Chromatographic techniques to determine conjugated linoleic acid isomers

Trends in Analytical Chemistry, Vol. 25, No. 9, 2006 Trends

Chromatographic techniques todetermine conjugated linoleic acidisomersMiguel Angel de la Fuente, Pilar Luna, Manuela Juarez

Conjugated linoleic acid (CLA) is a complex mixture of positional and

geometric isomers that contains a conjugated double-bond system and exhibits

health-promoting attributes. Although cis-9 trans-11 is the predominant

species in dairy foods and biological subtracts, there is a real need to determine

the CLA-isomer distribution, since the isomers may have different biological

functions. This review covers the latest advances in the analytical techniques,

mainly GC-MS and Ag+-HPLC, to determine the isomeric profile of CLA.

ª 2006 Elsevier Ltd. All rights reserved.

Keywords: Ag+-HPLC; Analysis; CLA; GC-MS; Isomer

Abbreviations: Ag+-HPLC, Silver-ion HPLC; CACI-MS/MS, Covalent adduct chemical

ionization tandem mass spectrometry; cis/trans, Chromatographic peaks including cis-

trans plus trans-cis CLA isomers; CLA, Conjugated linoleic acid; DMOX, Dimethyl-

oxazolyne; FAME, Fatty acid methyl esters; FID, Flame ionization detector; FFA, Free

fatty acids; FTIR, Fourier transform infrared spectroscopy; GC, Gas chromatography;

HPLC, High-performance liquid chromatography; MS, Mass spectrometry; MTAD,

4-methyl-1,2,4-triazolyn-3,5-dione derivatives; NMR, Nuclear magnetic resonance; RA,

Rumenic acid (cis-9, trans-11 C18:2); RRV, Relative retention volume; RV, Retention

volume; TLC, Thin-layer chromatography; TAG, Triacylglycerols

Miguel Angel de la Fuente*,

Pilar Luna, Manuela Juarez

Instituto del Frıo (CSIC),

Jose Antonio Novais 10,

Ciudad Universitaria s/n,

28040 Madrid,

Spain

*Corresponding author.

Tel.: +34 91 544 5607;

Fax: +34 91 5493627;

E-mail: [email protected]

0165-9936/$ - see front matter ª 20060165-9936/$ - see front matter ª 2006

1. Introduction

Conjugated linoleic acid (CLA) is a termused for a mixture of positional (6–8 to13–15) and geometric (cis-cis, trans-trans,cis-trans and trans-cis) isomers of linoleicacid that contain conjugated doublebonds. Two of the isomers, cis-9 trans-11(rumenic acid, RA) and trans-10 cis-12,could confer a number of beneficial bio-logical effects [1–3]. RA effects have beenidentified in a range of animal models andinclude anti-carcinogenesis, immuno-modulation and anti-atherosclerosis. Thetrans-10 cis-12 isomer is a potent inhibitorof milk-fat synthesis and has been impli-cated in diet-induced milk-fat depressionin dairy cows. However, there is accu-mulating evidence that this isomer mayadversely influence human health, inparticular concerning insulin sensitivity

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.04.012Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.04.012

and blood lipids as well as elicit pro-car-cinogenic effects in animal models of colonand prostate cancer [4,5]. It is also inter-esting to note that no such detrimentaleffects on health have been reported forthe more commonly employed 50:50mixtures of the trans-10 cis-12 and RA[4]. Other isomers, such as cis-9 cis-11 [6]and trans-9 trans-11 [7], have shown anti-tumoral properties but evidence is stillvery limited. By contrast, incorporatingcis-11 trans-13 into mitochondrial cardi-olipin could adversely affect the activity ofkey enzymes in the cellular energeticeconomy [8]. The multiple physiologiceffects reported for CLA could be the resultof multiple interactions of the biologicallyactive CLA isomers with numerous meta-bolic signaling pathways. Hence, studiesto define the beneficial and the detrimentaleffects of each individual CLA isomer areneeded and should be undertaken.

CLA isomers, naturally occurring infoods derived from ruminants, are pro-duced as intermediates of the biohydro-genation of polyunsaturated fatty acids,specifically linoleic (cis-9 cis-12 C18:2)and a-linolenic (cis-9 cis-12 cis-15 C18:3),by rumen bacteria. In addition to RA,present at about 75–90% of total CLA, amultiplicity of minor geometrical andpositional isomers, ranging from 7–9 to12–14, are also formed in the rumen. Inaddition, trans-vaccenic acid (trans-11C18:1), also a product of incompleterumen biohydrogenation, can be re-converted in mammalian tissues to RA. Incontrast to milk and biological material,which contains primarily RA, syntheti-cally prepared CLA mixtures produced byalkali isomerization of linoleic acid or oils

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Trends Trends in Analytical Chemistry, Vol. 25, No. 9, 2006

rich in this fatty acid, such as saflower oil, tend to con-tain a mixture with similar proportions of cis-9 trans-11and trans-10 cis-12 C18:2 together with minor amountsof cis-cis and trans-trans isomers.

The classical derivatization for gas chromatography(GC) and flame ionization detector (FID) analysis offatty acids is conversion into fatty acid methyl esters(FAME). This procedure allows determination of thetotal CLA content in different subtracts. However,separation, identification and quantification of CLAisomers offer a considerable analytical challenge,mainly in dairy foods and biological samples. From thesecond half of the 1990s, a lot of research in this fieldwas undertaken, and different reviews have covereddifferent aspects in detail [9–15]. At present, GC withmass spectrometry (MS) and silver-ion HPLC(Ag+-HPLC) with columns in series and a UV detectorof FAME are the most popular tools for elucidating theCLA-isomer profile. Although GC linked to Fouriertransform infrared spectroscopy (FTIR) can contributeto confirm the geometry (cis or trans) of the doublebonds [16,17] and nuclear magnetic resonance (NMR)spectroscopy [18,19] can also provide a great deal ofinvaluable information, the availability of these tech-niques is limited. This article summarizes the mainchromatographic techniques in CLA determination usedin the past decade, updates knowledge and reports thelatest advances in this field. We discuss the most recenttrends and developments, limitations and potential ofthese approaches.

