Sauvignon, Merlot, Shiraz and Petit Verdot cultivars. The ANTHRG index showed a different time evolution and Anth accumulation for the cultivars. The data obtained from the sensor were compared with the spectrophotometric analyses in order to assess the anthocyanoside content. The fluorescence-based sensor used in the present study represents a rapid and non-invasive tool for assessing grape phenolic maturity and quality, and may represent in perspective an alternative to destructive laboratory analyses, which are time-consuming and require an accurate sampling approach.
Identification of Fatty Acid Composition and Triglycerides Profile of Papaya Seed Oil from Different Varieties by Using LC-MS and GC-MS.
Shadi Samaran , Hamed Mirhosseini, Chin Pin Tan , Parviz Kavousi , Yaakub Che Man , University Putra Malaysia, Selangor, Malaysia
The major papaya varieties mostly cultivated in Malaysia are Sekaki, Eksotika, Eksotika II as well as Batu Arang. Since the papaya seed is mainly considered as the waste in food industry, the present study was conducted to investigate the effect of four different varieties of papaya (i.e. Sekaki, Eksotika, Eksotika II and Batu Arang) on fatty acid composition and triglycerids profile of papaya seed oil extracted by using solvent extraction technique. In this study, Liquid Chromatography-Mass Spectroscopy (LC/MS) and Gas Chromatography-Mass Spectroscopy (GC/MS) were used for qualitative analysis of fatty acid composition and triglycerids profile of papaya seed oil from different varieties. The results indicated the existence of 11 fatty acids in papaya seed oils obtained from all four varieties i.e. C16:0, C18:0, C20:0, C22:0, C16:1, C18:1, C20:1, C22:1, C18:2, C18:3, C22:4, C22:5 and C20:3; while the comparison analysis of fatty acids by GC/MS didn't show any significant (p > 0.05) difference between the amount of lauric acid methyl ester, myristic acid, methyl ester, palmitic acid, methyl ester, 7-hexadecenoic acid methyl ester and elaidic acid methyl ester in the extracted seeds oil.
A Simple, Modified GCMS Method to Quantify Oxidation Products in Edible Oils.
V. Jain, Oil-Dri Corporation of America, Chicago, IL, USA
Validation of FT-NIR Models for the Rapid Determination of Fatty Acid Composition.
M.M. Mossoba1, H. Azizian2, J.K.G. Kramer3, 1Food and Drug Administration, College Park, MD, USA, 2NIR Technologies Inc., Oakville, ON, Canada, 3Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, ON, Canada
Fourier transform near-infrared spectroscopy (FT-NIR) has recently been demonstrated to be a viable alternative to capillary gas chromatography (GC) for the rapid (1 min) determination of fatty acid composition for edible fats and oils. This method entailed the development of appropriate FT-NIR models using partial least squares analysis that required reliance on accurate GC determinations to correlate the fatty acid methyl ester (FAME) chromatographic profiles to FT-NIR spectral information. While GC is a complex and time consuming technique, subsequent FT-NIR routine measurements were time saving and cost effective in meeting regulatory food labeling requirements. An inter-laboratory study for the evaluation and validation of commercially available FT-NIR models was conducted. Several FT-NIR spectrometers from similar as well as different manufacturers were used in this preliminary multi-laboratory study on neat (underivatized) edible fats and oils. Several parameters were compared and used to evaluate the robustness of the FT-NIR models. These included transmittance and fiber optic transflectance measurements, as well as dependence of precision and accuracy on pathlength and temperature. The application of commercial FT-NIR models to the analysis of edible fats and oils will be discussed.
Antioxidant Phytochemicals in Pecan Kernel [Carya illinoensis (Wangenh) K. Koch] and Pecan Byproducts.
