A practicalguide to

the analysisof conjugated

linoleic acid

w.w.ChriotM

This article is by WiJ/iam W. Christie,MRS Lipid A1Ialysis Unit, Scottish

Crop Research Institute, Inoergcnorie,Dundee (DD2 5DA), Scotland; and

Jean LOllis Sibedio and Pierre[uaneda, INRA, Unite de NutritionLipidique, 17 me Sllll)~ 8. fl. 86510,

21065 Dlion Cedex, France.

Instrumentation

There is increasing interest in conjugat-ed linoleic acid (eLA) because of itspotential therapeutic properties.Natural CLA is known to consist of sev-eral geometrical isomers of which themost abundant is 9-ds,ll-trans-octadecadienoic acid, formed by biohy-drogenation in the rumen. CommercialCLA is produced by alkaline isomeriza-tion of linoleate-rich oils, such as sun-flower seed oil, and tends to contain anequimolar mixture of 9-cis,ll-trans-and 10-trans,12-cis-octadecadienoicacids, together with variable amounts(bur up to 30%) of both geometricaland positional isomers. In addition,these isomers can be elongated anddesarurared in animal tissues by theenzymes involved in the biosynthesis ofarachidonic acid to produce conjugatedanalogs, which may even be responsiblefor the biological activity of CLA (1). Inanalyzing CLA, it is therefore importantthat we should be able to separate andquantify these geometrical and position-al isomers, avoiding additional isomer-ization during any derivarizarion steps.Analytical methodology is especiallyimportant now that it is recognized thatthe various isomers may have very dif-ferent effects in biological systems. Werecommend especially the review byBanni and Martin (2) and the manychapters in a recent AOCS monograph(3); these should be consulted for thor-ough coverage of the literature. The pre-sent discussion is a personal account ofmethods in use in the authors' laborato-ries with selected references only.

In considering the analysis of CLA, itis useful to treat the subject from twopractical view points. Commercial CLAusually is supplied as the free acid withthe components of interest being pres-ent at high levels, so analysis is relative-ly straightforward. In animal tissues,eLA is in the esterified form and is pre-

'"

sent at low levels in general, so concen-tration steps may be required for char-acterization and analysis. However,some steps may be common to bothaspects. Capillary gas chromatography(CC) with a column of the type used forthe analysis of trans fany acids, e.g.,CP-Sil 88 or BPX-70 (100 m), will formthe basis of the preferred approach,with GC-mass spectrometry (MS) asthe 4,4-dimethyloxazoline (DMOX)derivatives and/or 4-methyl-I,2,4-tria-zoline-3,5-dione (MTAD) adducrs beingan invaluable adjunct. Silver-ion high-performance liquid chromatography(HPLC) has proved very useful for theseparation of geometrical and position-al isomers. In addition, with naturalsamples, concentration of CLA isomersby reversed-phase HPLC and silver-ionchromatography must be considered,prior (Q CC or GC-MS analysis.

Perhaps the single most comprehen-sive method for commercial CLA prepa-rations has proved co be 13CNMR(nuclear magnetic resonance) spec-troscopy (4), which permits the identifi-cation and quantification of all the posi-tional (7,9- co 11,13-18:2) and geomet-rical isomers (cis,trans-, trans.cis-,cis.cis-, and trans,trans-) present in suchsamples. This is by far the most com-plete single analysis of CLA, but unfor-tunately the methodology requires sub-sranrial amounts of sample and is notlikely to be applicable to tissue extractsat natural levels.

The following methods have beenfound satisfactory for derivarizanonand for the various chromatographicsteps in the authors' laboratories.Others must consider the nature of theirsamples, how much information theyrequire, what precision is necessary, andhow much time and effort they candevote to the analysis before decidingwhich approach to adopt.

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Instrumentation

'"

Gas chromatographic analysisBefore farry acids are analyzed by GC,they must first be converted to methylesters. There is now a substantial bodyof work to confirm that acid-catalyzedmethylation is undesirable in general forthe preparation of methyl esters, as itcauses geometrical isomerization withan increase in the relative proportionsof trans.trans isomers. However, thereappear to be no significant drawbacksto the use of base-catalyzed rransesreri-fication of lipids. Free fatty acids can bemethylated on a small scale by means ofa phase-transfer catalyzed method. Onthe other hand, trimethylsilyl-dia-zornerhane, which is often recommend-ed, can produce artifacts (e. Fernie, per-sonal communication). Alternatively,despite the caveat regarding acidicmethylation, mild boron trifluoride(BF3~ or sulfuric acid-methanol reac-tions can be employed provided thatscrupulous attention is paid to detail.