2. Standards

Analysis of CLA isomers has been hindered by a lack ofwell-characterized reference materials. The shortage ofcommercially available standards for most CLA isomershas frequently led to misidentifications. Most commercialCLA mixtures generally contain only four major posi-tional isomers (trans-8 cis-10, cis-9 trans-11, trans-10cis-12 and cis-11 trans-13) with smaller amounts of thecorresponding cis-cis and trans-trans isomers of eachpositional isomer. Only a few pure isomers (cis-9 trans-11, trans-10 cis-12, cis-9 cis-11, cis-11 trans-13 andtrans-9 trans-11) are available in the market.

Additional CLA isomers from 7–9 to 12–14 C18:2have been synthesized by combining sigmatropic rear-rangement with selenium-catalyzed geometric isomeri-zation of known CLA isomers [20]. Other authors[21,22] have generated a mixture of cis-6 trans-8, trans-7 cis-9, cis-9 trans-11 and trans-10 cis-12 by partialhydrogenation with hydrazine of c-linolenic acid (cis-6cis-9 cis-12 C18:3) followed by conjugation with KOH inethylene glycol (Fig. 1). Isolation of the different isomerswas performed by preparative Ag+-HPLC. Applying thesame procedure to a-linolenic acid, a mixture of cis-9

918 http://www.elsevier.com/locate/trac

trans-11, trans-10 cis-12, cis-12 trans-14 and trans-13cis-15 C18:2 was also synthesized [22].

All the geometric CLA FAME from positions 6–8 to13–15 were generated in mixtures by treating com-mercially available pure CLA compounds, a referencemixture, or synthetic preparations from a-linoleic and c-linolenic acids with I2 [23]. For example, the four geo-metric isomers (trans-trans, cis-cis, cis-trans and trans-cis)of 9–11 C18:2 are easily prepared by treating the com-mercially available RA with I2 and light. However,although all these synthetic preparations were developedto make standards for analytical purposes, their com-mercialization will require adaptation, and these isomersare not still found in the market [20–22].

3. Gas chromatography

3.1. Derivatization of fatty acidsCommercial CLA standards are usually supplied asmethyl esters or free acids. However, in dairy fat orbiological samples, CLA is esterified to form triacyl-glycerols (TAG). The traditional analysis of fatty acidsfrom lipids has involved derivatization to form less polarand more volatile compounds. Preparation of volatilederivatives is often the step before analyzing the profile offatty acids by GC. The most extend procedure consists intransform fatty acids from glycerides into methyl esters.

Alkali-catalyzed methylation methods (e.g., usingNaOCH3 or KOH in methanol at room temperature) areconsidered the most reliable for determining the distri-bution of CLA isomers because they cause no isomeri-zation and produce no methoxy artifacts [24].

Acid-catalyzed methods, employing BF3, HCl orH2SO4, favor extensive isomerization of conjugateddienes and contribute to forming allylic methoxy arti-facts [25]. These artifacts can hinder the chromato-graphic analysis. Increasing temperature and/orincubation time for either method decreased the RA andtrans-10 cis-12, but increased trans-9 trans-11, trans-10trans-12, as well as artifacts [26].

Ostrowska et al. [27] in studies by Ag+-HPLC alsoobserved that all acid catalysts were associated withsignificantly increased levels of trans-trans isomers ofCLA and lower levels of cis/trans (cis-trans plus trans-cis),mainly when HCl was used.

In the first half of the 1990s, acid-catalyzed methyl-ation with the BF3 procedure was used extensively toanalyze the CLA content of dairy foods. Some of thoseworks also showed a high content of CLA isomers otherthan RA, principally trans-trans [24].

In milk fat, with marked contents of short-chain fattyacids, it is advisable to use procedures that can minimizethe loss of volatile fatty acids (e.g., butyric (C4) andcaproic (C6)). KOH in methanol at room temperature ascatalyst is a reference procedure [28] to derivatize milk

cis-9 trans-11 C18:2 trans-10 cis-12 C18:2 cis-12 trans-14 C18:2trans-13 cis-15 C18:2

cis-6 trans-8 C18:2 trans-7 cis-9 C18:2 cis-9 trans-11C18:2

trans-10 cis-12 C18:2

cis-9 cis-12 C18:2 cis-9 cis-15 C18:2

cis-12 cis-15 C18:2

cis-6 cis-9 C18:2 cis-6 cis-12 C18:2cis-9 cis-12 C18:2

(hydrazine / MeOH)Partial HYDROGENATION

(KOH / ethylene glycol )CONJUGATION

(I2 + light)ISOMERIZATION

α-linolenic acid(cis-9 cis-12 cis-15 C18:3)

γ-linolenic acid(cis-6 cis-9 cis-12 C18:3)

12-14 C18:2

t-tc-tt-cc-c

Ag+-HPLC-Semipreparative

cis -12 trans-14 C18:2 trans-13 cis-15 C18:2

13-15 C18:2

t-tc-tt-cc-c

(I2 + light)ISOMERIZATION

6-8 C18:2

t-tc-tt-cc-c

cis-6 trans-8 C18:2 trans-7 cis-9 C18:2

7-9 C18:2

t-tc-tt-cc-c

Figure 1. Synthesis of CLA isomers according to the procedure proposed by Delmonte et al. [21,22] c: cis; t: trans.


fat before studying the profile of fatty acids. This methodreduces the loss of short-chain fatty acids and does notcause CLA isomerization. However, KOH in methanoldoes not react with free fatty acids (FFA) and does notcompletely methylate phospholipids, which makes thismethod unsuitable for matrices, such as ruminal liquid,or for tissues with high contents in those compounds. Ingeneral, this drawback is not a hurdle in dairy-fatstudies, except in research in milks with low bacterio-logical quality or cheeses with high levels of FFA. Inthese kinds of sample, acid-catalyzed methods at mildconditions can be recommended [29].

