Journal of the American Oil Chemists' Society

, Volume 90, Issue 3, pp 327–335

Analysis of CLA Isomer Distribution in Nutritional Supplements by Single Column Silver-Ion HPLC


    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
  • L. Giua
    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
  • G. Lombardi
    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
  • M. S. Simonetti
    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
  • P. Damiani
    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
  • F. Blasi
    • Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e NutrizioneUniversità degli Studi di Perugia
Original Paper

DOI: 10.1007/s11746-012-2176-x

Cite this article as:
Cossignani, L., Giua, L., Lombardi, G. et al. J Am Oil Chem Soc (2013) 90: 327. doi:10.1007/s11746-012-2176-x


Nutritional supplements containing conjugated linoleic acid (CLA) isomers, widely used to improve body composition and inhibit fat storage, should be thoroughly analyzed, as CLA effects are isomer specific. In this research, an improvement of silver-ion high performance liquid chromatography (Ag+-HPLC) was obtained by using a single-column instead of the most commonly used multi-columns. To develop the procedure, a standard CLA mixture has been derivatized with different chain length alcohols (from methanol to hexanol). Hexyl CLA showed better separation between CLA isomer pairs, in particular the resolution of the t10,c12: c9,t11 pair increased from 0.3 (methyl esters) to 0.8 (hexyl esters). Therefore, the CLA isomer composition of some commercial CLA products was determined by Ag+-HPLC analysis of hexyl esters. The main isomers in all supplements turned out to be c9,t11-CLA and t10,c12-CLA, the most anti-adipogenic isomer. t8,c10- and c11,t13-CLA were not detected. The nutritional supplements were also analyzed by high resolution gas chromatography of methyl esters to evaluate the fatty acid % composition and total CLA % content. The total CLA ranged from 79.4 to 94.4 %, and the t,t isomer from 1.4 to 7.3 %.


Nutritional supplementsConjugated linoleic acidHexyl estersAg+-HPLCHRGC


It is known that conjugated linoleic acid (CLA) isomers possess biological activity [1]. A great number of studies have shown the beneficial health effects of CLA isomers in animals and humans; it has been demonstrated that the effects of CLA may be isomer dependent and that c9,t11-CLA and t10,c12-CLA, the two main CLA isomers, have different effects on blood lipids and on metabolism in adipocytes. Both c9,t11-CLA and t10,c12-CLA have shown anti-carcinogenic [2] and anti-diabetogenic activities [3]. The t10,c12 isomer had in addition anti-adipogenic properties [4], in fact it turned to be more effective than the c9,t11 isomer in reducing incorporation of fatty acids (FA) into cell triacylglycerols (TAG). Besides t10,c12-CLA showed a greater potency in changing mice body composition and in reducing the expression of stearoyl-CoA desaturase in cultured 3T3-Li adipocytes [5]. However, it was also reported that dietary t10,c12-CLA isomer induces hyperinsulinemia and fatty liver in mice [6].

In any case, only a limited number of CLA supplementation studies have been conducted on humans, and the reported results are variable [7]. Some published human studies found small, but significant reductions in body fat with CLA supplementation. Other published human studies found no change in body fat, rather CLA supplements increased the risk factors for diabetes and cardiovascular diseases.

Endogenous production of CLA isomers by humans is very limited; therefore, a very large proportion of CLA found in the body tissue is from dietary origin, essentially meat and dairy products [8].

Ip et al. [9] estimated that a 70-kg man should consume 3.0 g of CLA/day to achieve maximum health benefits. At present, there is great interest in CLA as nutritional supplements and different products are now commercially available as free FA, alkyl esters [10] or TAG mixtures. It has been reported that the regain of fat-free mass was favorably and dose-independently affected by a 13-week consumption of 1.8 or 3.6 g CLA/day and consequently the resting metabolic rate was increased [11].

The c9,t11 and t10,c12 CLA are the two main isomers present at similar levels in commercial CLA preparations, showing only traces of c,c and t,t isomers with unsaturations in 9–11, 10–12, 11–13 positions [12].