Base-catalyzed methylation is recog-nized to be besr for esterified lipids asacid catalysis can cause isomerization ofCLA. The following method is recom-mended.

The lipid sample (up to 50 mg) is dis-solved in dry toluene (1 mL) in a test-tube, 0.5 M sodium rnerhoxide in anhy-drous methanol (2 mL) is added, andthe solution is maintained at 50°C for10 minutes. Glacial acetic acid (0.1 mL)is then added, followed by water (5mLI. The required esters are extractedinro hexane (2 x 5 mL), using a Pasteurpipette to separate the layers. The hex-ane layer is dried over anhydrous sodi-um sulfate and filtered, before the sol-vent is removed under reduced pressureon a rotary film evaporator. The sampleis dissolved in hexane (containing 50ppm burylared hydroxyrolene] for GCanalysis.

A longer reaction time is necessary

Volume 12 • febru.lry 200 I • Inform

for cholesterol esters containing CLA.Of course, with samples such as milk fatthat contain a high proportion of short-chain Iarry acids, it is advisable to usemodified methods to minimize the lossof butyric and hexanoic acids, especial-ly IS).

Free acids are best methylated on asmall scale by means of a phase-transfercatalyzed method, or by a mildBF3-methanol reaction (6), althoughsulfuric acid-methanol (1 'Yo) can beused in a similar way with care (e.Fernie, personal communication).

Phase transfer method. The sample(10 mg) is dissolved in dichloromerhane(1 mL) in a culture tube (125 x 16 mm)with a polytetrafluorethylene-lined cap(PTFE). To this is added 0.1 M tetra-butylammonium hydrogen sulfate in0.2 M aqueous sodium hydroxide (ImL) and methyl iodide (25 ilL). Aftermixing and continuous shaking for 60minutes, the layers are allowed to settle.The lower layer is taken by means of aPasteur pipette, and the solvent is evap-orated in a stream of nitrogen at 30°C.

BF3-methallol. The free fatty acid(up to 10 mg) is reacted with boron-trifluoride-methanol (1 ml.; 14%) for10 minutes at ambient temperature. Theesters are extracted into hexane (2 mL)and washed by water (2 x 5 mLI. Theupper phase is dried over anhydroussodium sulfate, then the solvent is evap-orated without heating in a stream ofnitrogen.

Capillary columns of the Carbowaxtype are the standard in most laborato-ries for routine analysis of farry acids,but they are of limited value for theanalysis of CLA. The two main compo-nents, 9-cis, II-tralls- and 10-trOlIS,I2-cis-18:2, are well separated from eachother; but not from other positional iso-mers. The various types of geometricalisomers give distinct peaks, but within

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Fisurt! I. Gas chromatosraphic separation of con]usated linolenic acid methyl esters on aBPX70column (120 m x 0.25 mm;0.25 prn film thickness; SGE ltd., Melbourne,Austnlia).The carrier sas was hydrogen (linear las velocity at leO'C" 15.2 cmfs).SpUtiessinjection was employed, and the column temperature was programmed: 60·C forI minute, then increased at lO-C/min to 170'C and held at this tempen.ture for a furtherSOmin.

these groups, positional isomers are notfully resolved, unfortunately. For rea-sons rhar are not at all clear, with allpolar phases. cis,trans- elute beforecis,cis- before tranS,trans- isomers. Evenwith these limitations, a single chro-matographic run with almost any polarstationary phase will at least give anapproximate figure for the total contentof CLA relative to other components. Itmay be worth noting that lipid analystsappear to demand much higher stan-dards of precision than is possible inmost fields of biochemistry and nutri-tion.

As mentioned above. long columnsof the rype favored for analysis of trans-monoenoic fatty acids are required forresolution of CLA isomers, and goodseparations have been reported for CP-S1188 and BPX-70 columns; an exampleof rhe larrer is illustrated in Figure 1.CLA isomers elute long after nonccnju-gated dienes, and the important posi-tional isomers emerge in the order

9c,IIt < 8t,lOe < lle,13t < lOt, I2e.The authors have observed that it is noreasy to obtain such separations repro-ducibly, and in particular the separationof the first two isomers is rarely easy.

There is little to confuse analysis ofcommercial CLA samples, but whenCLA is fed in nutritional experiments,other fatty acids may be present in tis-sues that confuse the picture. For exam-ple, 2 LO or 20:2 fatty acids may occurnaturally and they elute in the sameregion, of the chromatogram as thecis,cis- and trans,trans-isomers, espe-cially. GC-MS may then be useful, bothto locate the double bonds and to iden-tify any nonconjcgared fatty acids thatco-chromatograph with those of inter-est. Several types of nitrogen-containingderivatives have been developed forGC-MS in general, and of these,DMOX derivatives have proved themost useful for conjugated dienes(reviewed by Spitzer in Reference 3).They are prepared by heating the fatty

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acid or lipid derivative with 2-amino-2-methylpropanol as follows.