D.M.A. Molina-Quijada1, L.A. Medina-Juárez2, N. Gámez-Meza2, 1Posgrado en Biociencias. Universidad de Sonora, Hermosillo, Sonora, Mexico, 2Depto. Investigaciones Científicas y Tecnológicas de la Universidad de Sonora, Hermosillo, Sonora, Mexico
The total phenolic and total flavonoids contents in pecan kernel cake and pecan husk extracts (70% methanol) were estimated in two pecan cultivars (Western and Wichita). Their antioxidant activities were evaluated through ABTS and DPPH. Significantly higher levels (p<0.05) of total phenolics (16.6 and 17.0 mg GAE/g), total flavonoids (14.4 and 13.7 mg QE/g) and antioxidant activities (409.5 and 402.6 μmol TEAC/g) were observed for the pecan kernel cake extracts in two cultivars (Western and Wichita, respectively). However, the pecan husks from two cultivars (Western and Wichita) showed significant levels of total phenolics (13.0 and 14.1 mg GAE/g), total flavonoids (10.9 and 11.8 mg QE/g) and antioxidant activity (284.7 and 317.3 μmol TEAC/g, respectively). The inhibition percent determined by DPPH was 94% for pecan kernel cake extracts and 88.9% for pecan husk extracts. These results suggest that pecan husk could potentially be considered as an excellent and readily available source of natural antioxidants.
Trans-trans CLA Fatty Acid Isomer Separation from CLA-rich Oil by Ag+ HPLC.
Utkarsh Shah, Jack Lay, Andrew Proctor, University of Arkansas, Fayetteville, Arkansas, USA
A CLA (conguated linoleic acid) rich soy oil has been produced by photo-isomerization of linolieic acid, containing 20% CLA. Most CLA is as trans, trans isomers, but positional isomers have not been determined. Recent studies showed trans, trans CLA- rich soy oil had significant anti-heart disease and anti-diabetes effects. The objectives of this study are to determine the chromatographic conditions for isolation of trans, trans, CLA isomer to allow biological activity evaluation. CLA rich soy oil was saponified to obtain free fatty acids and then extracted by partitioning into hexane. 2 mg of FA in hexane were then injected into two Ag+ columns in series (250 x 10 mm) with a mobile phase of Hexane with 2.5% Acetic acid and 0.025% Acetonitrile at a flow rate of 4.72 ml/min. CLA which are conjugated dienes were observed at 233 nm and collected. Trans-trans CLA isomers were identified using ATR-FTIR and GC-FID. This study showed that trans-trans CLA isomers present in CLA-rich oil are mixture of two isomers which were not resolved by GC-FID and RP-HPLC. We anticipate using preparative Ag+ HPLC to isolate larger quantities of trans, trans CLA for nutrition studies.
Nutraceutical Content of Georgia-grown Pomegranate Cultivars.
D. Rajasekar, C.C. Akoh, K.G. Martino, D.D. MacLean, University of Georgia, Athens, GA, USA
Pomegranate juice is well recognized for its phytonutrient content. The objective of this study was to evaluate the effect of processing on juice yield and antioxidant properties of fourteen pomegranate cultivars grown in Georgia. Folin-Ciocalteau method was used to determine the total polyphenols. Antioxidant capacity was studied using ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), and oxygen radical absorbance capacity (ORAC) assays. The juice yield ranged between 20-41% based on fresh weight of the fruit. Total polyphenols ranged from 28.88 – 88.75 mg GAE/100 g of FW. Cultivar Cranberry and R-26 had the highest antioxidant capacity. The total monomeric anthocyanins were found to vary between 6.18–158.56 mg cyanidin-3-glucoside equivalents/L. The organic acids, sugars, phenolic acids, and flavonoids were quantified using HPLC. Citric acid was found to be the main organic acid, followed by malic acid in all the cultivars. This study would enable pomegranate growers in Georgia to determine a suitable cultivar that could be grown to produce maximum juice yield with good nutritional properties.
Measurement of Conjugated Linoleic Acid in CLA-Rich Potato Chips by ATR-FTIR.
J. Kadamne, C. Castrodale, A. Proctor, University of Arkansas, Fayetteville, AK, USA
We have made CLA-rich potato chips by frying in CLA-rich soy oil. Measurement of CLA in chips by extracting oil and GC-FID or FTIR analysis is tedious and time consuming. The objective of this study was to develop a rapid FTIR method to determine the CLA content of oil in potato chips, directly from the chips. Photoisomerized soy oil samples were diluted with soy oil and a total of 100 soy oil samples with total CLA levels ranging from 0.89-24.4% were made. Potato chips were fried using each of the 100 300 g CLA rich soy oil dilutions at 175°C for approximately 3 min. Duplicate GC-FID fatty acid analysis were conducted on oil extracted from each batch of potato chips. The chip samples were ground and duplicate ATR-FTIR spectra of each sample were averaged. Calibration models were developed using PLS regression analysis to correlate total CLA and CLA isomers from GC-FID fatty acid data in potato chips with corresponding FTIR spectral features. The models were full cross validated and tested using samples that were not used in the calibration sample set. Calibrations for total CLA; trans, trans CLA isomers; cis-9, trans-11 CLA had coefficients of determinations (R2v) of 0.96, 0.96 and 0.95, and corresponding root-mean-square error of prediction (RMSEP) of 1.79, 1.54 and 1.17 respectively. The ATR-FTIR technique showed great potential to determine CLA levels in potato chips.