3.2. CLA analysis on capillary columnsGC-FID is by far the most widely used method for theanalysis of fatty acids and it remains the only tool em-ployed by many researchers to determine total CLAcontent. Identification is often based solely on compari-son of retention times, with limited availability of stan-dards, and therefore, it may be tentative at best. The100-m highly polar cyanosilicone capillary columnsavailable under different commercial names (CP Sil 88,

SP-2560 and BPX-70) are the best for trying to resolvemost of the closely related isomers of CLA. On 100-m CPSil88 column, CLA isomers eluted in a GC region justafter a-linolenic methyl ester [30]. The elution order ofthe CLA isomers with this column was first all the cis/trans, followed by the cis-cis, and finally all the trans-trans positional isomers. For the same positional isomer,the cis-trans elutes before the trans-cis geometric isomer.

Theoretically it would be possible to resolve the fourgeometrical isomers in all positional species. Fig. 2 showsthe gas chromatograms of all the standard CLA isomersfrom 6–8 to 13–15 CLA, in relative retention time/c-linolenic acid scale. They were previously synthesizedand isolated, as Delmonte et al. described [21,22]. Ascan be seen, most geometric isomers belonging to thesame positional configuration were discriminated. Infact, when dealing only with a mixture of two majorisomers (e.g., RA plus trans-10 cis-12 in commercialstandards), one should be able to resolve them. Never-theless, when up to eight isomers are present in roughlysimilar proportions, as produced by I2 isomerizing acommercial CLA mixture of four isomers (trans-8 cis-10,

http://www.elsevier.com/locate/trac 919

Figure 2. Gas chromatograms of the CLA isomers from 6–8 to 13–15 with relative retention time (RRT)/c-linolenic acid (GLA) scale.c: cis; t: trans (Reproduced with permission from [22]).


cis-9 trans-11, trans-10 cis-12 and cis-11 trans-13), theycan barely be seen to separate, but are not clearly re-solved by GC. Furthermore, such a separation is impos-

67.50 68.00 68.50 69.00

c9, t11

t7, c9

t9,c11

t10,c12

t11, c

c9, c1

2

3

4

Figure 3. GC-MS (total ion) partial chromatograms showing the profiles ofat (solid line) and a standard mixture (dotted line). CLA isomers of standatrans-10 cis-12; 5: cis-9 cis-11; 6: trans-11trans-13; 7: trans-8 trans-10 + tpermission from [53]).


sible when one isomer predominates, as is usually thecase with RA in natural products. Whenever the relativeconcentration is uneven, a number of CLA isomers willbe masked by the predominant isomers. Of particularconcern is the trans-7 cis-9, the second most abundantCLA isomer in dairy products, which is not resolved fromRA by GC using these 100-m capillary columns.

3.3. Application of GC-MSGC-MS combines high-resolution separation with selec-tive and sensitive detection, so it is a valuable tool foridentifying CLA isomers if suitable derivatives are used.FAME can supply useful information about the molecu-lar weight and the degree of unsaturation of the fattyacid. In order to discriminate CLA from unknown orinterfering fatty acids, high-resolution selected-ionrecording MS of the molecular ion (m/z = 294) can beused. Qualitatively, the mass spectra of CLA FAME iso-mers are indistinguishable from one another and frommethylene-interrupted octadecadienoic acid methylesters, such as linoleate. However, because the 294 ionis selective for C18:2 methyl esters, it could be useful fordiscriminating CLA species from other fatty acids thatelute in the same region of the chromatogram. The CLAregion by GC with a CP-Sil 88 100-m column wasshown to be rather free of interfering FAME except forone peak corresponding to C21 (Fig. 3). This acid hasbeen reported as co-eluting in GC analysis with trans-10

min69.50 70.00 70.50

13

11

t12 ,t14

t10, t12

t9, t11

t8, t10

t11, t13

5

6

7

C21

f conjugated fatty acid (CLA) methyl esters from processed cheeserd mixture: 1: cis-9 trans-11; 2: trans-8 cis-10; 3: cis-11 trans-13; 4:rans-9 trans-11 + trans-10 trans-12. c: cis; t: trans (Reproduced with


cis-12 [31] or in the cis-cis area [32,33] in the liver lipidof pigs and milk fats, respectively. Some signals, lowerthan those for minor CLA isomers, corresponding to themolecular ion for C20:2 isomers were also apparent inthe trans-trans CLA region [31].

To distinguish between different CLA positional isomers,specific derivatives of the conjugated diene system havebeen assayed. Spitzer [34] reviewed derivatization tech-niques that produce structurally useful data from CLA.He recommended transforming unsaturated fatty acidsinto dimethyloxazolyne (DMOX) derivatives after adding2-amino-2-metil-1-propanol, or forming 4-methyl-1,2,4-triazolyn-3,5-diones (MTAD) through Diels-Alderreactions. Other derivatives of pyrrolidides and picolinylesters have also been used [12].

DMOX derivatives are strongly preferred, even thoughtheir preparation requires higher temperatures thanMTAD. CLA DMOX separations are influenced by bothgeometry and position of the double bonds of the CLAisomers, with the result that these derivatives providesuperior CLA-isomer resolution compared to MTAD [10].Furthermore, the chromatographic separations observedfor FAME are retained or improved upon by the sepa-rations observed for the corresponding DMOX derivatives[35,36].

