research article comparison of two derivatization methods

Research ArticleComparison of Two Derivatization Methods forthe Analysis of Fatty Acids and Trans Fatty Acids inBakery Products Using Gas Chromatography

Jumat Salimon Talal A Omar and Nadia Salih

School of Chemical Sciences amp Food Technology Faculty of Science and Technology Universiti Kebangsaan Malaysia43600 Bangi Selangor Malaysia

Correspondence should be addressed to Jumat Salimon jumatukmmy

Received 18 December 2013 Accepted 14 January 2014 Published 25 February 2014

Academic Editors T Kaneta and C Pistos

Copyright copy 2014 Jumat Salimon et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Two different procedures for the methylation of fatty acids (FAs) and trans fatty acids (TFAs) in food fats were compared usinggas chromatography (GC-FID) The base-catalyzed followed by an acid-catalyzed method (KOCH

3HCl) and the base-catalyzed

followed by (trimethylsilyl)diazomethane (TMSndashDM)methodwere used to prepare FAmethyl esters (FAMEs) from lipids extractedfrom food products In general both methods were suitable for the determination of cistrans FAs The correlation coefficients (119903)between the methods were relatively small (ranging from 086 to 099) and had a high level of agreement for the most abundantFAs The significant differences (119875 = 005) can be observed for unsaturated FAs (UFAs) specifically for TFAs The results from theKOCH

3HCl method showed the lowest recovery values (119877) and higher variation (from 84 to 112) especially for UFAs The

TMS-DMmethod had higher 119877 values less variation (from 90 to 106) and more balance between variation and RSD valuesin intraday and interday measurements (less than 4 and 6 resp) than the KOCH

3HCl method except for C120 C140 and

C180 Nevertheless the KOCH3HCl method required shorter time and was less expensive than the TMS-DM method which is

more convenient for an accurate and thorough analysis of rich cistrans UFA samples

1 Introduction

Oils and fats in foods are composed of four different types ofFAs polyunsaturatedmonounsaturated saturated and TFAs[1] Naturally all unsaturated FAs in vegetable oils are inthe cis form whereas a large proportion of unsaturated FAsisomerize to their TFA counterparts during the industrialhydrogenation of vegetable oils [2] Therefore dietary fatsmade with fully andor partially hydrogenated oils which areused in foods to improve texture and stability for a longershelf life contain TFAs [3 4] Likewise bakery productsmade with hydrogenated oils and fats such as biscuits cakescookies crackers and breads contain TFAs [5 6]

Results published in recent years indicate the importanceof food FA composition in human nutrition and health [5 7]In general it is recommended to increase the intake of n-3 polyunsaturated FAs (PUFAs) and to decrease the intakeof saturated fatty acids (SFAs) and TFAs because TFAs

affect cholesterol levels in much the same ways as saturatedand trans fats increase your risk of developing coronaryartery and heart diseases [8 9] This association between thedietary consumption of some FAs and increased risk of somediseases has led to the implementation of new regulations thatrequire the declaration of FAs including TFA content onthe labels of conventional foods and dietary supplements inseveral countries [2 3 10] Therefore it is important to haveaccurate and precise techniques for the identification andquantification of FAs and TFAs in foods of natural origin orin foods formed during the processing of fats and oils [1 11]that is performed due to consumer demand for improved fatquality in foods [12]

In recent years GC has been used for the separation andanalysis of geometric and positional isomers Although GCmass spectrometry and other technical methods have beendeveloped to quantitate C8ndashC26 chain-length FAs the GCanalysis of FAs with FID remains the most frequently used

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 906407 10 pageshttpdxdoiorg1011552014906407

2 The Scientific World Journal

method [1 13ndash17]The quantification of FAs in fats and oils byGC involves transforming the analytes into more volatile andnonpolar derivatives after extracting the lipids from the foodproduct before GC analysis [14] The most important stagefor the GC-FID determination of FAs is sample preparationwhich usually requires derivatization of the FAs to increasethe volatility of the substances to improve separation and toreduce tailing [18]Moreover the speed of analysis sensitivityand accuracy are important parameters in GC that may beimproved with derivatization [18 19]

Sample preparation including the derivatization of FAshas been carefully reviewed by several authors [19ndash21] Themost commonly usedmethod for the determination of FAs isconversion of the FAs into their corresponding methyl esters(FAMEs) Many different methylation approaches have beendescribed in the literature and some methods have beenestablished for preparing FAMEs from lipids extracted fromvarious food samples acid- or base-catalyzed transmethy-lation borontrifluoride (BF

3) methylation after hydrolysis

methylation with diazomethane and silylation [18ndash20 22ndash24] In general these methods involve two steps first thesamples are heated with sodium hydroxide in methanol andsecond the free FAs (FFAs) are esterifiedwithmethanolic BF

3

[23] or methanolic KOH [24] However each method has itsown advantages and disadvantages [16 25]

In general the base-catalyzedmethod for the direct trans-esterification of lipids has been reported to bemore applicablefor nutrition analysis because it is easy to use and uses lessaggressive reagents than othermethods [22 24 26] Howeverthis method has resulted in poor recoveries of FAMEsbecause FFAs might remain partially unreacted [27] andbecause FFAs are notmethylated under these conditions [26]Therefore some studies have suggested that the repeatabilityrecovery with low variation and the highest concentrationdetected are improved for the most abundant FAs when thecombined base- and acid-catalyzedmethod is used comparedto the base- or acid-catalyzed methods alone [20 26 2829] Nevertheless using acid-catalyzed methods is usuallyundesirable because it is likely to lead to changes in theconfiguration of the double bond characteristics and toproduce artifacts [20 25 30]

An alternative method used by a number of laboratoriesto improve the accuracy of analysis is base hydrolysis followedby methylation of the resulting FFAs with diazomethanehowever the disadvantage of this method is that diazometh-ane needs precautions during extraction [21 31 32] In con-trast the esterification by TMS-DMhas been reported to be aconvenient alternative to diazomethane because it is safer tohandle and does not produce artifacts [33 34] Furthermoremethylation by TMS-DM after the saponification process hasbeen shown to be more accurate for cistrans PUFA analysisin seafood [31] and conjugated linoleic acid (CLA) isomersin ruminant meat tissues [32] when compared to othermethylation reagents However the hydrolysis or presence oftrace water leads to poor recoveries of FAMEs [16 27]

There is a need to investigate the concentration of FA andTFA isomers in all lipid fractions from food fats and theirproducts such as biscuits cakes crackers wafers and breadto monitor the low levels of FAs and TFAs and to control

labeling authenticity Therefore it is possible to apply theadvantages of sodium methoxide (NaOCH