Selection of Vegetal Oil Ingredients Based on Sensory Profile Ageing.
J.C. Mifsud, M. Lamboy, C. Schneider, M. Parada, M. Bonnefille, Alpha M.O.S. America Inc., Hanover, MD, USA
Vegetal oils are common ingredients entering the composition of many food products. Hence, it is important that their sensory properties do not negatively affect the overall sensory features of the final product, especially over ageing.An Electronic Nose was used to analyze the complex headspace of 3 vegetal oils (palm, soybean, colza) aged at ambient temperature and under daylight conditions. Samples were analyzed at day 0, then at days 5, 10 and 15.Prior to analysis, oil samples were incubated for 20 minutes at 60°C in order to generate a homogeneous headspace phase, of which a fraction was injected onto the E-Nose.A Principal Component Analysis model was set up based on instrumental measurement, showing a clear differentiation of the various oils and different behaviors over photo-oxidation and ageing.The analyzer-integrated database of Kovats indices allowed to identify the main odorant compounds: the important presence of numerous alkanes is typical of a high level of oxidation.The electronic nose thus brought the ability to rapidly compare the sensory quality of oil products. By building quality control cards, it is also possible to check the batch to batch conformity of ingredients and final products and assure the consistency of testing methods.
Detection of Residual Hexane in Edible Oils using Flash Gas Chromatography with Headspace Injection.
J.C. Mifsud, M. Lamboy, C. Schneider, M. Parada, M. Bonnefille, Alpha M.O.S. America Inc., Hanover, MD, USA
Hexane solvent is used to extract oil from oleaginous grains. After the extraction step, the solvent must be removed in order to guarantee a safe product for consumers.Many countries set regulations on the permitted hexane concentration in edible oils (e.g. NF EN ISO 9832 sets the maximum authorized concentration at 1 mg/kg in Europe).Gas chromatographic, most often with direct injection, is a common technique used for determining the hexane content in oils.This study proposes to analyze and quantify very low amounts of hexane in extracted vegetal oils using flash gas chromatography with headspace injection mode.Quantitative determination was achieved with a gas chromatograph featuring an embedded trap for pre-concentration before injection and 2 metal columns of different polarities coupled to two Flame Ionization Detectors.The method was calibrated using an alkane solution (n-hexane to n-hexadecane) in order to convert retention time in Kovats indices, then identify and quantify the volatile components.Oil samples were heated at 60°C for 20 minutes and a headspace fraction of 5mL was injected onto the instrument.The method was fast (analysis time was less than 90 seconds) and sensitive, showing a detection limit of 0.1mg/kg.
Rapid Determination of Total Saturated Fat and Total trans Fat Contents by FT-NIR for Regulatory Compliance.
M.M. Mossoba1, H. Azizian2, J.K.G. Kramer3, P. Delmonte1, A.R.F. Kia1, F.J. Bueso1, J.I. Rader1, 1Food and Drug Administration, College Park, MD, USA, 2NIR Technologies Inc., Oakville, ON, Canada, 3Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, ON, Canada
Since total saturated fat and total trans fat labeling requirements became mandatory in the US, Canada, and many other countries, there has been an urgent need for both rapid and accurate analytical methodologies that would facilitate the verification of compliance with the various regulations. Capillary gas chromatography (GC) has been the industry standard because it provides detailed information on fatty acid composition. However, GC is complex and time consuming. Consequently, the potential of applying Fourier transform near-infrared spectroscopy (FT-NIR) for the rapid (1 min) determination of these fat and oil constituents has recently been demonstrated to be a viable alternative analytical method. This novel approach entailed the development of FT-NIR models based on accurate GC data and the application of partial least squares analysis to observed FT-NIR spectra. Currently commercially available FT-NIR models permit the rapid and accurate determination of the complete fatty acid composition of fats and oils using observed FT-NIR spectral information. This capability allows the rapid determination of the total saturated fat and trans fat contents of edible fats and oils as well as the rapid verification of the amounts declared on the Nutrition Facts labels of various commercial products.