In relation to this, it is therefore important to have reliable and precise techniques for identification and quantification of particular CLA isomers in food and nutritional supplements. Use of the tandem-column Ag+-HPLC method led to an enhanced chromatographic resolution of the CLA isomers in a commercial mixture [13]. Three silver-ion high performance liquid chromatography (Ag+-HPLC) columns in series appeared to be the best compromise to obtain satisfactory resolution of most CLA isomers found in natural products, such as beef and milk fat [14, 15]. Because of the multiplicity of geometrical and positional CLA isomers, complete separation and accurate analysis of complex mixtures are needed. A combination of high resolution gas chromatography (HRGC) and Ag+-HPLC was found to be necessary to resolve all CLA isomers [16].

Several researchers investigated the effects of the nutritional supplements [11, 17, 18], but only few researchers analyzed these products. Yu et al. [19] analyzed some nutritional supplements using HRGC analysis coupled with a flame ionization detector (FID). Other authors [20] analyzed commercial CLA products for their CLA content and isomer composition using GC, Ag+-HPLC and spectroscopic techniques. Two important quality issues for nutritional CLA products are the total CLA content, and the CLA isomer distribution.

In the present study, CLA isomer separation by Ag+-HPLC has been developed by derivatization with different chain length alcohols (from methanol to hexanol). Successively, six nutritional CLA supplements were analyzed as hexyl esters in order to determine the isomer distribution. CLA supplement samples have been also analyzed by HRGC.

Experimental Procedures


A mixture of CLA isomers (catalog nO5507, linoleic acid ≤1 %), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD, 95 %), CLA as fatty acid methyl esters (FAME) (catalog nO5632, linoleic acid methyl ester ≤1 %), n-propanol, n-pentanol, n-hexanol, 1,3-hexadiene, glyceryl trilinoleate (catalog nT9517, ≥98 %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dichloromethane was from BDH (Poole, England). Methanol, ethanol, diethyl ether and n-hexane (analytical and HPLC grade) were purchased from Panreac (Barcelona, Spain). n-Butanol, n-pentane, petroleum ether and sulfuric acid were from JT Baker (Deventer, Holland). Sodium sulfate anhydrous was purchased from Riedel-de Haën (Seelze, Germany). Formic acid was purchased from Carlo Erba (Milan, Italy). Acetonitrile of HPLC grade was from Laboratory Supplies-England.


Six nutritional CLA supplements (S1–S6) in soft gel capsules were purchased online. They were stored in a cool dry place.

The composition of each nutritional supplement, from a lipid point of view, reported on the packaging, was the following:

Safflower oil, glycerin, water, caramel, gelatin; produced in USA


CLA, glycerin, purified water, natural tocopherol mixture, gelatin; produced in EU


Converted safflower oil, glycerin, flavorings, anti-oxidants (ascorbyl palmitate, α-tocopherol), gelatin; produced in USA


CLA titrated at 95 % from sunflower and saffron oils, capsule (gelatin, glycerin, titanium dioxide); produced in Canada


CLA, glycerin, anti-oxidant E306, gelatin. Saturated FA 0.28 g, monounsaturated FA 0.48 g, polyunsaturated FA 3.24 g, total CLA 3.20, t10,c12 + c9,t11 2.96 g; produced in Spain


Vegetable oil, capsule (gelatin, glycerin, separating agent: silicon dioxide, water); produced in Germany


Thin Layer Chromatography (TLC) Analysis of Six Nutritional CLA Supplements

The S1–S6 CLA supplements (10 mg/mL in hexane), together with the following reference compounds, CLA as free FA, CLA as FAME and glyceryl trilinoleate, were spotted on silica gel plates (SIL G-25, 0.25 mm, 20 × 20 cm, Macherey–Nagel, Germany). A mixture of petroleum ether/diethyl ether/formic acid (70:30:1, v/v/v) was used as the developing solvent, then the TLC plates were visualized with iodine vapor.

FAME Preparation and HRGC-FID Analysis of Nutritional CLA Supplements

The S1–S5 CLA supplements (200 μL, 10 mg/mL) were esterified in the presence of methanol (200 μL), pentane (1 mL) and sulfuric acid (10 μL). The reaction was carried out at 50 °C for 30 min. After esterification reaction, the samples were washed five times with water to remove acid residues. The organic phase was dried over anhydrous Na2SO4 and then concentrated under a nitrogen stream [21]. The adopted methylation method was adequate because no isomerization phenomena have been reported [22].