3) as a useful

reagent for the fast transformation of FAs into FAMEs [18 35]along with using the TMS-DM reagent for the completemethylation of all FFAs which can be more reliable andproduce a higher accuracy In the current study to verify theaccuracy of measuring the concentrations of FAs and TFAs infood fats of bakery products the repeatability and recoveryusing a method based on the derivatization of lipid extractby base-catalyzed followed by TMS-DMwere compared withthe combined base- and acid-catalyzed methylation method(KOCH

3HCl) In addition the advantages disadvantages

and applicability to determine the complex mixture of FAsand TFAs in various types of bakery products are discussed

2 Materials and Methods

21 Standards and Reagents Nine FA and FAME standards(C120 C140 C160 C180 C181 C181t9 C182 C182t912and C183) were purchased from Fluka (purity ge99 (GC)Sigma-Aldrich Germany) the internal standard (IS) C150(Pentadecanoic acid) was purchased from Sigma (Sigma-Aldrich Germany) and the purity of all reagents was greaterthan 99 All chemicals (methanol toluene glacial aceticacid hydrochloric acid potassium hydroxide and sodiumhydroxide) were of analytical reagent grade and purchasedfrom Systerm (SystermMalaysia) except for n-hexane whichwas of higher purity (Systerm Malaysia for GC ge99) Theesterifying agent TMS-DM (2M) in n-hexane was purchasedfrom Sigma (Sigma-Aldrich Germany)

22 Food Samples Eight commercial food items were usedfor analysis and comparison in this study The samplesincluded different bakery and fast-food products such ascrackers bread with filling cakes wafers cookies andbiscuits as these products mainly contain FAs and TFAsThe samples were purchased from several Malaysian localsupermarkets including national and imported brands andall of those samples were coded with a letter (from A to H)

23 Sample Preparation and Lipid Extraction Each samplewas ground and placed in an oven at 50∘C until completedryness before analysis The total lipids were extracted usingthe Soxhlet Method for cereal fats [28] Approximately 10 g ofhomogenized sample was weighed into a cellulose extractioncartridge and the Soxhlet apparatus containing the cartridgewas fitted to a distillation flask containing 150mL of n-hexane with (50 ppm) butylated hydroxytoluene (BHT) anda few antibumping granules After 3 hours the mixture wasdried with Na

2SO4and filtered through fluted filter paper

The oil was recovered after stripping the solvent in a rotaryevaporator Finally the extracted lipids were dried undernitrogen (N

2) weighedand stored at minus20∘C until analysis

24 Preparation of Fatty Acid Methyl Esters (FAMEs) AfterSoxhlet extraction all lipid extracts weremethylated and con-verted into FAMEs using two different methylation methodsApproximately 015 g of each fat extract (in triplicate) wastransferred to a screw-cap test tube (10mL) and 1mL of

The Scientific World Journal 3

CH2O

CH2O

O

O

O

C

C

C

R

R998400

R998400998400

CHO

Triacylglycerol

KOCH3HClmethod

(b)

Δ60∘C

NaOCH3

CH3OH

KOCH3HClmethod

(a)

Δ70∘C

KOH

CH3OH

CH3O

O

C R

CH3O

O

C R

O

C RHO

(FAME)

(FAME)

(FFA)

Methanol toluene2 1

10 M

HClH3C Si

CH3

CH3

N2

TMS-DM Δ50∘C

Figure 1 Diagram for the procedures of the method (a) (KOCH3HCl) and method (b) (TMS-DM)

a solution containing 10mg5mL (C150) in methanol wasadded as an IS The mixtures were reduced to dryness undernitrogen (N

2) before derivatization using two different

methodologies (Figure 1) and the procedures were perfor-med as described in the following sections

241 Base-Catalyzed Followed by the Acid-Catalyzed Method(KOCH

3HCl) The mixtures were redissolved in 2mL of n-

hexane and 1mL of 2Mmethanolic KOH solutionwas addedto the samplesThe tubes were capped and vigorously shakenfor 30 s and boiled for 2min in a water bath at 70∘C Then12mL of HCl (10M) was added and the solution was gentlystirred After phase separation 1mL of n-hexane was addedThe upper phase containing the FAMEs was transferred intoan analysis vial and 10 120583L of the solution was injected intothe GC-FID

242 Base-Catalyzed Method Followed by TMS-DM Themixtures were redissolved in 2mL of n-hexane and 1mL of2M NaOCH

3was added The content was placed in a water

bath at 60∘C for 5min Drops of concentrated glacial aceticacid were added to each tube to neutralize the NaOH Thesamples were reduced to dryness under N

2and dissolved

in 1mL of methanol toluene (2 1 vol) Next TMS-DM wasadded in a molar excess of 2M in n-hexane (100 120583L) at50∘C for 10min without capping the tubes Drops of glacialacetic acid were added until the yellow color disappeared to

remove any unreacted TMS-DM and the reaction mixturewas diluted with 1mL of a 05 NaCl solution To extractthe FAMEs 1mL of n-hexane (containing 50 ppm BHT) wasadded and the tubes were vortexed for 30 s After the solutionsettled the organic layers containing the methyl esters weretransferred to a vial for GC

25 GC Analysis The samples were manually injected (1 120583L)into a gas chromatograph (Shimadzu GC-17A Kyoto Japan)equipped with a flame ionization detector (FID) for sepa-ration and quantification of the FAMEs The analysis wasperformed using a BPX-70 fused silica capillary column(30M 025mm id 02-120583m film thickness Melbourne Aus-tralia)The runwas under an optimized temperature programas follows initial column temperature was 100∘C and wasprogrammed to increase at a rate of 10∘Cminminus1 up to 160∘Cand then at 3∘Cminminus1 up to 220∘C This temperature wasmaintained for 5min increased at 10∘Cminminus1 to a finaltemperature of 260∘C and held for 5min The injector anddetector temperatures were 260∘C and 280∘C respectivelyHeliumwas used as the carrier gas at a flow rate of 1mLminminus1with a split ratio of 60 1

26 The General Measurement Procedures

261 Calibration and Quantification A standard mixturecontaining all FAMEs and the IS (C150) was used to prepare


Inte

nsity

47500

37500

17500

27500

minus2500

7500

0

10 2030

40(min)

1 2 3 45 6 7 98

9

8

7521

4

6

3

10 1112

101112

IS

IS

(a) KOCH3HCl

(b) TMS-DM

Figure 2 GC-FID chromatograms of the total FAs for a sample of bakery products (biscuit) derivatized by the KOCH3HCl method (a) and

TMS-DM method (b) 1 = C120 2 = C140 3 = C160 4 = C180 5 = C181 t9 6 = C181 7 = 182 9t12t 8 = C182 9 = C183 10 = C200 11 =C220 12 = C240 IS = C150

five working standard sets by diluting the stock solution withn-hexane Calibration curveswere constructed from the anal-ysis of the working standards in triplicate using the same GCconditions as those used for quantitative purposes Accordingto the Multiple Point IS method [36] a calibration plot ofeach compound was run by using the ratio of the peak areaof the FAME standards to the peak area of the IS againstthe ratio of the concentration of the FAME standards to theconcentration of the IS