To the lipid sample (up to 2 rng] in a[CSt tube is added 2-amino-2-methyl-l-propanol (0.25 g). The vessel is flushedwith nitrogen, stoppered, and placed ina heating block, at 190DC overnight. Oncooling, diethyl ether/hexane (I: I,voUvol; 5 ml.) is added to the rube, fol-lowed by water (5 ml.]. The organiclayer is washed with distilled water (3rnl.I and dried carefully over anhydroussodium sulfate. The sample is taken 10

dryness in a gentle stream of nitrogen at30DC, and is dissolved in a small volumeof hexane for GC-MS analysis. A fewcrystals of anhydrous sodium sulfatehelp to stabilize the derivatives.

DMOX derivatives can be subjectedto GC analysis under similar conditionsto methyl esters, and [hey afford com-parable resolution. However, in view ofthe harsh conditions required for prepa-ration of the derivatives, we suggest thata rigorous test of the methodology withpure standards be required if they are tobe used quanrirarively,

A valuable alternative consists in theuse of a Diels-Alder reaction to preparethe MTAD adducts of the conjugatedfatty acids. The reagent reacts almostinstantaneously with the conjugateddouble bond system, and the adducrshave excellent mass spectrometric prop-erties that enable location of the conju-gated double bonds, as illustrated inFigure 2 for the MTAD adduct ofmethyl 9,ll-octadecadienoate. The ionsat mix = 250 and 322 represent cleavageon either side of the six-membered ringthat contains the carbons of the originalconjugated double bond system.

The reaction is carried out as fol-lows: The CLA methyl ester (220 pg;'1.15 mM) and MTAD (425 IJg; 5.8mM) in dichlorornerhane (650 IJL) aremixed in a test-tube at O°C by agitating

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150

Instrumentation

Abundance ""• ~32290250~

eoCH:jCH21~{CH21~OOCH:Jzc

N-N

ec o",z)..o 322

eo N zso,"

CH,

30 W

'"20 T 3rto 'I' I. ~",1': '" Helf '" 'f. cos ":'. ,so "" >5" 200 "" ooc "" '00 ...

Fllure 1. Mus spectnlm of the HTAD ..dduct of methyl 9,II-octadecadlenooate (HTAO •".methyl_I.l ..._tri~oline_].5.oione).

for less than 10 seconds. The reaction isslopped immediately by addition of 1,3-hexadiene, followed by agitation for afew seconds. Excess reagents areremoved by a stream of nitrogen at30°C, and the sample is redissolved indichloromethane for analysis byGC-MS.

By means of GC-MS with selectiveion monitoring, excellent results havebeen obtained with commercial eLAthat complemented the NMR results(4), On the other hand, with eLA atnatural levels in tissues, it is advisable toobtain a concentrate before applyingthe dcrivarizurion procedure, as we havesometimes observed some nonspecificreaction with polyunsaturated fattyacids.

NOT only have these methods beenused to characterize conjugated dienesin CLA samples, but they have alsobeen employed to prove the structuresof CLA metabolites formed in tissues,such as 5,8,11,13- and 5,8,12,14-eicosarerraenoic acids (1).

Silver-ion chromatographyIn silver-ion thin-layer chromatography(TLC), conjugated dienes tend to elute

Volume 12 • February 200 I - Inform

close to monoenes. Much better resolu-tion is possible by silver-ion HPLCusing columns packed with ion-exchange media loaded with silver ions.There have been three approaches to theproblem, but these have not been com-pared objectively in the laboratory so itis not yet possible to state which is best.

The first approach was pioneered byAdlof, but then greatly improved byYurawecz, Kramer, and colleagues, whoreviewed the topic in Reference 3. Theyutilized a mobile phase of hexane con-taining a small amount of acetonitrile toseparate the methyl ester derivatives,using the specific ultraviolet (UV)absorbance of the conjugated doublebonds for detection and quantification.By coupling as many as six columns inseries (though two were adequate formost practical purposes), some remark-able separations were achieved, both ofpositional and geometrical isomers.Trans,trans-isomers eluted first, fol-lowed by cis, trans then cis.cis, andwithin each group many positional iso-mers were dearly resolved. As an exam-ple, Figure 3 illustrates a sepnrarion of acommercial CLA sample. In thisinstance, the peaks were identified by

collecting them and converting each tothe MTAD adduct for GC-MS.