Determining Levels of Acrylamide by UPLC-MS/MS in Fried Foods and Oils.
B. McNew, J. Flook, T. Patterson, A. Syed, C. Kahl, D. Meyer, Dow AgroSciences, Indianapolis, IN, USA
Acrylamide is a toxic compound formed from the reaction of asparagine with carbonyls during cooking. A reliable and sensitive method using liquid chromatography (UPLC) coupled with electrospray ionization mass spectrometry in positive ion mode was developed to determine concentration levels of acrylamide in restaurant fry oils and food products. Acrylamide-d3 was used as the internal standard and the linearity of response to acrylamide was 0.5 ng/mL to 1000 ng/mL with a correlation coefficient ≥ 0.9. Acrylamide was evaluated by monitoring specific transitions in MRM mode. A comparative study was done to determine the influence of fry oil composition on the levels of acrylamide formed in fried food and oil. The results of the study show a stronger correlation with food type than with oil type. The factors that contribute to the variability in acrylamide levels in prepared foods will also be discussed.
Comparison of Polymer Formation of Various Vegetable Oils.
J. Flook, T. Patterson, J. Behrman, A. Syed, C. Kahl, D. Meyer, Dow AgroSciences, Indianapolis, IN, USA
The rate of polymer formation in various vegetable oils under oxidizing conditions was examined. Vegetable oils were oxidized under standardized conditions using a Rancimat set to 150°C with continuous purging with air. Samples were then removed at 30 minute intervals for analysis. Aliquots were analyzed for polymer content by Size Exclusion HPLC using ELSD detection. The fatty acid composition of the oil was determined by GC-FID of fatty acid methyl esters. The results show that the level of polyunsaturated fat directly impacts the rate at which oils undergo oxidative polymerization. Samples of corn, low linoleic soybean, mid-oleic sunflower, and regular soybean oils, all high in polyunsaturated fats, formed polymers faster than the oils with low levels of polyunsaturated fats. Canola and sunflower oils that are high in monounsaturated fats, (primarily oleic acid) and low in polyunsaturated fats formed polymers at a slower rate than the more polyunsaturated oils. Highly saturated fats were resistant to polymerization. The results indicated that the formation of polymers were most strongly related to the levels of linolenic acid in the oils.
A Comparative Study of Antioxidant Phytochemicals of Pomace Extracts from Industrial Carignane Grape (Vitis vinifera) by Different Solvent Systems.
N. Gámez-Meza1, S. Agustín-Salazar2, H. Soto-Valdez3, L.A. Medina-Juárez1, 1Depto. Investigaciones Científicas y Tecnológicas de la Universidad de Sonora., Hermosillo, Sonora, Mexico, 2Posgrado en Biociencias. Universidad de Sonora., Hermosillo, Sonora, Mexico, 3Centro de Investigacin en Alimentacion y Desarrollo A.C., Hermosillo, Sonora, Mexico
The objective of this study was to investigate how commonly used solvent systems affected the extraction and antioxidant activity of several phytochemicals from grape pomace (seed, skin, and stick). The total phenolic and flavonoids contents were measured. The antioxidant activities were tested by free radicals scavenging methods (DPPH and ABTS). The results showed that 60% methanol-70% acetone extract exhibited the highest total flavonoids content (12.53±1.03 mg QE/g); however, the 60% methanol extract presented the highest DPPH-free radical scavenging activity with a total phenolic content of 17.60±0.59 mg GAE/g. Moreover, seed extracts showed the highest total phenolic content, total flavonoids content and antioxidant activities. The 70% methanol extract from seed showed the highest total phenolic content (29.87±0.92) and antioxidant activity (872±15 mg ET/g). Total flavonoids contents from seed extracts no presented significance difference. Resveratrol, rutin and myricetin were the main compounds in all extracts at different levels. The results showed that it possible to maximize the extraction of phenolic compounds depending of the sample.
Nutritive and Therapeutic Values of Bottle Gourd Seed, a Potential Source of Edible Oil with Anti-cancer Properties.