DMOX derivatives of fatty acids are very useful forlocating double bonds, especially in polyunsaturated andconjugated systems, and have confirmed the positions ofdouble bonds in CLA isomers in a range of materials[32,33,37–39]. Roach [40] described the distinctiveelectron impact mass spectra of the most common CLApositional isomers (from 7–9 to 12–14) and Fritscheet al. [41] characterized the more unusual 13–15 and6–8 C18:2 species. All mass spectra of CLA DMOXderivatives contain intense ion at m/z 113 and 126 anda molecular ion at m/z 333. They are also characterizedby a loss of 15 from the molecular ion and successivelosses of 14, except for the conjugated diene system,which may be located in the chain by its characteristicloss sequence of 12, 14 and 12. Carbons in the chainallyl of the conjugated diene system are favored radicalsites and produce more abundant fragmentation thanother positions in the chain [10,40]. These abundant

Table 1. Diagnostic ions for dimethyloxazolyne derivatives of conjugated(Adapted from [10])

Positional isomer Allylic ion Losses of 12

6–8 140 154, 166, 17–9 154 168, 180, 18–10 168 182, 194, 29–11 182 196, 208, 2

10–12 196 210, 222, 211–13 210 224, 236, 212–14 224 238, 250, 213–15 238 252, 264, 2

fragment ions, flanking a loss sequence of 12, 14 and12, facilitate assignment of the positions of the doublebonds in the carbon chain. Roach et al. [10] highlightedseven diagnostic ions per isomer giving a total of 28 ionsthat could be useful for identifying positions rangingfrom 6–8 to 13–15 C18:2 (Table 1).

Some diagnostic ions are very characteristic and theirabundance can be useful to detect the presence of somepositional isomers. This tactic led to finding the trans-7cis-9 CLA isomers in different food and biological mate-rials [38]. The elution sequence of CLA isomers on100-m CP Sil 88 column, together with the CLA DMOXspectra also provided evidence for the existence of traceamounts of cis-7 trans-9 C18:2 geometrical isomer inewes milk fat [33]. However, most of the diagnostic ionsused to distinguish isomers of CLA are also found in theDMOX derivative mass spectra of many other fatty acids.Thus, identification of a specific CLA isomer as its DMOXderivative requires sufficient chromatographic separa-tion from other congeners and co-extractives to obtain arepresentative mass spectrum of the isomer. Further-more, although geometrical configuration can beinferred from chromatographic behavior, geometricalisomers cannot be differentiated on the basis of theirmass spectra.

GC-MS of DMOX derivatives would require the com-bined use of other techniques for CLA-isomer identifica-tion, mainly to determine cis or trans configuration. GClinked to FTIR is potentially a powerful technique for thedetermination of the geometrical configuration of doublebonds in fatty acids. In the context of CLA, it enables theidentification of cis/trans, cis-cis and trans-trans isomers.Although it does not distinguish between cis-transand trans-cis isomers, assignments can be made withreasonable certainty when GC retention data are takeninto account.

Acetonitrile covalent adduct chemical ionization tan-dem mass spectrometry (CACI-MS/MS) has been shownto be an alternative method for identifying double-bondpositions and geometry in methyl esters of CLA [42–44].The (1-methyleneimino)-1-ethenylium ion (m/z = 54),generated by self-reaction of acetonitrile under CACI-MSconditions, reacts with unsaturated fatty acids to yield

linoleic acid positional isomers ranging from 6–8 to 13–15 C18:2

, 14 and 12 Allylic ion Allylic ion

80, 192 220 23494, 206 234 24808, 220 248 26222, 234 262 27636, 248 276 29050, 262 290 30464, 276 304 31878, 290 318 332



an [M + 54]+ ion. Collisionally activated dissociation ofthe [M + 54]+ ion yields two diagnostic ions corre-sponding to bond cleavage at specific locations inunsaturated fatty acids.

Acetonitrile CACI-MS/MS diagnostic ions expected forCLA with different double-bond positions (3–5 to 14–16)were determined in standards by Michaud et al. [42].These ions correspond to C-C cleavage at a position thatis vinylic to either side of the conjugated diene unit, andproduce the a diagnostic ion containing the ester group,and the x diagnostic ion containing the terminal methylgroup. In CLA with mixed double-bond geometry, them/z = 54 ion adds preferentially across the cis doublebond, for steric reasons, resulting in a higher abundanceof the diagnostic ion in which C-C cleavage occursvinylic to the original trans double bond [43]. Thus, thea/x ion-abundance ratio is characteristic of the double-bond geometry of the CLA. As can be seen in Table 2, thea/x ratio is an order of magnitude less in trans-cis thancis-trans isomers.

Acetonitrile CACI-MS/MS alone can therefore dis-criminate CLA into three categories of double-bondgeometry: cis-trans, trans-cis and, finally, cis-cis plus

Table 2. a/x ratios of diagnostic ions for methyl esters of cis-transand trans-cis conjugated linoleic acid positional isomer standardsranging from 7–9 C18:2 to 12–14 C18:2 (Adapted from [42])

Positional isomer a/x diagnostic ion ratios

cis-trans trans-cis

7–9 10 0.38–10 5.3 0.49–11 4.9 0.1

10–12 11 0.111–13 6.8 0.212–14 5.8 0.1

Table 3. Overview of the main analytical procedures to determine conjug

Matrices/type of sample Sample treatment Fat

Dairy products Fat extraction FAMilk/cheese Fat extraction FADairy products/Biological tissues/Animal fats/Human milk/Soybean oil