The retention times of the selected FAME standards wereused to identify individual FAs and TFAs in food fat samplesThe concentration of FAMEs in the samples was determinedusing the area ratio and calibration plots For both methodsthe absolute and relative contents of 9 FAs were calculatedfrom all 8 bakery products

262 Comparison of the Precision and Accuracy The studyincluded a comparison between both methods in terms ofprecision and accuracy for each analytical procedure accord-ing to guidelines for the validation of chromatographicmeth-ods [37] The precision of the methods was checked throughthe repeatability (intraday) and reproducibility (interday)and both values were expressed as relative standard deviation(RSD) The values for intraday RSD were calculated usingthe measured data from a single day and interday RSDvalues were calculated using the measured data from threesuccessive days

The accuracy of both methods was verified using a recov-ery assay The recovery was established by spiking theextracted fats of four selected samples with a FA standard attwo different concentrations (Std1 and Std2) and assaying thesample in triplicateThe concentrations of the FAs in the non-spiked samples were subtracted from the concentrations inthe spiked samples and the recovery percentages (119877) were

calculated by dividing the calculated concentrations by theexpected concentrations

27 Statistical Evaluation A paired 119905-test was used to com-pare the differences between the mean values for the contentof each FA measured using both methods (significance level119875 le 005) To evaluate the precision of both methods theintraday and interday RSD values for each componentof all samples were calculated and the calculation of themeans and standard deviations (SD) was performed usingMicrosoft Excel (Professional Edition 2007 Microsoft Cor-poration Redmond WA USA) The correlation coefficients(119903) between both methods were calculated for each FA as ameasure of concordance

3 Results and Discussion

31 Analysis of Selected Samples

311 Identification of FAMEs As research on cistrans UFAsand other FAs in food products becomes more popular itis essential to provide correct information about the com-position and the performance of quantitative analysis usingthe proper application of the methylation procedure [30]Therefore in this current study eight different bakery andfast-food products with varying FA and TFA contents wereanalyzed using two derivatization procedures (describedabove) to prepare FAMEs for GC analysis in triplicate tocompare the two methods and to discuss their advantagesand disadvantages FAMEs in the samples were identified byconducting a comparison of similar peak retention times(Rt) using pure FAME standards Figure 2 shows typical GC-FID chromatograms of total FAs in a sample of biscuits


determined using both methylation procedures as previouslyoutlined

The chromatograms for bothmethods show that all peaksrepresenting all components were well resolved with a goodseparation between the FA and TFA peaks within 37minand this result indicates that peak overlap was not affected bythe peaks of the major constituents in both methods whichis unlike some of the chromatograms produced by othermethods [38] However it is possible that there are somerelative differences between the areas of some FA peaks forboth methods

Furthermore no strange peaks or artifacts that interferedwith the FA chromatographic peaks were detected in bothchromatograms although this result was more obvious inthe chromatogram of the TMS-DM method In general thisresult also confirms earlier reports stating that TMS-DM didnot produce any methoxy artifacts associated with the basecatalysts [27 32 39 40]

312 Quantification of FAMEs For both methods the con-centrations of all nine FAs studied including TFAs wereanalyzed and calculated for all eight food samples in absolute(g100 g) and relative (ww percentage) contents Tables 1 and2 present the means of the absolute (g FA 100 gminus1 sample)and relative ( of total identified FA) FA contents in all sam-ples using the base-catalyzed followed by the acid-catalyzedmethod (KOCH

3HCl) and base-catalyzed method followed

by methylation with TMS-DM respectivelyAs observed in Tables 1 and 2 higher concentrations

for all cis and trans FAs were observed following the TMS-DMmethod compared to the KOCH

3HCl method whereas

C120 and C160 were at slightly lower concentrations forsome of the samples (no significant differences) followingthe TMS-DM method than for the KOCH

3HCl method

Less significant differences between the two methods wereobserved for the absolute and relative contents of cis-UFAsfor most of the samples The relative proportions also showthat significant differences between the two methods arefound for the TFAs All other FAs showed no statisticallysignificant differences for the relative composition betweenthe two derivatization methods

On the other hand most researches which are interestedin the analysis of fats in bakery products and food samplesusually focus on a couple of major isomers C181 cis-9 withC181 trans-9 as well as C182 cis-912 with C182 trans-912 [2841ndash43] The current study found that the KOCH

3HCl and

TMS-DMmethods gave results that were significantly differ-ent for these FAs and contained greater significant differencesfor C181 trans-9 and C182 trans-912 and less significantdifferences for C181 cis-9 and C182 cis-912 However in eachcase the TMS-DM method rendered a higher percentagewhich conforms to the higher concentration of detectablecomponents

32 The Correlation Coefficient The correlation coefficientsbetween both methods were calculated for each FA Table 3presents the correlation coefficients between both methodsfor all FAs studied

0

2

4

6

8

10

12

0 5 10 15

NaO

CH3H

Cl (g

100

g)

TMS-DM (g100 g)

R2

= 09847 X = Y

Figure 3 Comparison of C181 cis-9 measurements (g100 g) byGC-FID based on the KOCH

3HCl and TMS-DM derivatization

procedures

0

04

08

12

16

0 05 1 15 2 25

NaO

CH3H

Cl (g

100

g)

R2

= 09668 X = Y

TMS-DM (g100 g)

Figure 4 Comparison of C181 trans-9 measurements (g100 g) byGC-FID based on the KOCH


procedures

For two of the FAs C181 cis-9 andC181 trans-9 all 8meanmeasurements of the TMS-DM and KOCH

3HCl methods

were plotted (119883 = 119884) to demonstrate the degree of agreementbetween the two methods as shown in Figures 3 and 4respectivelyThe correlation coefficient was 098 for C181 cis-9 and 096 for C181 trans-9

For the low-level FAs (C140 C182 trans-912 and C183)the correlation coefficients (089 086 and 089 resp)between themethodswere relatively small In addition a highlevel of agreement between the twomethodswas observed fortwo of the most abundant FAs (C160 and C181 cis-9) wherethe correlation coefficients were high (099 and 098 resp)

33 Comparison of Accuracy To evaluate the accuracy forboth procedures the recovery percentage (119877) values were


Table 1 The mean of the absolute (g FA 100 gminus1 sample) and relative ( of total identified FA) content of each FA determined using theKOCH3HCl method

Fatty acidsDetected concentration [g FA 100 gminus1] ( Total FA)

SamplesA B C D E F G H

C120 mdash mdash [1035] (4223) [148] (648) [1009] (3980) mdash [504] (3111) mdash

