ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
RAPID MICROWAVE ASSISTED PREPARATION OF FATTY ACID METHYL ESTERS FOR THE ANALYSIS OF FATTY ACID PROFILES IN FOODS
© 2015 N. P. Brunton*, C. Mason**, M. J. Collins, Jr**
*Department of Agriculture and Food Science, University College Dublin (UCD) Belfield, Dublin 4, Ireland 1E-mail: nigel.brunton@ucd.ie **CEM Corporation P.O. Box 200, Matthews, NC 28106, USA Received 03.06.2014; in final form 09.02.2015
A microwave (MW) assisted rapid high through-put method for the preparation of fatty acid methyl esters (FAME) for the analysis of fatty acid profiles in a selection of foods was evaluated by comparing fatty acid profiles with those resulting from conventional FAME preparation. The microwave method gave fatty acid profiles in close agreement with those arising from conventional methods and the protocol gave acceptable recoveries (98—102%) and repeatability (RSDs for replicate analyses 0.56—5.2%). In comparison to conventional methods the MW assisted method was simple, rapid and universally effective across foods ranging from dairy products to ready meals. EU regulation (No. 1169/2011) requiring declaration of the saturated, mo-nounsaturated and polyunsaturated content of foods is placing pressure on processors and contract laboratories to analyse fatty acid profiles using relatively low through-put conventional methods. MW assisted preparation of FAMEs offers analysts a high-through-put, rapid and universal method to help overcome this potentially costly and labour intensive regulatory hurdle.
Keywords: FAMEs analysis, fatty acid analysis, fatty acid profiling, fatty acid GC, fat analysis GC.
DOI: 10.7868/S0044450215100047
Preparation of fatty acid methyl esters and their separation and quantification using gas chromatography is by far the most commonly used method for the quantification of fatty acids in foods and other natural matrices [1—3]. Fatty acid methyl esters are usually prepared by firstly saponifying esterified fatty acids to free fatty acids and then re-esterifying them to form methyl esters. The procedure was first established by James and Martin [4] and subsequently optimised by themselves and other authors [5, 6] with the aim of reducing the polarity of the free fatty acids thus making them easier to separate and quantify for the then newly developed gas-liquid chromatography systems. The saponification step is usually carried out by heat treatment of the foodstuff in the presence of a strong alkali such as potassium hydroxide. The proceeding esterifi-cation condenses the carboxyl group of the free fatty acid and the hydroxyl group of an alcohol. This step is usually carried out in the presence of a catalyst (such as boron triflouride) which promotes the reactivity of the oxygen atom on the carboxyl group by protonating it. An alcohol (usually methanol) then combines with the protonated acid to yield an ester with the loss of water and the catalyst is removed with the water. The methyl esters are then extracted into an organic sol-
vent for separation and quantification by gas chromatography. Both the saponification and esterification steps ofFAME preparation involve often lengthy incubation, and the subsequent removal of the often toxic catalysts that add to the labour and time required to carry out a procedure which is used routinely in many food analytics laboratories. BF3 is a commonly used acid catalyst for methylation but it is harmful, and the use of boron and fluorine is often restricted due to environmental laws. In addition, the methanolic BF3 reagent has a limited shelf life [7]. Therefore there is a desire amongst analysts to simplify and speed up the procedure, and indeed some simplified rapid methods have been developed [8, 9]. However many of these still require the use of a catalyst and cannot be automated. Indeed further impetus has been added to the search for a rapid and simple method for FAME preparation by the publication of EU Regulation No. 1169/2011 which will come into effect in December 2014, which will require food producers to display saturated, mo-nounsaturated, polyunsaturated, and cholesterol contents on their foodstuffs [10]. The legislation requires food producers to give more detailed information on many of the macronutrients present in their foodstuffs. For carbohydrates, protein and salt, analysis is
Table 1. Fatty acid methyl ester derivatization methods used in the present study and the products to which they were applied
Product Reagents Reference
Milk and butter Sodium hydrocarbonate followed by boron triflouride [18]
Infant formula Saponification: 0.5% sodium methylate in methanol. Methylation: boron triflouride in methanol [19]
Cooked chicken breast slices, cooked ham slices, uncooked sausages Saponification: 5 M KOH in methanol:water (50 : 50, v/v). Methylation: 2 M trimethylsilyldiazomethane in K-hexane [20]
Spaghetti Bolognese, lasagne, chicken curry Saponification: methanolic NaOH at 100°C for 5 min. Esterification: BF3—methanol reagent [21]
straight-forward and rapid, whilst analysis of fatty acid profiles requires up to a full day work-up followed by gas chromatographic analysis of fatty acid methyl ester derivatives. The through-put limiting steps in FAME preparation are usually considered to be the saponification and esterification steps in which the energy required for the saponification is supplied by heat and the lowering of the activation energy for the esterification by the catalyst. It is well known however that microwaves can be used as an energy source for many chemical reactions and could therefore be used to supply the energy for the trans-esterification of fatty acids for GC analysis [11]. Indeed there is intense interest in the use of microwave to assist in the preparation of FAME's for biodiesel production [12—16]. Methods for the MW assisted preparation of FAME's for analysis of fatty acid profiles have been published as far back as 1998 [17], however the technique has received little attention, possibly due to concerns over microwave induced losses of very volatile compounds. This is despite the fact that recent interest in microwave assisted synthesis has led to the development of sophisticated, high through-put equipment capable of providing the energy to drive many reactions which previously could only take place via a catalytic route.