Fat extraction DM

Biological tissues Fat extraction MTpic

Milk Fat extraction FADairy products/Human milk/Biological tissues Fat extraction FADairy products/Biological tissues/Animal fats/Ruminal fluid

Fat extraction FA

Beef and pork fat blend Pre-fractionation ofFAME by TLC

p-mest

Pig adipose tissue and muscle Fat extraction + mildsaponification

FFA

Ag+-HPLC: Silver-ion HPLC; CACI-MS/MS: Covalent adduct chemical iderivatives; FAME: Fatty acid methyl esters; FFA: Free fatty acids; FID: FlamGC: Gas chromatography; MS: Mass spectrometry; MTAD: 4-methyl-1,2,4


trans-trans. Double-bond geometry in cis-cis and trans-trans isomers could be deduced from GC relative reten-tion times. Together with GC retention time data, ace-tonitrile CACI-MS/MS provided positive identification for13 CLA FAME in a milk-fat sample [44]. Subsequentacquisition of CLA standards and publication of the massspectra demonstrated that every peak in the milk-fatsample was properly assigned without standards.

Table 3 summarizes the main GC approaches reportedin the last 10 years.

4. CLA analysis by Ag+-HPLC

Over the decades, silver-ion or argentation chromatog-raphy has become an important methodology in frac-tionation and characterization of lipids. Argentationchromatography can separate fatty acids according tothe configuration as well as the number and position oftheir double bonds. A fairly comprehensive listing ofrecent applications of silver ion chromatography (TLCand HPLC) to the analysis of CLA in a variety of sub-strates was presented by Adlof [11].

This overview also includes topics such as CLA-enriched TAG or the application of semi-preparativeAg+-HPLC to isolate multigram quantities of CLAisomers. We discuss Ag+-HPLC of CLA FAME, otherderivatives and FFA. We emphasize FAME analysis be-cause the use of this derivative is easy in that it is usedfor general analysis of fatty acid moieties. The comple-mentary use of this technique with GC is currently themost effective way to separate and determine individualisomers proportion of CLA. In stable silver-ion columnsfor HPLC, the silver ions are linked via ionic bonds tophenylsulfonic acid moieties, which are bound to a silica

ated linoleic acid isomers in different type of matrices

ty acid derivatives Separation + detection References

ME GC + FID [23,30,31,37,53]ME GC + MS [23,31,33]OX GC + MS [33,37–39]

AD/pyrrolidides/olinyl esters

GC + MS [10,12]

ME GC + CACI-MS/MS [44]ME/DMOX GC-FTIR [16,17,32,37,38]ME Ag+-HPLC+ UV

(233 nm)[23,33,37,45–48,50,53]

ethoxy-phenacylers

Ag+-HPLC + UV(270 nm)

[52]

(No derivatization) Ag+-HPLC + UV (233 nm) [27,51]

onization tandem mass spectrometry; DMOX: Dimethyloxazolynee ionization detector; FTIR: Fourier transform infrared spectroscopy;-triazolyn-3,5-diones derivatives; TLC: Thin-layer chromatography.


matrix. This type of column has been used extensivelyfor the separation of CLA isomers.

The use of Ag+-HPLC to complement GC in the assayof CLA was first reported by Sehat et al. [45]. Thismethod was based on the work of Adlof [46], who usedan acetonitrile:hexane elution system. General require-ments for CLA analysis by Ag+-HPLC were standardizedat the end of the 1990s [37,45,47]. The systems used forisocratic separations of CLA FAME were equipped withcommercial ChromSpher 5 Lipids silver-impregnatedcolumns, the mobile phase being 0.1% acetonitrile inhexane.

CLA FAME are selectively detected by their charac-teristic UV absorbance at 233 nm and the identificationsof isomers in HPLC chromatograms are based on co-injections of known reference materials obtained fromcommercial sources or synthesized. The Ag+-HPLC pro-file was shown to separate the different trans-transcompounds followed by a chromatographic zone wherecis/trans (cis-trans plus trans-cis) eluted (Fig. 4). Al-though these geometrical isomers were not resolved,species differing in positional double bonds eluted sepa-rately. Finally, after the cis/trans area, cis-cis CLAisomers should be located. Theoretically, these isomerscould be individually determined. However, in somesubtracts, such as milk fat, a wide peak could mask thecis-cis area [33,47]. This peak was attributed to theabsorption of methyl oleate when this fatty acid is foundin high amounts. The use of a dual UV wavelengthdetector meant that it was possible to detect simulta-neously and selectively CLA at 233 nm and other fatty

30 40 50

trans, trans

7,9

8,109,11

10,12

11,13

12,14?

?

12,1411

Figure 4. Ag+-HPLC profile with three capillary columns in series of ewes(broken line). Asterisk represents methyl oleate. (Reproduced with permiss

acids at 205 nm. The existence of other minor interfer-ences in the CLA area have also been reported [15].

Operating from one to six Ag+-HPLC columns in series[47,48] progressively improved the resolution of themethyl esters of CLA isomeric mixtures from natural andcommercial products. However, it would appear that theuse of three columns is the best compromise to achieve,in a timely manner, resolution of most CLA isomers inbiological matrices. Long periods of time to elute allcompounds and mobile-phase consumption would dis-suade the analyst from using more than three columns.This approach should be used to resolve only specificcritical pairs of isomers. Furthermore, three columnscould resolve the 11–13 pair of cis/trans geometric CLAisomers.

The major problem with Ag+-HPLC is the retention-volume (RV) drift that occurs over time. This technique,which provides a wide separation of CLA isomers, isgaining more acceptance, but it is not yet in commonuse because the non-reproducibility of RV makes iden-tification of peaks difficult. Factors influencing RV andpeak shapes on individual CLA isomers include precon-ditioning and column temperature, column sampleloading, double-bond positions relative to each other andto the fatty acid carbonyl group as well as double-bondgeometry and, mainly, solvent composition.