C140 [016a](123a) [027a] (188) [238a] (1106) [065] (289) [394a] (1555) mdash [075a] (463) mdash

C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)

C181 tran-9 mdash mdash [121a] (511a) mdash [032a] (101a) [012a] (059a) [045a] (277a) [108a](490a)

C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)

C182 trans-912 mdash [002a] (095a) mdash [003a] (011a) [014a] (048a) [009a] (043a) mdash [012a](060a)

C182 [117a](697a) [141a] (729a) [010a] (042a) [172a] (876a) [082a] (284a) [218] (1090a) [081a] (50a) [134a]

(709a)C183 mdash [006a] (027a) [004a] (017a) [007a] (028a) [045a] (155a) mdash mdash mdashaSignificant differences (119875 le 005) [mdash] not detected

Table 2 The mean of the absolute (g FA 100 gminus1 sample) and relative ( of total identified FA) content of each FA determined using theTMA-DMmethod




C140 [020b](109b) [036b] (162) [289b] (1223) [069] (286) [473b] (1636) mdash [081b] (504) mdash

C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)

C181 tran-9 mdash mdash [183b] (775b) mdash [045b] (154b) [019b] (089b) [056b] (347b) [174b](790b)

C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)

C182 trans-912 mdash [004b] (019b) mdash [005b] (017b) [033b] (113b) [014b] (066b) mdash [018b](085b)

C182 [183b](962b) [201b] (899b) [013b] (057b) [229b] (943b) [124b] (428b) [214] (1023b) [099b] (617b) [175b]

(795b)C183 mdash [008b] (035b) [005b] (022b) [009b] (038b) [056b] (206b) mdash mdash mdashbSignificant differences (119875 le 005) [mdash] not detected

calculated The (119877) values of both methods and FAs wereestablished from the complete analysis (in triplicate) of fourfood samples fortified with FA standards at two levels (std1and std2) In Table 4 mean values of 119877 for both methods arepresented

As observed in Table 4 the lowest 119877 values at the twostudied levels were those for the KOCH

3HCl method

However for most samples the 119877 values in this methodwere slightly higher for C120 C160 and C180 The values

decreased when lower concentrations were used Moreoverthese data show a high range of values obtained from thismethod (between 84 and 112) On the other hand theTMS-DM method showed higher 119877 values except for somesaturated FAs in most of the samples which showed 119877 valuesslightly lower than the other method Moreover an increasedlevel of homogeneity was observed because the values rangedbetween 90 and 106 at the two levels Accordingly theKOCH

3HCl method showed the lowest recovery values and


Table 3 Correlation coefficients between the KOCH3HCl methodand TMS-DMmethod

Fatty acids Correlation coefficients (119903) for g100 gC120 091C140 089C160 099C180 095C181 trans-9 096C181 098C182 trans-912 086C182 094C183 089

highest variationThese results suggest that the conditions ofthis process might not be sufficient to methylate all lipids

34 Comparison of Precision The repeatability (intradayRSD) and reproducibility (interday RSD) of the replicationson real samples were used to measure the precision of bothmethods The repeatability of both methods was establishedfrom four (119899 = 4) complete analyses of each sample underthe same conditions on one day and the reproducibility wasestablished from three (119899 = 3) complete analyses of each sam-ple repeated for three consecutive daysThe repeatability andreproducibility data are shown in Tables 5 and 6 respectivelyand the results are expressed as a relative standard deviation(RSD )

For most samples except for TFAs the KOCH3HCl

method showed intraday RSD values lower than 5 TheC181 trans-9 and C182 trans-912 had greater relative vari-ation The interday RSD for this method had values under6 except for TFAs (especially for C182 trans-912) whichshowed greater values

The TMS-DMmethod had the lowest RSD with intradayRSD values less than 4 Most of the FAs and TFAs studiedhad the lowest RSD variation values which ranged between032 and 301 except for C140 and C180 Most ofthe interday RSD values for the TMS-DM method rangedbetween 1 and 5 The highest values for most of thesamples were those observed for C140 and C183

In general the results from the KOCH3HCl method

showed the lowest recovery values especially for cistransUFAs and the highest intraday and interday variation valuesfor TFAsThe rest of the FAs studied had acceptable variationvalues These results match to some extent with those byNeo et al [6] and Phillips et al [23] Although the base-catalyzed method for the direct transesterification of lipidsis more applicable for routine analysis of some food samplesbecause it is easy to use and does not isomerize cistransUFAs [18] FFAs and some lipid classes such as those foundin sphingolipids are not methylated under these conditions[30] Therefore this method has resulted in poor recoveriesof FAMEs [27] Some studies have proven that the combinedbase-catalyzedmethod and acid-catalyzedmethod comparedto the base-catalyzed method alone has led to better results

in accuracy and precision of the analysis by improvingthe repeatability and 119877 values [20 26 28] Neverthelessother studies that used the acid-catalyzed method haveindicated that BF

3 HCl and other acidic catalysts will

change the double-bond configuration of cistrans FAs (egoctadecadienoic isomers CLA)Thus acidic catalysts are notrecommended for lipid samples that have a mixture of thesestructures such as bakery dairy and ruminantmeat products[30] In addition it has been reported that when using apaste date or concentrated reagent of acids the productionof artifacts as well as the loss of PUFAs may result [18 20]In summary the use of HCl in methanol and other acidiccatalysts is not recommended because the reactions take along time and require high temperatures and the reagentsmust be prepared often [20 25 30] Hence the KOCH

3HCl

method under milder conditions may not be sufficient toobtain complete methylation and these factors may explainthe poor results observed for UFAs and TFAs in comparisonwith other methods However this method is faster easyto use less expensive and more environmentally friendlythan the TMS-DM method Thus the KOCH

3HCl method

could bemore applicable for routine analysis and study of thegeneral composition of FAs in some food samples

In contrast the TMS-DM method showed the best bal-ance between recovery and variation values especially forthe cistrans UFAs when compared to the second methodIt also had the lowest intraday and interday variation formost FAs and TFAs This finding is most likely because ofthe use of TMS-DAM as an alternative to an acid catalystTMS-DM is an ideal derivatization reagent and a convenientalternative source of diazomethane which is known to besafer to handle andmore stable [40 44] It converts carboxylicacids to methyl esters in high yields with short incubationtimes and forms few by-products (N

2) [39] Furthermore the

esterification by TMS-DAM has been reported to be effectiveand accurate for the analysis of FA isomers in various foodsamples such as the analysis of cistrans PUFAs in seafood[31] and CLA isomers in ruminant meat tissues [27 32]Otherwise the base catalyst (NaOCH

3) is a useful reagent

for the fast transmethylation of FAs linked to TAG is stablefor a long time and performs better on lipids rich in FAswith PUFAs TFAs and CLA [14 18 19 35] In addition to allthese advantages TMS-DM and NaOCH