S.S.M. Allam, Food Technology Research Institute, Giza, Egypt
In seeking for a new untraditional source of edible oil to fulfill the shortage in oil production in Egypt, bottle gourd seed proved to be one of the most important source containing high amount of oil (46.5%) with high quality characteristics (PV of 0.31 meq/kg oil and 0.66% FFA) and a high oxidative stability; 14.6 hr at 100°C by the Rancimat apparatus.The nutritive value of Lagenaria siceraria (bottle gourd) grown in Egypt was studied and the results show that, the bottle gourd is rich in protein (24.59%) and their amino acids were analyzed. Fatty acids profile of bottle gourd seed oil was identified and it have a moderate percentage of SFA (34.61%) represented in 23.12% palmitic acid and 11.49% stearic acid. It also contained a high amount of USFA (63.39%) represented in 21.55% oleic acid (MUSFA) and 41.74% linoleic acid (PUSFA). Physicochemical properties and sterol composition were also determined. Bottle gourd seed oil was examined for potential cytotoxicity by SRB assay. Results attested that bottle gourd seed oil exhibited an efficient cytotoxicity against Hep2, MCF7 and Hepg2 carcinoma cell line with IC50 values of 9.8, 9.7 and 5.3 mL for larynx, breast and liver carcinoma cell lines, respectively.
Characterization of Castor Oil by HPLC and Charged Aerosol Detection.
M.A. Plante, B. Bailey, C. Crafts, I. Acworth, ESA - A Dionex Company, USA
Castor oil is a natural oil that, in its native state, has many uses ranging from personal care (laxative, cosmetics, topicals), to chemical (raw materials), and to industrial (lubricants, hydraulic fluids, dielectric fluids, textiles, paints, coatings). The composition of castor oil is unique in that it contains a triglyceride, RRR, composed of the omega-9 unsaturated fatty acid, ricinoleic acid (R) (12-hydroxy-9-cis-octadecenoic acid). Ninety percent of the total triglycerides is RRR.The castor bean is the source of castor oil, but it also contains ricin, a highly toxic protein. As a result, the castor plant is being genetically manipulated to eliminate the production of ricin and increase the oil yield. To provide a measurement of oil quantity and quality for this research, we have developed an HPLC charged aerosol detection method that can be used to accurately determine the amount of triglycerides that is present in a castor oil sample, and to fully characterize individual lipid components. Presented here is a simple, reliable, and direct method for the characterization of castor oil from different commercial sources. Results are obtained with only sample dilution and analysis.
Effect of Extraction Conditions on Fatty Acid Composition and Triacylglycerol Profile of Jackfruit Seed Oil.
Bahareh Tabatabaee Amid 1, Hamed Mirhosseini1, Parviz Kavousi1, Farhad Farivar2, Tan Chin Ping 1, 1Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, 2Business Training Center, 137, 18th Bahar St., sajjad Blv., Mashhad, Iran
The main objective of present work was to evaluate the influence of type of solvent on extraction efficiency, colour intensity, fatty acid composition and triacylglycerol profile of jackfruit seed oil. Petroleum ether plus methanol, hexane plus methanol, ethanol plus methanol and 60% hexane plus 40%isopropoanol and methanol were employed in this study. The same volume of methanol was considered for all the treatments in order to bind the moisture and subsequently improve the extraction efficiency. The highest and least extraction efficiency was obtained by using solvent EM and HIPM, respectively. As shown in the results, the extraction using ethanol and methanol significantly (p < 0.05) resulted in the highest colour intensity compared to the oils extracted by using the other solvents PEM, HM and HIPM. The major fatty acid compositions identified by using gas chromatography-mass spectrometry (GC-MS) were: palmitic (P, C16:0), stearic (S, C18:0), oleic (O, C18:1), linoleic (L, C18:2), linolenic (Ln, C18:3), gadoleic (G, C20:1), arachidic (A, C20:0), behenic (B, C22:0) and lignoceric (Li, C24:0). The main triacylglycerol profile of jackfruit seed oil composed of LLL, POP, LLO, LnOO, OOO, POO, POS, PLP, OLO, PLS and PLL.
Extraction and Characterization of Papaya Seed Oil using Solvent Extraction Technique.