To obtain reproducible results between Ag+-HPLCruns, potential sources of error, such as solvent compo-sition, should be addressed. The solubility of acetonitrilein hexane is less than 5% because of the difference inpolarity of these two solvents, resulting in an unstable

min60 70

cis, trans / trans, cis

9,11

,1310,12

8,107,9

methyl oleate

*

milk fat FAME using UV detector at 233 nm (solid line) and 205 nmion from [33]).


Figure 5. Ag+-HPLC chromatograms of all geometric CLA isomersfrom 6–8 to 13–15 C18:2. cis-9 trans-11 was added to each posi-tional mixture for reference. RRV: Relative retention volume;c: cis; t: trans (Reproduced with permission from [50]).


solution. Increasing the amount of acetonitrile in hexanedecreases the retention time of FAME and small varia-tions in the percentage of acetonitrile in the elutionsolvent cause a big variation in RV of CLA isomers.lthough this drift would not affect the relative resolutionof the CLA isomers, the retention time of the FAMEisomers can increase with successive runs. Alternatively,the addition of diethyl ether to the mobile phase wassuggested [21] because it partly stabilizes the solventmixture. However, this elution system (0.1% aceto-nitrile/0.5% diethylether in hexane) did not totallyeliminate retention-time shift.

More recently, Muller et al. [49] evaluated 13 solventsystems with respect to stability of retention times andresolution using a commercial CLA mixture and twoChromSpher 5 Lipids columns in series. The eluent system,0.2% propionitrile in hexane, showed the higheststability compared with the reference acetonitrile system.This enhanced stability was attributed to better solubilityof propionitrile in hexane. Unfortunately, propionitrile isa toxic reagent and did not give as good a resolution ofCLA as acetonitrile. To improve resolution with thiseluent system, use of a third column was suggested[49].

Another approach to circumvent this problem, basedin relative retention volumes (RRV), has been proposedby Delmonte et al. [15,50]. They observed that, by usingtoluene as a reference to approximate the dead volumein the Ag+-HPLC system and RA as a retention reference,the chromatographic data could be recalculated into areproducible format using the formula:

RRVi ¼ ðRVi � RVtolueneÞ=ðRVRA � RVtolueneÞ

where RRVi would represent the RRV of an individualCLA isomer.

This application is based on the assumption thatchanges in composition of the mobile phase will affectthe elution of all the CLA isomers in the same way.Expressing the elution of all the CLA isomers as theirRRV greatly helped to standardize each CLA isomer,resulting in coefficients of variation lower than 2% in awide variety of subtracts [50]. Ag+-HPLC chromato-grams, transformed using that equation are shown inFig. 5. Isomers cis-10 trans-12 and trans-10 cis-12 havethe same RRV. For a given position, from 6–8 to 9–11,the RRV of the trans-cis isomer is higher than that of thecorresponding positional cis-trans isomer, whereas, from11–13 to 13–15, the cis-trans isomers elute after thetrans-cis isomer.

As an alternative mobile phase, Delmonte et al. [50]assayed 2% acetic acid/hexane. It produced a different,but complementary, pattern of elution for CLA FAME.The advantages included discrimination of the two cis/trans 10–12 isomers and partial resolution for the cis-6trans-8 isomer from the trans-7 cis-9 isomer. Howeverthis procedure took longer for a chromatographic run.


Ag+-HPLC was adapted to analyze CLA as FFA, thuseliminating the need to methylate prior to assay[27,51]. Using mobile phases previously optimized forthe analysis of the methylated CLA, retention time wasexcessive and a competing acid was required. Forslightly shorter run times, a mobile phase of 2.5%acetic acid and 0.025% acetonitrile was chosen as theoptimum mobile phase for analysis. This proceduredemonstrated smaller RV and better resolution than theCLA FAME separation in a single ChromSpher 5 Lipidscolumn. Although we know that this methodology hasnot assayed in samples naturally enriched with FFA,such as ruminal fluid, it could be very useful for thistype of subtract.

Another alternative procedure to analyze CLA isomersby Ag+-HPLC was based on the conversion of fatty acidsto aromatic esters, especially phenacyl and p-meth-oxyphenacyl derivatives [52]. The results of thisapproach confirmed the beneficial effect of usingp-methoxyphenacyl esters for the separation of isomericCLA. Using only a single column, rather than multiplecolumns required by others, and a step-wise gradient ofhexane/dichloromethane/acetonitrile with UV detectionat 270 nm, it was possible to separate satisfactorilysaturated CLA, cis-monoenes and methylene-interruptedC18:2 in a single chromatographic run. Nevertheless,the non-specificity of this procedure is a drawback. Non-CLA esters that are present in the sample will be detectedas sensitively as CLA esters, so the sample will requireboth clean-up after ester preparation and isolation ofCLA esters from non-CLA esters prior to Ag+-HPLC bypreparative liquid chromatography.


Table 3 also summarizes the main Ag+-HPLCapproaches reported in the past 10 years.

5. Summary

Analysis of CLA can be simple or complex, depending onthe requirements of the analyst. The classical derivati-zation for GC-FID analysis of fatty acids by conversioninto FAME with alkaline catalysts allows determinationof the total CLA content in different subtracts. However,at present, there is a real need to determine CLA-isomerdistribution, since the isomers may have different bio-logical functions.

GC using 100-m highly polar capillary columns withMS is an invaluable adjunct to detect different CLA iso-mers. GC-MS of the DMOX derivatives permits the iden-tification of positional isomers of CLA with double bondslocated at carbons 6–8 to 13–15, whereas GC withacetonitrile CACI-MS/MS or FTIR could be used to con-firm double-bond geometrical configuration.