The second approach consisted inthe separation of CLA isomers as the p-methoxyphenacyl esters with di-chlorometha ne/hexa ne/aceton it ri Iemixtures as mobile phase (7). In thisinstance, only a single chromato-graphic column was required. Asdetection was by the absorbance ofthe p-methoxyphenacyl moiety at 270nm, all farry acids were detected and quan-tified, not simply the conjugated dienes.

Finally, good resolution of CLA asthe free acids has recently been repon-ed, with hexane/acetonitrile/acetic acidmixtures as the mobile phase with spe-cific detection of the conjugated doublebonds at 234 nm (8). This proceduremay be of special value for commercialCLA samples, supplied as the free acids,since no derivarization step is required.

Reversed-phase HPLCwith second-derivative UV detectionConjugated dienes exhibit a distinct UVabsorbance in the region of 230-135nrn, while isolated double bonds absorbat 206-210 nm. However, the latter caninterfere with the analysis of CLA iso-mers in tissues, since they tend to bepresent at such low levels. Banni andcoworkers were able to overcome thisproblem by taking the differential of thefirst derivative spectrum, thus calcular-ing a second derivative with rwo distinctpeaks with minima at 234 and 242 nm.This gave a much more sensitive andaccurate estimate of the conjugateddiene content of fatty acids, since theBeer-Lam ben law is unaffected by dif-ferentiation. When combined withreversed-phase HPLC it was possible toseparate and quantify the metabolites ofCLA as well as CLA per se (2). Thetechnique has also been used in serieswith MS (6).

Concentration of naturalCLA isomers for further analysisIn order to have sufficient material foranalysis with tissue samples containingCLA at low levels, a preconcenrrarionstep may be required. This can beaccomplished either by reversed-phaseHPLC or by silver-ion chromatographyor better, by using the two techniquessequentially.

In reversed-phase chromatography,CLA isomers elute close to linoleate andcan be collected preparatively by collect-ing a single broad CI8 diene fraction.Many analysts add water to the mobilephase or use acetonitrile/water gradi-ents. However, we prefer to use acetoni-trile alone, either at a constant flow rareor with a flow gradient, as this makes iteasier to recover the required esters byevaporation of the mobile phase. Mostcolumns of the ocradecylsilyl (ODS) typecan be used, but one of us employs base-stabilized Hichrom RPB [Hichrom Lrd.,Reading, United Kingdom), as it canalso be used with DMOX derivativesand picolinyl esters (but nor free fattyacids) (9). For methyl esters a more con-ventional ODS phase, such as NucleosilC18 (Hypersil ThermoQucst, Lcs Ulis,France), is suitable. Evaporative light-scattering detection with a stream split-ter can be used, or UV detection ar 206(isolated double bonds) or 230 nm (con-jugated double bonds), or refractiveindex detection. Keeping the columntemperature constant aids reproducibili-ty, bur is not essential. On 3 standardanalytical column (4.6-mm diam.),about 1 mg of sample C3n be separatedin micro-preparative mode, but up to 20mg can be chromatographed on apreparative column (lO-mm diam.), asillustrated in Figure 4.

Silver-ion TLC and HPLC methodsare available to obtain a concentrate ofeLA, but a simple solid-phase exrrac-

IS'

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.. ~--Figure). Silve ...ion chromatography of a commercial conjugated linolenic: add mixture(Sigma.Aldrich Inc.). Two Chroms.pher lipids columnl (150 x 4.6 mm i.d.; Varian Inc.)were used in 5eries wtth a mobile phue of hexane/acetonitrile (99.9:0.1, vollvol) at a nowrate of I mUmin, with ultraviolet detection at 234 nm (Source: Dobson, G.,/.Am. 011Chem. Soc. 15:1)1-142, 1998).

0

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0 , , , , , , ,.s , , , , , , ,, , , , , ,, , , , , ,0

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u u u • u _ u _--F1 :: 22:6, 20:5; F2:: 22:5, 20:4, 18:3; F3:: 22:4, 20:3, 18:2, CLA,16:1, 14:0;F4:: 20:2,18:1,16:0; F5:: 18:0

Figure 4. Reversed·phase leparation of a fatty acid methyl elten, including CLA. ANudeosil C 18 column (250 • 10 mm I.d.; 5 ...m particlel) was used with acetonitrile asmobile phue, and ultraviolet detection at 234 nm. Methyl esten (20 mg) in acetone wereinjected, with acetonitrile as mobile phue and a now rate of 4 mUm;n. The fractiOfl COf'-responding to the cia dienes may also contain some 14:0, 16: I, and certUn polyunsatu.rated fatty adds (Source:Jtl;l(leda., P., and J.L 5ebedlo, J. Chromcrtov. 8 124:21l-219, 1999).