3do not change the

original FA distribution or alter the geometric configurationof the double bonds during the transformation reactions [1819 27 31] Artifacts are not produced when NaOCH

3is used

as a transesterification agent [30 35] and TMS-DM does notproduce the methoxy artifacts associated with base-catalyzedmethod [31 32 39 40] Accordingly this method resultedin less FA and TFA losses than using the acid-catalyzedmethylation method as well as a complete methylation ofFFAs

In general both methods were found to be suitable forthe determination of FA profiles and for quantifying relativelydifferent levels of cis and trans FAs in several bakery productssuch as biscuits cookies crackers wafers cakes and breadHowever no single method can fulfill the derivatizationrequirement for all types of food samples Consequently agood alternative could be to have both methods available for


Table 4 The recovery percentage (119877 calculated from four samples studied) at two addition levels for both methods employed

Sample Std119877 for KOCH3HCl (119877 for TMS-DM)

Fatty acidsC120 C140 C160 C180 C181 t9 C181 C182 t9 t12 C182 C183

A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)

a119877 recovery Std standard solution t trans fatty acids

Table 5 Intraday variation (RSD ) for four studied samples by both methods employed

Fatty acidsSample (119899 = 4 RSD )a

A B C Di ii i ii i ii i ii

C120 248 204 198 175 295 149 255 248C140 321 362 260 150 177 185 313 179C160 214 119 205 032 290 228 432 098C180 258 092 188 059 307 388 234 203C181 trans-9 503 114 423 202 627 217 592 301C181 344 226 110 089 355 199 190 127C182 trans-912 684 256 541 101 468 201 677 299C182 406 156 377 189 260 255 315 093C183 258 302 442 240 099 086 467 211aRSD relative standard deviation (i) the KOCH3HCl method (ii) the TM-SD method

Table 6 Interday variation (RSD ) for four studied samples by both methods employed





use in the laboratory The KOCH3HCl method is ideal for

the routine and fast analysis of samples that do not contain acomplexmixture of FAs and TFAs and the TMS-DMmethodis ideal for a more thorough analysis of rich cistrans UFAsamples such as bakery dairy and ruminant meat productsand for monitoring low levels of FAs and TFAs as well ascontrolling labeling authenticity

For both methods the appropriate use of an IS duringthe procedure might partially correct the recovery valuesfor both methods and compensate for any partial hydrolysisthat may occur during the course of the reactions [27]Moreover according to Eder [45] and Christie and Han [15]the extraction of FAMEs should be performedmore than onetime for complete recovery whichmay assist in improving theefficiency and accuracy of the performance by increasing therecovery values for both methods Otherwise lipid oxidationinvolves the attack of free radicals (formation by oxygen) toadjacent positions of double bonds [27] and these factorsare controlled in the TMS-DM method with the additionof the antioxidant agent BHT during FAME extraction andbefore storage whereas the KOCH

3HCl method has been

originally validated without using antioxidants and there wasno indication for the need to use antioxidants with thismethod

4 Conclusions

The results of the absolute concentration of most FAs inseveral bakery products such as biscuits cookies crackerswafers cakes and bread by GC-FID using two derivatizationprocedures (KOCH

3HCl and TMS-DM) have good agree-

ment However higher concentrations of all cis and transUFAs were detected when following the TMS-DM methodcompared to the KOCH

3HCl method and the results of

the relative proportion of the TFAs were significantly higherwhen estimated using the TMS-DM method compared tothe KOCH

3HCl method The results from the KOCH

3HCl

method showed the lowest 119877 values especially for cistransUFAs and it almost had acceptable intraday and interdayvariation values except for the TFAs Overall this methodunder the milder conditions used may not be sufficient toobtain complete methylation Nevertheless this method isfaster less expensive andmore environmentally friendly thanthe TMS-DMmethod In contrast the TMS-DMmethod hadbetter recovery values and the lowest intraday and interdayvariation values compared to the other method It presentsthe best balance combination between recovery and variationvalues especially for UFAs and TFAs It is likely that usingNaOCH

3along with TMS-DM as an alternative to the acid

catalyst in this method could result in a decrease in the lossof FAs and TFAs as well as a complete methylation of FFAswhen compared to the acid-catalyzed methylation methodAccordingly the KOCH

3HCl method could be appropriate

for the routine analysis and compositional study of FAs insome food samples whereas the TMS-DM method could beavailable for more accurate quantification of food samplescontaining a complex mixture of FAs and TFAs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the UniversitiKebangsaan Malaysia for funding (ldquoCode DPP-2013-045rdquoand ldquoUKM-AP-2011-17rdquo) and the direct contributions of thesupport staff from the School of Chemical Sciences and FoodTechnology the Faculty of Science and Technology UKM tothis study

References

[1] S J Lehotay and J Hajslova ldquoApplication of gas chromatogra-phy in food analysisrdquo Trends in Analytical Chemistry vol 21 no9-10 pp 686ndash697 2002

[2] A Aro J van Amelsvoort W Becker et al ldquoTransfatty acidsin dietary fats and oils from 14 European countries theTRANSFAIR studyrdquo Journal of Food Composition and Analysisvol 11 no 2 pp 137ndash149 1998

[3] S Satchithanandam C J Oles C J Spease M M Brandt MP Yurawecz and J I Rader ldquoTrans saturated and unsaturatedfat in foods in the United States prior to mandatory trans-fatlabelingrdquo Lipids vol 39 no 1 pp 11ndash18 2004

[4] J SalimonA TalalOmar andN Salih ldquoAn accurate and reliablemethod for identification and quantification of fatty acids andtrans fatty acids in food fats samples using gas chromatographyrdquoArabian Journal of Chemistry 2013

[5] S L Elias and S M Innis ldquoBakery foods are the major dietarysource of trans-fatty acids among pregnant women with dietsproviding 30 percent energy from fatrdquo Journal of the AmericanDietetic Association vol 102 no 1 pp 46ndash51 2002

[6] Y P Neo C H Tan and A Ariffin ldquoFatty acid composition offive Malaysian biscuits (cream crackers) with special referenceto trans- fatty acidsrdquoASEANFood Journal vol 14 no 3 pp 197ndash204 2007

[7] P C Calder and P Yaqoob ldquoOmega-3 polyunsaturated fattyacids and human health outcomesrdquo BioFactors vol 35 no 3pp 266ndash272 2009

[8] K Backholer and A Peeters ldquoReduction of trans-fatty acidsfrom foodrdquo The Journal of the American Medical Associationvol 308 no 18 pp 1858ndash1859 2012

[9] F A Kummerow ldquoThe negative effects of hydrogenated transfats and what to do about themrdquo Atherosclerosis vol 205 no 2pp 458ndash465 2009