However, GC alone is not capable of separating theisomers of CLA known to occur in food and biologicalmatrices. Within the last decade, Ag+-HPLC with theacetonitrile:hexane elution system has become one of themost widely used complementary methods in CLAanalysis. This technique has played and will continue toplay a prominent role in isolating and separating indi-vidual CLA isomers.

The availability of new CLA standards and the appli-cation of RRV and/or an alternative eluent system toAg+-HPLC separations could greatly improve identifica-tions of CLA isomers in the future.

Acknowledgement

The authors acknowledge the financial support for theresearch project AGL2005-04727-C02-01 from theMinisterio de Educacion y Ciencia and the projectS-0505/AGR/000153 from the Comunidad Autonomade Madrid.

References

[1] M. Belury, Annu. Rev. Nutr. 22 (2002) 505.

[2] K.W. Lee, H.J. Lee, H.Y. Cho, Y.J. Kim, Crit. Rev. Food Sci. Nutr.

45 (2005) 135.

[3] M.W. Pariza, Am. J. Clin. Nutr. 79 (2004) 1132S.

[4] K.W.J. Wahle, S.D. Heys, D. Rotondo, Prog. Lipids Res. 43 (2004)

553.

[5] S. Tricon, G.C. Burdge, C.M. Williams, P.C. Calder, P. Yaqoob,

Proc. Nutr. Soc. 64 (2005) 171.

[6] P. Tanmahasamut, J. Liu, L.B. Hendry, N. Sidell, J. Nutr. 134

(2004) 674.

[7] K.L. Lai, A.P. Torres-Duarte, J.Y. Vanderhoek, Lipids 40 (2005)

1107.

[8] F.L. Hoch, Biochim. Biophys. Acta 1113 (1992) 71.

[9] J. Fritsche, H. Steinhart, Fett-Lipid 100 (1998) 190.

[10] J.A.G. Roach, M.M. Mossoba, M.P. Yurawecz, J.K.G. Kramer,

Anal. Chim. Acta 465 (2002) 207.

[11] R.O. Adlof, in: J.L. Sebedio, W.W. Christie, R.O. Adlof (Editors),

Advances in Conjugated Linoleic Acid, Vol. 2, American Oil

Chemists’ Society, Champaign, IL, USA, 2003, p. 37.

[12] G. Dobson, in: J.L. Sebedio, W.W. Christie, R.O. Adlof (Editors),

Advances in Conjugated Linoleic Acid, Vol. 2, American Oil

Chemists’ Society, Champaign, IL, USA, 2003, p. 13.

[13] J.K.G. Kramer, C. Cruz-Hernandez, Z. Dheng, J. Zhou, G. Jahreis,

M.E.R. Dugan, Am. J Clin. Nutr. 79 (2004) 1137S.

[14] C. Cruz-Hernandez, Z. Deng, J. Zhou, A.R. Hill, M.P. Yurawecz,

P. Delmonte, M.M. Mossoba, M.E.R. Dugan, J.K.G. Kramer,

J. AOAC Int. 87 (2004) 545.

[15] P. Delmonte, M.P. Yurawecz, M.M. Mossoba, C. Cruz-Hernandez,

J.K.G. Kramer, J. AOAC Int. 87 (2004) 563.

[16] M.M. Mossoba, Eur. J. Lipid Sci. Technol. 103 (2001) 624.

[17] M.M. Mossoba, M.P. Yurawecz, P. Delmonte, J.K.G. Kramer,

J. AOAC Int. 540 (2004) 540.

[18] A.L. Davis, G.P. McNeill, D.C. Caswell, Chem. Phys. Lipids 97

(1999) 155.

[19] M.S.F.L.K. Jie, Eur. J. Lipid Sci. Technol. 103 (2001) 594.

[20] F. Destaillats, P. Angers, Eur. J. Lipids Sci. Tech. 105 (2003) 3.

[21] P. Delmonte, J.A.G. Roach, M.M. Mossoba, K.M. Morehouse,

L. Lehmann, M.P. Yurawecz, Lipids 38 (2003) 579.

[22] P. Delmonte, J.A.G. Roach, M.M. Mossoba, G. Losi,

M.P. Yurawecz, Lipids 39 (2004) 185.

[23] K. Eulitz, M.P. Yurawecz, N. Sehat, J. Fritsche, J.A.G. Roach,

M.M. Mossoba, J.K.G. Kramer, R.O. Adlof, Y. Ku, Lipids 34 (1999)

873.

[24] J.K.G. Kramer, V. Fellner, M.E.R. Dugan, F.D. Sauer,

M.M. Mossoba, M.P. Yurawecz, Lipids 32 (1997) 1219.

[25] M. Yamasaki, K. Kishihara, I. Ikeda, M. Sugano, K. Yamada,

J. Am. Oil Chem. Soc. 76 (1999) 933.

[26] Y. Park, K.A. Albright, Z.Y. Cai, M.W. Pariza, J. Agric. Food Chem.

49 (2001) 1158.

[27] E. Ostrowska, F.R. Dunshea, M. Muralitharan, R.F. Cross, Lipids

35 (2000) 1147.

[28] ISO, International Standard ISO 15884-IDF 182, International

Organization for Standardization, Geneva, Switzerland, 2002.

[29] W.W. Christie, J.L. Sebedio, P. Juaneda, Inform 12 (2001) 1.

[30] J.K.G. Kramer, C.B. Blackadar, J. Zhou, J. Am. Oil Chem. Soc. 37

(2002) 823.

