A Direct Method for Fatty Acid Methyl Ester (FAME) Synthesis

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A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet meat tissues, oils and feedstuffs J. V. O'Fallon, J. R. Busboom, M. L. Nelson and C. T. Gaskins J ANIM SCI published online February 12, 2007

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://jas.fass.org/content/early/2007/02/12/jas.2006-491.citation

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Journal of Animal Science

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Running head: Direct FAME synthesis

A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet meat tissues, oils and feedstuffs

J. V. OFallon, J. R. Busboom, M. L. Nelson, and C. T. Gaskins1

Department of Animal Sciences, Washington State University, Pullman 99164-6351

Correspondence: 135 Clark Hall (phone 509-335-6416; fax 509-335-4246; e-

mail:gaskins@wsu.edu).

Published OnlineDownloaded February 12, 2007 on May 4, 2012 First on from jas.fass.org by guest as doi:10.2527/jas.2006-491

Journal of Animal Science

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Direct Fame synthesis 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 The analysis of fatty acids has become increasingly important because more people Introduction ABSTRACT: A simplified protocol to obtain fatty acid methyl esters (FAME) directly

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from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 hr at 55 C in 1N KOH in methanol containing C13:0 as internal standard. The KOH is neutralized and the free fatty acids are methylated by H2SO4 catalysis for 1.5 hr at 55 C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a GC vial for subsequent gas chromatography. All reactions are conducted in a single screw cap Pyrex tube for convenience. The method meets a number of criteria for fatty acid analysis including not isomerizing conjugated linoleic acid (CLA) or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyopholyzed tissue samples, in addition to oils, waxes and feedstuffs. The method saves time, effort and is economical when compared to other methods. Its unique performance, including easy sample preparation, is achieved because water is included in the FAME reaction mixtures rather than eliminated.

Key words: fatty acid analysis, FAME synthesis, longissimus muscle, conjugated linoleic acid (CLA), fish oil, feedstuffs

have become aware of their nutritional and health implications. Because of this, a method

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Direct Fame synthesis 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 for analyzing fatty acids that provides both rapid and reliable results is of great value. Many methods are currently used to analyze fatty acids (e.g., Morrison and Smith, 1964;

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Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuernberg et al., 2002; Budge and Iverson, 2003; Cooper et al., 2004). These methods, however, are not necessarily convenient, nor direct, and often must be optimized for reaction conditions, including the catalyst and the temperature (Lewis et al., 2000; Park et al. 2002; Shahin et al., 2003). On the other hand, the ideal method, as noted by Palmquist and Jenkins (2003) in discussing challenges encountered in developing fatty acid methods, would determine the total fatty acid concentration in tissues, oils, and feed samples by converting fatty acid salts, as well as the acyl components in all lipid classes such as triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple direct one-step esterification procedure. In this paper we present a method that is established upon a surprising conception, i.e., we add water to the fatty acid methyl ester (FAME) synthesis reagents. Until now, FAME synthesis methods have rigorously avoided water as a matter of standard procedure. However by adding water, the dynamics of sample preparation and methyl ester formation can be revisited and the ideal outcome of FAME synthesis discussed by Palmquest and Jenkins (2003) mentioned above becomes possible. Although the method described herein requires two steps, it does so in one reaction tube. By introducing water into FAME synthesis, the drudgery of special sample preparation, e.g., freeze-drying, is no longer necessary. A sample is analyzed in the state it is obtained. Thus, for example, meat and milk samples can be analyzed directly without any prior dehydration step. The presence of water in the reagents allows for tissue sample

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Direct Fame synthesis 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 Methylation Agents and Sample Selection Materials and Methods dissolution and facilitates the total extraction of fatty acids not possible in the absence of water. Importantly, water does not interfere with the methylation of any fatty acid. The

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method uses familiar and economical chemicals, namely, methanol, potassium hydroxide, and sulfuric acid, but uses these methylating reagents in the presence of water. As a result of these characteristics, the direct FAME synthesis method is convenient, reliable and efficient. In short, the objective of this paper is to develop a method to directly methylate fatty acids from muscle tissue, oils, and feedstuffs in aqueous solution.

To assess certain features of our method, we compared direct FAME synthesis to two methylating agents routinely used for FAME synthesis, namely, the base catalyst sodium methoxide and the acid catalyst boron trifluoride. Samples used with sodium methoxide and boron trifluoride did not contain water, as this would compromise the performance of these two methylating agents. The direct FAME synthesis method, however, always contained water, even if the sample did not, because its reagents contained water, i.e., the direct FAME synthesis reaction mixture contained 13.2% water due to the water in the potassium hydroxide and sulfuric acid reagents. The samples we used in this manuscript were chosen for distinct reasons. The Supelco fatty acid standard mixture was chosen because it contained short and long chain

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Direct Fame synthesis 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Hexane (OmniSolv) was purchased from EM Science, Cherry Hill, NY. Absolute methanol and potassium hydroxide were obtained from J.T. Baker Chemical Co., Phillipsburg, NJ. Chloroform and sulfuric acid were purchased from Fisher Scientific, Tustin, CA. Sodium methoxide and boron trifluoride-methanol were obtained from Sigma-Aldrich, St. Louis, MO. The Supelco standard FAME mixture (47885-U) was Materials saturated, monounsaturated, and polyunsaturated fatty acids in defined amounts and thus

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served as a primary test of the feasibility of our method. Fish oil was chosen because it is an important source of the long chain polyunsaturated omega-3 esterified eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) fatty acids. Conjugated linoleic acid (CLA), as the free acid, was chosen because it is of medical importance as perhaps the only fatty acid that can directly inhibit cancer in animal models (Belury, 2002) and because current FAME synthesis methods often cause undesirable isomerizations of this fatty acid (Kramer et al., 1997). Beef longissimus muscle was chosen because of our special interest in beef fatty acids and it serves as a direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty acids present in meat tissue. To address the problem of refractory samples, we included wax esters, cholesteryl lipid derivatives and alkyl methane sulfonates, as these are difficult fatty acid derivatives to analyze (Palmquist and Jenkins, 2003). Finally, as a concluding demonstration of the versatility of the direct FAME synthesis method, we included a variety of oils, feedstuffs and foods.

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Direct Fame synthesis 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 FAME Synthesis with Sodium Methoxide or Boron Trifluoride Longissimus muscle (1g) was extracted with chloroform/methanol (2:1, vol:vol), containing C13:0 as internal standard, according to the method of Folch et al.(1956), using a Brinkmann polytron at room temperature. The extraction mixture was then Folch Extraction of Fatty Acids from Longissimus Muscle obtained from Supelco, Bellefonte, PA. Spring Valley fish oil capsules were distributed by Leiner Health Products, Carson, CA. Tonalin 1000-CLA (conjugated linoleic acid)

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capsules were obtained from Nature Bounty, Bohemia, NY. All other fatty acid standards were purchased from Nu-Chek Prep. Inc., Elysian, MN. Beef longissimus muscle samples (n=20) were obtained from department owned animals processed at an abattoir. Nuts and sundry food items were purchased from local grocery stores. Coffee bean grinders were purchased from Mr. Coffee, Inc., Cleveland, OH. Pyrex screw-cap culture tubes (16 x 125 mm) were obtained from Corn