[10] M Brandt J Moss and M Ferguson ldquoThe 2006-2007 foodlabel and package survey (FLAPS) nutrition labeling trans fatlabelingrdquo Journal of Food Composition and Analysis vol 22 ppS74ndashS77 2009

[11] S Filip R Fink J Hribar and R Vidrih ldquoTrans fatty acids infood and their influence onhumanhealthrdquoFoodTechnology andBiotechnology vol 48 no 2 pp 135ndash142 2010

[12] F Priego-Capote J Ruiz-Jimenez and M Castro ldquoIdentifica-tion and quantification of trans fatty acids in bakery prod-ucts by gas chromatography-mass spectrometry after focusedmicrowave Soxhlet extractionrdquo Food Chemistry vol 100 no 2pp 859ndash867 2007

[13] S Jumat S Mamot R Suria and A Mohamad Oil and FatAnalysis UKM Press Bangi Malaysia 2006


[14] I Brondz ldquoDevelopment of fatty acid analysis by high-perform-ance liquid chromatography gas chromatography and relatedtechniquesrdquo Analytica Chimica Acta vol 465 no 1-2 pp 1ndash372002

[15] WW Christie and X Han Lipid Analysis Isolation SeparationIdentification and Lipidomic Analysis Oily Press BridgewaterUK 2010

[16] L S Basconcillo and B E McCarry ldquoComparison of three GCMS methodologies for the analysis of fatty acids in Sinorhizo-bium meliloti development of a micro-scale one-vial methodrdquoJournal of Chromatography B vol 871 no 1 pp 22ndash31 2008

[17] P Gunc Ergonul I Akata F Kalyoncu and B Ergonul ldquoFattyacid compositions of six wild edible Mushroom speciesrdquo TheScientific World Journal vol 2013 Article ID 163964 4 pages2013

[18] W W Christie ldquoPreparation of ester derivatives of fatty acidsfor chromatographic analysisrdquo Advances in Lipid Methodologyvol 2 pp 69ndash111 1993

[19] P Delmonte A-R F Kia Q Hu and J I Rader ldquoReview ofmethods for preparation and gas chromatographic separationof trans and cis reference fatty acidsrdquo Journal of AOAC Interna-tional vol 92 no 5 pp 1310ndash1326 2009

[20] M C Teale Omega 3 Fatty Acid Research Nova Science NewYork NY USA 2006

[21] E M Antolın D M Delange and V G Canavaciolo ldquoEvalua-tion of fivemethods for derivatization andGC determination ofa mixture of very long chain fatty acids (C240 minusminusC360)rdquo Journalof Pharmaceutical and Biomedical Analysis vol 46 no 1 pp194ndash199 2008

[22] S KoohiKamali C P Tan and T C Ling ldquoOptimization ofsunflower oil transesterification process using sodiummethox-iderdquoThe Scientific World Journal vol 2012 Article ID 475027 8pages 2012

[23] K M Phillips D M Ruggio and K R Amanna ldquoOptimiza-tion of standard gas chromatographic methodology for thedetermination of trans fat in unlabeled bakery productsrdquo FoodAnalytical Methods vol 3 no 4 pp 277ndash294 2010

[24] M Petrovic N Kezic and V Bolanca ldquoOptimization of the GCmethod for routine analysis of the fatty acid profile in severalfood samplesrdquo Food Chemistry vol 122 no 1 pp 285ndash291 2010

[25] R G Ackman ldquoRemarks on official methods employing borontrifluoride in the preparation of methyl esters of the fatty acidsof fish oilsrdquo Journal of the American Oil Chemistsrsquo Society vol75 no 4 pp 541ndash545 1998

[26] K Nuernberg D Dannenberger K Ender and G NuernbergldquoComparison of different methylation methods for the analysisof conjugated linoleic acid isomers by silver ion HPLC in beeflipidsrdquo Journal of Agricultural and Food Chemistry vol 55 no3 pp 598ndash602 2007

[27] M Juarez O Polvillo M Conto A Ficco S Ballico and SFailla ldquoComparison of four extractionmethylation analyticalmethods to measure fatty acid composition by gas chromatog-raphy in meatrdquo Journal of Chromatography A vol 1190 no 1-2pp 327ndash332 2008

[28] L J E DanceODoran KHallett DDannenberger GNuern-berg and K Nuernberg ldquoComparison of two derivatisationmethods for conjugated linoleic acid isomer analysis by Ag+-HPLCDAD in beef fatrdquo European Journal of Lipid Science andTechnology vol 112 no 2 pp 188ndash194 2010

[29] S Kravic Z Suturovic J Svarc-Gajic Z Stojanovic and MPucarevic ldquoDetermination of trans fatty acids in foodstuffsby gas chromatography-massspectrometry after simultaneous

microwave-assisted extraction-esterificationrdquo Journal of the Ser-bian Chemical Society vol 75 no 6 pp 803ndash812 2010

[30] Y Li and B A Watkins ldquoUNIT D1 2 analysis of fatty acids infood lipidsrdquo in Current Protocols in Food Analytical ChemistryJohn Wiley amp Sons 2001

[31] M Juarez A Juarez N Aldai C Aviles and O Polvillo ldquoVal-idation of a gas-liquid chromatographic method for analysingsamples rich in long chain n-3 polyunsaturated fatty acidsapplication to seafoodrdquo Journal of Food Composition and Anal-ysis vol 23 no 7 pp 665ndash670 2010

[32] N Aldai B E Murray A I Najera D J Troy and K OsoroldquoDerivatization of fatty acids and its application for conjugatedlinoleic acid studies in ruminant meat lipidsrdquo Journal of theScience of Food and Agriculture vol 85 no 7 pp 1073ndash10832005

[33] M Yamasaki K Kishihara I IkedaM Sugano and K YamadaldquoA recommended esterification method for gas chromato-graphic measurement of conjugated linoleic acidrdquo Journal of theAmerican Oil Chemistsrsquo Society vol 76 no 8 pp 933ndash938 1999

[34] A Presser and A Hufner ldquoTrimethylsilyldiazomethanemdashamild and efficient reagent for the methylation of carboxylicacids and alcohols in natural productsrdquo Monatshefte furChemieChemical Monthly vol 135 no 8 pp 1015ndash1022 2004

[35] C D W Marais The Determination of Cis and Trans FattyAcid Isomers in Partially Hydrogenated Plant Oils University ofStellenbosch Stellenbosch South Africa 2007

[36] V J Barwick Preparation of Calibration Curves A Guide to BestPractice VAM Teddington UK 2003

[37] I Taverniers M de Loose and E van Bockstaele ldquoTrends inquality in the analytical laboratory II Analytical method vali-dation and quality assurancerdquo Trends in Analytical Chemistryvol 23 no 8 pp 535ndash552 2004