[31] J.A.G. Roach, M.P. Yurawecz, J.K.G. Kramer, M.M. Mossoba,

K. Eulitz, Y. Ku, Lipids 35 (2000) 797.

[32] J.K.G. Kramer, N. Sehat, M.E.R. Dugan, M.M. Mossoba,

M.P. Yurawecz, J.A.G. Roach, K. Eulitz, J.L. Aalhus, A.L. Schaefer,

K. Yu, Lipids 33 (1998) 549.

[33] P. Luna, J. Fontecha, M. Juarez, M.A. de la Fuente, J. Dairy Res. 72

(2005) 415.

[34] V. Spitzer, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer,

M.W. Pariza, G.J. Nelson (Editors), Advances in Conjugated

Linoleic Acid, Vol. 1, American Oil Chemists� Society, Champaign,

IL, USA, 1999, p. 110.

[35] M.M. Mossoba, R.E. McDonald, J.A.G. Roach, D.D. Fingerhut,

M.P. Yurawecz, N. Sehat, J. Am. Oil Chem. Soc. 74 (1997)

125.

[36] J.L. Sebedio, P. Juaneda, G. Dobson, I. Ramilision, J.C. Martin,

J.M. Chardigny, W.W. Christie, Biochim. Biophys. Acta 1345

(1997) 5.

[37] N. Sehat, J.K.G. Kramer, M.M. Mossoba, M.P. Yurawecz,

J.A.G. Roach, K. Eulitz, K.M. Morehouse, Y. Ku, Lipids 33

(1998) 963.

[38] M.P. Yurawecz, J.A.G. Roach, N. Sehat, M.M. Mossoba,

J.K.G. Kramer, J. Fritsche, H. Steinhart, Y. Ku, Lipids 33 (1998)

803.



[39] M.Y. Jung, M.O. Jung, J. Agric. Food Chem. 50 (2002) 6188.

[40] J.A.G. Roach, in: M.P. Yurawecz, M.M. Mossoba, J.K.G. Kramer,

M.W. Pariza, G.J. Nelson (Editors), Advances in Conjugated

Linoleic Acid, Vol. 1, American Oil Chemists� Society, Champaign,

IL, USA, 1999, p. 126.

[41] J. Fritsche, M.P. Yurawecz, R. Pawlosky, V.P. Flanagan,

H. Steinhart, Y. Ku, J. Sep. Sci. 24 (2001) 59.

[42] A.M. Michaud, M.P. Yurawecz, P. Delmonte, B.A. Corl,

D.E. Bauman, J.T. Brenna, Anal. Chem. 75 (2003) 4925.

[43] A.M. Michaud, P. Lawrence, R. Adlof, J.T. Brenna, Rapid

Commun. Mass Spectrom. 19 (2005) 363.

[44] J.T. Brenna, in: M.M. Mossoba, J.K.G. Kramer, J.T. Brenna,

R.E. McDonald (Editors), New techniques and application in lipid

analysis and lipidomics, American Oil Chemists� Society Press,

Champaign, IL, USA, 2006, p. 157.

[45] N. Sehat, M.P. Yurawecz, J.A.G. Roach, M.M. Mossoba,

J.K.G. Kramer, Y. Ku, Lipids 33 (1998) 217.

[46] R.O. Adlof, in: R.E. McDonald, M.M. Mossoba (Editors), New

techniques and application in lipid analysis, American Oil

Chemists� Society Press, Champaign, IL, USA, 1997, p. 256.

[47] N. Sehat, R. Rickert, M.M. Mossoba, J.K.G. Kramer,

M.P. Yurawecz, J.A.G. Roach, R.O. Adlof, K.M. Morehouse,

J. Fritsche, K. Eulitz, H. Steinhart, Y. Ku, Lipids 34 (1999) 407.

[48] R. Rickert, H. Steinhart, J. Fritsche, N. Sehat, M.P. Yurawecz,

M.M. Mossoba, J.A.G. Roach, K. Eulitz, Y. Ku, J.K.G. Kramer,

J. High Res. Chromatogr. 22 (1999) 144.

[49] A. Muller, K. Dusterloh, R. Ringseis, K. Eder, H. Steinhart,

J. Sep. Sci. 29 (2006) 358.


[50] P. Delmonte, A. Kataoka, B.A. Corl, D.E. Bauman, M.P. Yurawecz,

Lipids 40 (2005) 509.

[51] R.F. Cross, E. Ostrowska, M. Muralitharan, F.R. Dunshea, J. High

Res. Chromatogr. 23 (2000) 317.

[52] B. Nikolova-Damyanova, S. Momchilova, W.W. Christie, J. High

Resol. Chromatogr. 23 (2000) 348.

[53] P. Luna, M.A. de la Fuente, M. Juarez, J. Agric. Food Chem. 53

(2005) 2690.

Manuela Juarez is Research Professor of the Spanish Council for

Scientific Research (CSIC). She is a scientist, whose research is

focused on the development and the application of novel analytical

techniques for the determination of lipids in dairy foods. In the past

35 years, she has been working in dierent analytical aspects of food

science. She has published more than 150 scientific papers about

these subjects.

Miguel Angel de la Fuente received his PhD in Chemistry from the

Universidad Autonoma de Madrid in 1996. Currently, he is Scientist in

the Dairy Product Department of the Instituto del Frıo under the

supervision of Professor M. Juarez. He is involved in dierent research

projects concerning conjugated linoleic acid (CLA) analysis. In the past

15 years, he has been working in the field of dairy chemistry, mainly

devoted to analytical problems.

Pilar Luna joined Prof. Manuela Juarez�s group in 2002 and has

completed her PhD. She has worked on CLA analysis in dierent dairy

foods, applying several chromatographic techniques.

Documents

Chromatographic techniques to determine conjugated linoleic acid isomers