Hamed Mirhosseini, Shadi Samaran, Parviz Kavousi, Yaakub Che Man, Tan Chin Ping , Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
Papaya seeds obtained from the most popular variety in Malaysia named as Ì” SekakiÌ• were used in conventional solvent extraction to extract the papaya seed oil. The main objective of present study was to investigate the effect of 4 different solvents on fatty acid composition and triacylglycerol profile of the extracted papaya seed oil. Petroleum ether plus methanol, hexane plus methanol, ethanol plus methanol and 60% hexane plus 40%isopropoanol and methanol were employed in this study. Regarding the extraction yield, the mixture solvent of Methanol-Ethanol significantly showed the lowest amount (P<0.05) among other solvents (i.e. Methanol-Petroleum Ether, Methanol-Hexane-Isopropanol as well as Methanol-Hexane). In terms of fatty acid composition identified by gas chromatography-mass spectroscopy (GS-MS), Oleic acid (C18:0) was the major fatty acid in the fatty acid profile of all extracted papaya seed oils with more than 70%, thus indicating the high oxidative stability of proposed papaya seed oil. Moreover triacylglycerol profile of the extracted papaya seed oil detected by High performance liquid chromatography (HPLC) mainly composed of glycerol-oleate-oleate-oleate (OOO) and 1-palmitoyl-dioleoyl glycerol (POO).
Effect of Sun-drying and Air-drying on Antioxidant Phytochemicals of Red Pepper (Capsicum annuum L.).
A.K. Blanco-Rios2, N. Gámez- Meza1, L.A. Medina-Juárez1, 1Departamento de Investigaciones Científicas y Tecnológicas de la Universidad de Sonora, Hermosillo, Sonora, Mexico, 2Posgrado en Biociencias, Hermosillo, Sonora, Mexico
Red pepper (Capsicum annuum L.), is widely used in a variety of commercial products in powder form because of the characteristics of color and taste that provide to foods. However, it is known that drying operations may cause a marked loss of phytochemicals. For this reason, the aim of this study was to compare the effect of two different drying methods, sun-drying (120 h at direct sunlight, 35-40 °C) and hot air-drying (24 h at 70 °C), on composition and antioxidant activity of phenols, flavonoids, and tocopherols of red pepper. The results showed a similar loss of phenols (45.37 and 46.22%) and flavonoids (79.86 and 83.8%) when two drying methods were used. However, the reduction of antioxidant activity (ABTS•+ and DPPH•) was lower (13.99 and 17.76%) when sun-drying was used rather than air-drying (51.1 and 70.0%). Among tocopherols only α-tocopherol was found in fresh red pepper, which decreased 25%. The antioxidant activity of α-tocopherol fraction decreased 38.9% (ABTS•+) and 86.7% (DPPH•). These results show that the effect of air-drying was significantly greater than sun-drying on antioxidant activity of red pepper, implying that temperature had greater impact than drying time.
Performance Evaluation of a Heated 9-Bounce Internal Reflection Portable Mid-FTIR Spectrometer for the Rapid Determination of Total trans Fat.
M.M. Mossoba1, J.K.G. Kramer2, H. Azizian3, J. Kraft4, P. Delmonte1, A.R.F. Kia1, F.J. Bueso1, J.I. Rader1, 1Food and Drug Administration, College Park, MD, USA, 2Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, ON, Canada, 3NIR Technologies Inc., Oakville, ON, Canada, 4The University of Vermont, College of Agriculture and Life Sciences, Burlington, VT, USA
The performance of a miniature mid-FTIR spectrometer equipped with a novel 9-reflection diamond ATR crystal has been evaluated and applied to the rapid (5 min) determination of the total trans fat content. The procedure used for the determination of total trans fat by mid-infrared spectroscopy has been recently standardized as AOCS Cd 14e-09. This method entails the measurement of the height of the negative second derivative of the trans absorption band at 966 cm-1 which is uniquely characteristic of all isolated double bonds with trans configuration. Observed data indicated that this methodology and portable instrumentation can lead to approximately an order of magnitude enhancement in sensitivity. The minimum level of trans fat and oil in a product is approximately 1.8% (as percent of total fat). This is the level that needs to be measured with confidence to meet the current declaration requirement of zero trans fat, or 0.5 g trans fat per serving, on the US Nutrition Facts label. Calibration curves (0.1-60 %) for low and high trans fat contents, and data on the determination of accuracy standards as well as commercial fats and oils measured with the miniature system will be reported. Results will also be compared to those obtained on a conventional bench top mid-FTIR system as well as by GC and FT-NIR.