Volume 12 - February 200 I - inform

152

rion method adapted from a publishedprocedure can be recommended (10). Itis conveniently applied to the C 18 dienefraction isolated by reversed-phaseHPLC as above.

An Isolute sex solid-phase extrac-tion column (500 rug] (InternationalSorben Technologies Lrd., Hegoed,United Kingdom) or equivalent,wrapped to the level of the top of theadsorbent bed in aluminum foil, ispreconditioned by elution with ace-tonitrile (2 mL), A solution of silvernitrate (20 mg) in aceronirrile/warer(0.25 mL; 10: I, vol/vol) is thenallowed [Q percolate through it. Thecolumn is flushed with acetonitrile (5mL), acerone (5 mL), anddichloromerhane (10 mL) by applyingslight pressure from a pipette bulb,and is then ready for use. The methylester sample (0.1 to 0.25 mg) IS

applied to the column In

dichloromethane (0.1 ml) and iswashed onto the column with a similarvolume of fresh solvent. Solvent mix-tures are allowed to flow under gravi-ty (approximately 0.5 mLlmin).Saturated fatty acids arc eluted withdichloromerhane (5 ml), while theClNmonoene fraction is eluted withdichloromethane/acetoue (9:1, vol. vel,5 ml). Linoleare remains on the col-umn. Fractions are collected manually,and they C3n be analyzed by GC afterevaporation of the solvent.

Instrumentation

Dairy Cows,]. Nlltr. 129:1579-1584(19991.

6. Banni, S. ItW. Day, R.W. Evans, EP.Corongui, and B. Lombardi, liquidCh r o rn a r o g r a ph ic-Ma s sSpectrometric Analysis of ConjugatedDiene Fatty Acids in a PartiallyHydrogenated Fat,]. Am. Oil Chem.Soc. 71:1321-1325 (1994).

7. Nikolova-Damyanova, a., S.Momchilova, and W.W. Christie,Silver-Ion High-Performance liquidChromatographic Separation ofConjugated Linoleic Acid Isomers,and Other Fatty Acids, AfterConversion to p-Methoxyphen3cylDerivatives, J. High Resolut,Chromatogr. 23:348-352 (2000).

8. Cross, R.E, E. Osrrowska, H.Muralitharan, and ER. Dunshee,Mixed Mode Retention and the Useof Competing Acid for the Ag+-HPlC Analysis of UnderivatizedConjugated linoleic Acids, l- HighReeolut. Cbromatogr. 23:317-323(20001·

9. Christie, W.W., GasChromatography-Mass Spect-rometry Methods for StructuralAnalysis of Farry Acids, Lipids33:343-353 (1998).

10. Christie, W.w., Silver-IonChromatography Using Solid-PhaseExtraction Columns Packed with aBonded-Sulfonic Acid Phase, l-Lipid Res. 30:1471-1473 (1989).0

AcknowledgmentThis work has been funded in pan bythe Scottish Executive Rural AffairsDepartment, and in parr by the EU pro-ject No. FAIR 3671.

ReferencesI. Sebedio, J.L, P. juaneda, G. Dobson,

L Ramilison, j.C. Martin, J.M.Chardigny, and W.W. Christie,Metabolites of Conjugated Isomersof Linoleic Acid (CLA) in the Rat,Biochim, Biophys. Acta 1345:5-10(1997).

2. Banni, S., and j.-c. Martin,Conjugated linoleic Acid andMetabolites, in Trans Fatty Acidsin Human Nutrition, edited by j.LSebedio and W.W. Christie, OilyPress, Dundee, 1998, pp. 261-302.

3. Yurawecz, M.P., M.M. Mossoba,].K.G. Kramer, M.W. Panza, andC.]. Nelson, Advances inConjugated Linoleic Acid Research,Vol. 1, AOCS Press, Champaign,1999.

4. Davis, A.L, G.P. McNeill, and D.C.Caswell, Analysis of ConjugatedLinoleic Acid Isomers by C-13NMR Spectroscopy, Chem. Phys.Lipids 97:155-165 (1999).

5. Chouinard, nv, L. Comeau, D.M.Barbano, L.E. Metzger, and D.E.Bauman, Conjugated Linoleic AcidsAlter Milk Fatty Acid Compositionand Inhibit Milk Fat Secretion in

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