In the present study we examine the use of one such system to aid in the preparation of fatty acid methyl esters in a range of foodstuffs. FAME's were also prepared for these foods using conventional catalytic methods and fatty acid profiles prepared using two methods are compared in order to evaluate the efficacy, accuracy and ease of use of the microwave assisted method.
EXPERIMENTAL
Reagents. Potassium hydroxide, acetyl chloride, methanol, salt, pentane and Supelco 37 FAME standard mix were purchased from Sigma-Aldrich (Ireland). Three samples of all products for FAME analysis were purchased from a local supermarket on three separate occasions.
Conventional preparation of FAME's. As outlined in the discussion analysts have been using FAME de-rivitization to analyse fatty acids in foods since the late
1950's and a wide variety of procedures have been used in different foodstuffs. Therefore since the aim of the present study was to compare MW assisted FAME preparation with conventional methods on a wide range of foods, a selection of conventional methods for FAME preparation was used depending on the foodstuff under investigation as no universal method appeared to be available. Table 1 lists the conventional methods used and the reagents involved in the deriviti-zation, detailed descriptions of the methods are available from the cited references.
Fatty acids were expressed as percent of total fat content of each of the foodstuffs. Total fat content was determined by extracting 1 g of sample with 30 mL of chloroform—methanol (2 : 1) containing 0.005% (v/v) butylated hydroxy toluene shaking for two hours following an overnight extraction at 4°C. The final extract was dried over sodium sulphate and it's weight determined.
Microwave assisted preparation of FAME's. Microwave assisted FAME preparation was carried out using a MARS 6 Express 40 position Microwave Reaction System (CEM Corporation, Matthews, NC, USA). Reactions took place in PFA 55 ml reaction vessels. For FAME preparation 1 g of wet/0.5 g dry/1 mL of liquid (e.g., milk)/3 drops oil were added to the reaction vessel containing a 10 mm stir bar. To this 10 mL of potassium hydroxide (2.5%) in methanol was added and the reaction vessel was heated in the MARS 6 Express system to 90°C over 4 min and held at this temperature for 10 min. The reaction vessels were then removed from the spindle wheel and cooled on ice for 5 min or until they had reached room temperature before they were opened. The methylation was then carried out by adding 15 mL of 5% acetyl chloride in MeOH solution and heating to 120 °C over 4 min and holding at this temperature for 2 min. The reaction tubes were removed again and cooled on ice to room temperature. To the cooled tubes 10 mL of pentane was added and the reaction tubes were mixed by placing over a heater stirrer for 2 min. Following this, 15 mL of a saturated salt solution (30%, w/v) was added, and the solution was mixed again as described above. After it had separated the top pentane layer was removed and aliquoted into amber GC vials (1.5 mL) containing sodium sul-
Table 2. Fatty acid profiles for a selection of dairy products as determined using microwave assisted or conventional (CON) derivatization to fatty acid methyl esters following by GC-FID separation and quantification
Fatty acid* Milk Infant formula Butter
MW CON MW CON MW CON
C4:0 2.47 3.5 ND** ND ND ND
C6:0 1.98 2.23 0.07 0.05 ND ND
C8:0 1.18 1.37 0.78 0.53 ND ND
C10:0 3.18 3.03 0.59 0.59 2.94 3.77
C10:l 0.51 ND ND ND ND ND
C12:0 3.21 4.11 7.09 7.63 2.97 4.38
C14:0 10.97 11.62 3.42 3.0
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