[38] MMMossoba J KGKramer VMilosevicMMilosevic andH Azizian ldquoInterference of saturated fats in the determinationof low levels of trans fats (below 05) by infrared spectroscopyrdquoJournal of the American Oil Chemistsrsquo Society vol 84 no 4 pp339ndash342 2007


[40] C M Murrieta B W Hess and D C Rule ldquoComparison ofacidic and alkaline catalysts for preparation of fatty acid methylesters from ovine muscle with emphasis on conjugated linoleicacidrdquoMeat Science vol 65 no 1 pp 523ndash529 2003

[41] W M N Ratnayake and J L Beare-Rogers ldquoProblems ofanalyzing C18Cis- and trans-fatty acids of margarine on the SP-2340 capillary columnrdquo Journal of Chromatographic Science vol28 no 12 pp 633ndash639 1990

[42] P Delmonte and J I Rader ldquoEvaluation of gas chromatographicmethods for the determination of trans fatrdquo Analytical andBioanalytical Chemistry vol 389 no 1 pp 77ndash85 2007

[43] J M Shin Y Hwang O Tu et al ldquoComparison of differentmethods to quantify fat classes in bakery productsrdquo FoodChemistry vol 136 no 2 pp 703ndash709 2013

[44] J Rosenfeld ldquoApplication of analytical derivatizations to thequantitative and qualitative determination of fatty acidsrdquo Ana-lytica Chimica Acta vol 465 no 1-2 pp 93ndash100 2002

[45] K Eder ldquoGas chromatographic analysis of fatty acid methylestersrdquo Journal of Chromatography B vol 671 no 1-2 pp 113ndash131 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


method [1 13ndash17]The quantification of FAs in fats and oils byGC involves transforming the analytes into more volatile andnonpolar derivatives after extracting the lipids from the foodproduct before GC analysis [14] The most important stagefor the GC-FID determination of FAs is sample preparationwhich usually requires derivatization of the FAs to increasethe volatility of the substances to improve separation and toreduce tailing [18]Moreover the speed of analysis sensitivityand accuracy are important parameters in GC that may beimproved with derivatization [18 19]

Sample preparation including the derivatization of FAshas been carefully reviewed by several authors [19ndash21] Themost commonly usedmethod for the determination of FAs isconversion of the FAs into their corresponding methyl esters(FAMEs) Many different methylation approaches have beendescribed in the literature and some methods have beenestablished for preparing FAMEs from lipids extracted fromvarious food samples acid- or base-catalyzed transmethy-lation borontrifluoride (BF

3) methylation after hydrolysis

methylation with diazomethane and silylation [18ndash20 22ndash24] In general these methods involve two steps first thesamples are heated with sodium hydroxide in methanol andsecond the free FAs (FFAs) are esterifiedwithmethanolic BF

3

[23] or methanolic KOH [24] However each method has itsown advantages and disadvantages [16 25]

In general the base-catalyzedmethod for the direct trans-esterification of lipids has been reported to bemore applicablefor nutrition analysis because it is easy to use and uses lessaggressive reagents than othermethods [22 24 26] Howeverthis method has resulted in poor recoveries of FAMEsbecause FFAs might remain partially unreacted [27] andbecause FFAs are notmethylated under these conditions [26]Therefore some studies have suggested that the repeatabilityrecovery with low variation and the highest concentrationdetected are improved for the most abundant FAs when thecombined base- and acid-catalyzedmethod is used comparedto the base- or acid-catalyzed methods alone [20 26 2829] Nevertheless using acid-catalyzed methods is usuallyundesirable because it is likely to lead to changes in theconfiguration of the double bond characteristics and toproduce artifacts [20 25 30]

An alternative method used by a number of laboratoriesto improve the accuracy of analysis is base hydrolysis followedby methylation of the resulting FFAs with diazomethanehowever the disadvantage of this method is that diazometh-ane needs precautions during extraction [21 31 32] In con-trast the esterification by TMS-DMhas been reported to be aconvenient alternative to diazomethane because it is safer tohandle and does not produce artifacts [33 34] Furthermoremethylation by TMS-DM after the saponification process hasbeen shown to be more accurate for cistrans PUFA analysisin seafood [31] and conjugated linoleic acid (CLA) isomersin ruminant meat tissues [32] when compared to othermethylation reagents However the hydrolysis or presence oftrace water leads to poor recoveries of FAMEs [16 27]

There is a need to investigate the concentration of FA andTFA isomers in all lipid fractions from food fats and theirproducts such as biscuits cakes crackers wafers and breadto monitor the low levels of FAs and TFAs and to control

labeling authenticity Therefore it is possible to apply theadvantages of sodium methoxide (NaOCH

3) as a useful

reagent for the fast transformation of FAs into FAMEs [18 35]along with using the TMS-DM reagent for the completemethylation of all FFAs which can be more reliable andproduce a higher accuracy In the current study to verify theaccuracy of measuring the concentrations of FAs and TFAs infood fats of bakery products the repeatability and recoveryusing a method based on the derivatization of lipid extractby base-catalyzed followed by TMS-DMwere compared withthe combined base- and acid-catalyzed methylation method(KOCH

3HCl) In addition the advantages disadvantages

and applicability to determine the complex mixture of FAsand TFAs in various types of bakery products are discussed

2 Materials and Methods

21 Standards and Reagents Nine FA and FAME standards(C120 C140 C160 C180 C181 C181t9 C182 C182t912and C183) were purchased from Fluka (purity ge99 (GC)Sigma-Aldrich Germany) the internal standard (IS) C150(Pentadecanoic acid) was purchased from Sigma (Sigma-Aldrich Germany) and the purity of all reagents was greaterthan 99 All chemicals (methanol toluene glacial aceticacid hydrochloric acid potassium hydroxide and sodiumhydroxide) were of analytical reagent grade and purchasedfrom Systerm (SystermMalaysia) except for n-hexane whichwas of higher purity (Systerm Malaysia for GC ge99) Theesterifying agent TMS-DM (2M) in n-hexane was purchasedfrom Sigma (Sigma-Aldrich Germany)

22 Food Samples Eight commercial food items were usedfor analysis and comparison in this study The samplesincluded different bakery and fast-food products such ascrackers bread with filling cakes wafers cookies andbiscuits as these products mainly contain FAs and TFAsThe samples were purchased from several Malaysian localsupermarkets including national and imported brands andall of those samples were coded with a letter (from A to H)

23 Sample Preparation and Lipid Extraction Each samplewas ground and placed in an oven at 50∘C until completedryness before analysis The total lipids were extracted usingthe Soxhlet Method for cereal fats [28] Approximately 10 g ofhomogenized sample was weighed into a cellulose extractioncartridge and the Soxhlet apparatus containing the cartridgewas fitted to a distillation flask containing 150mL of n-hexane with (50 ppm) butylated hydroxytoluene (BHT) anda few antibumping granules After 3 hours the mixture wasdried with Na

2SO4and filtered through fluted filter paper

The oil was recovered after stripping the solvent in a rotaryevaporator Finally the extracted lipids were dried undernitrogen (N

2) weighedand stored at minus20∘C until analysis

24 Preparation of Fatty Acid Methyl Esters (FAMEs) AfterSoxhlet extraction all lipid extracts weremethylated and con-verted into FAMEs using two different methylation methodsApproximately 015 g of each fat extract (in triplicate) wastransferred to a screw-cap test tube (10mL) and 1mL of


CH2O

CH2O

O

O

O

C

C

C

R

R998400

R998400998400

CHO

Triacylglycerol

KOCH3HClmethod

(b)

Δ60∘C

NaOCH3

CH3OH

KOCH3HClmethod

(a)

Δ70∘C

KOH

CH3OH

CH3O

O

C R

CH3O

O

C R

O

C RHO

(FAME)

(FAME)

(FFA)

Methanol toluene2 1

10 M

HClH3C Si

CH3

CH3

N2

TMS-DM Δ50∘C











2and dissolved







Inte

nsity

47500

37500

17500

27500

minus2500

7500

0

10 2030

40(min)

1 2 3 45 6 7 98

9

8

7521

4

6

3

10 1112

101112

IS

IS

(a) KOCH3HCl

(b) TMS-DM




















3HCl method whereas


3HCl method



3HCl and



0

2

4

6

8

10

12

0 5 10 15

NaO

CH3H

Cl (g

100

g)

TMS-DM (g100 g)

R2

= 09847 X = Y



procedures

0

04

08

12

16

0 05 1 15 2 25

NaO

CH3H

Cl (g

100

g)

R2

= 09668 X = Y

TMS-DM (g100 g)



procedures


3HCl methods










C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)


C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)









C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)


C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)






3HCl method

















3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


CH2O

CH2O

O

O

O

C

C

C

R

R998400

R998400998400

CHO

Triacylglycerol

KOCH3HClmethod

(b)

Δ60∘C

NaOCH3

CH3OH

KOCH3HClmethod

(a)

Δ70∘C

KOH

CH3OH

CH3O

O

C R

CH3O

O

C R

O

C RHO

(FAME)

(FAME)

(FFA)

Methanol toluene2 1

10 M

HClH3C Si

CH3

CH3

N2

TMS-DM Δ50∘C











2and dissolved







Inte

nsity

47500

37500

17500

27500

minus2500

7500

0

10 2030

40(min)

1 2 3 45 6 7 98

9

8

7521

4

6

3

10 1112

101112

IS

IS

(a) KOCH3HCl

(b) TMS-DM




















3HCl method whereas


3HCl method



3HCl and



0

2

4

6

8

10

12

0 5 10 15

NaO

CH3H

Cl (g

100

g)

TMS-DM (g100 g)

R2

= 09847 X = Y



procedures

0

04

08

12

16

0 05 1 15 2 25

NaO

CH3H

Cl (g

100

g)

R2

= 09668 X = Y

TMS-DM (g100 g)



procedures


3HCl methods










C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)


C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)









C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)


C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)






3HCl method

















3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Inte

nsity

47500

37500

17500

27500

minus2500

7500

0

10 2030

40(min)

1 2 3 45 6 7 98

9

8

7521

4

6

3

10 1112

101112

IS

IS

(a) KOCH3HCl

(b) TMS-DM




















3HCl method whereas


3HCl method



3HCl and



0

2

4

6

8

10

12

0 5 10 15

NaO

CH3H

Cl (g

100

g)

TMS-DM (g100 g)

R2

= 09847 X = Y



procedures

0

04

08

12

16

0 05 1 15 2 25

NaO

CH3H

Cl (g

100

g)

R2

= 09668 X = Y

TMS-DM (g100 g)



procedures


3HCl methods










C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)


C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)









C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)


C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)






3HCl method

















3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









3HCl method whereas


3HCl method



3HCl and



0

2

4

6

8

10

12

0 5 10 15

NaO

CH3H

Cl (g

100

g)

TMS-DM (g100 g)

R2

= 09847 X = Y



procedures

0

04

08

12

16

0 05 1 15 2 25

NaO

CH3H

Cl (g

100

g)

R2

= 09668 X = Y

TMS-DM (g100 g)



procedures


3HCl methods










C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)


C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)









C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)


C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)






3HCl method

















3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







C160 [921](4835) [928] (4343) [253] (1070) [887] (3805) [416] (1641a) [976] (4870a) [591a] (3648a) [778]

(4118)

C180 [079a](495) [084a] (496) [365a] (1724) [165a] (859) [107a] (422) [094] (469) [031a] (191a) [087a]

(461)


C181 [662a](3870a) [897a] (4104a) [110] (565) [794a] (3483a) [202a] (797a) [673a] (3421a) [183a] (1130a) [786a]

(4106a)









C160 [895](4708) [934] (4170) [229] (969) [876] (3608) [420] (1454b) [941] (4504b) [497b] (3070b) [861]

(3914)

C180 [086b](450) [099b] (443) [418b] (1770) [206b] (844) [123b] (425) [105] (504) [037b] (230b) [112b]

(509)


C181 [722b](3791b) [956b] (4270b) [113] (479) [887b] (3647b) [285b] (986b) [797b] (3814b) [219b] (1351b) [863b]

(3922b)






3HCl method

















3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of














3HCl


3HCl method







3do not change the


3is used







A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





A1 1068 877 1108 973 959 978 869 932 995

(1043) (928) (1049) (979) (1020) 10312 (989) (958) (988)

2 1059 872 1094 955 922 940 837 908 981(1032) (896) (1058) (943) (987) (1049) (938) (923) (960)

B1 981 968 1124 915 934 971 910 887 1041

(967) (1017) (1060) (898) (952) (1033) (970) (946) (1056)

2 965 958 1063 924 914 941 887 834 1015(954) (983) (1054) (907) (921) (1018) (951) (934) (1031)

C1 924 9361 1069 935 837 9775 836 859 103 6

(934) (1007) (1052) (898) (923) (1022) (937) (926) (1045)

2 911 918 1041 915 839 971 826 842 1040(912) (992) (1032) (892) (912) (1042) (895) (912) (1062)

D1 1041 977 1021 965 909 940 866 1012 890

(1019) (1026) (1007) (980) (988) (991) (1034) (1041) (973)

2 981 968 961 965 879 931 840 982 850(984) (1012) (965) (972) (943) (982) (984) (1042) (952)
















4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







4 Conclusions







3HCl








Acknowledgments


References

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article comparison of two derivatization methods

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