The S6 CLA supplement (200 μL, 10 mg/mL in hexane) was transesterified using methanolic KOH as reported in a previous paper [21].

FAME obtained from S1 to S6 supplements were dissolved in n-hexane (500 μL) and analyzed by HRGC-FID. Chromatograms were obtained with a Dani GC1000 gas-chromatograph (Norwalk, CT, USA) equipped with a split/splitless injector port and a FID. The fused silica WCOT capillary column CP-Select CB for FAME (50 m × 0.25 mm i.d., 0.25 μm film thickness, Varian, Superchrom, Milan, Italy) was used. The chromatograms were acquired and processed using Clarity integration software (DataApex Ltd., Prague, Czech Republic). The chromatographic conditions were the following: the injector and detector temperature was 250 °C; the oven temperature was 180 °C, held for 6 min then increased to 250 °C at 3 °C/min; the final temperature was held for 10 min. The carrier gas (He) flow rate was 1.0 mL/min.

Fatty acids were identified by comparing the retention times of their methyl esters with the standard FAME mixture. Repeated injections of standard solutions were carried out to test the analytical precision. The relative standard deviations (RSDr %) were less than 5 % for all the FA, both considering the intra-day precision, calculated on 5 repeated injections, and the inter-day precision, evaluated over 5 days.

The percentage of each FA was calculated using the peak area corrected with the response correction factors, as reported by Christie [21]. Each HRGC-FID analysis was carried out in triplicate.

Esterification of Standard CLA Mixture

The standard CLA mixture (100 μg) was subjected to sulfuric acid esterification by reaction with 100 μL of six different alcohols (methanol, ethanol, n-propanol, n-butanol, n-pentanol and n-hexanol) in the presence of sulfuric acid (10 μL). To obtain methyl, ethyl and propyl CLA esters, the reactions were carried out in pentane (1 mL) at 50 °C for 1 h. To obtain butyl, pentyl and hexyl CLA esters the reactions were carried in pentane (1 mL) at 65 °C for 15 min. After the esterification reaction, the samples were washed five times with water to remove acid residues, anhydrificated with Na2SO4 anhydrous, dried and then dissolved in n-hexane.

All alkyl esters were analyzed by Ag+-HPLC as reported in the following paragraph.

Ag+-HPLC Analysis

CLA alkyl esters from the standard mixture and FA hexyl esters obtained from the S1 to S6 supplements were dissolved in n-hexane and then 20 μL samples were analyzed by Ag+-HPLC.

The HPLC analysis was carried out using a Shimadzu LC-10AD VP liquid chromatography pump (Kyoto, Japan), a silver-impregnated ChromSpher 5 Lipids column (5 μm, 250 × 4.6 mm i.d.; Varian, Milan, Italy) and a Spectra System UV 6000LP detector (Thermo Separation Products, San Jose, CA), operating at 232 nm. The column temperature was maintained at 20 °C, in a Shimadzu CTO-10AS column oven. The chromatograms were acquired and processed using Class-VP software (Shimadzu). The Rheodyne injector (7725i Model; Rohnert Park, CA, USA) had a 20 μL injection loop. The samples injected into the column were subjected to isocratic elution (0.6 mL/min) using a mobile phase, freshly prepared, composed of 0.1 % acetonitrile in n-hexane. Each HPLC analysis was carried out in triplicate.

Validation of the Ag+-HPLC Method

To evaluate the response linearity parameters, the CLA standard derivatized as hexyl ester at four different concentrations (0.002–0.016 μg/μL) was injected in triplicate. Calibration lines for each of the four analytes (c11,t13, t10,c12, c9,t11 and t8,c10 CLA isomers) were constructed and the least square method was used to calculate the regression equations. The detection (LOD) and the quantification (LOQ) limit values were estimated according to the standard deviation response and slope method [23].

To perform the repeatability assay, the hexyl ester CLA standard solution (0.016 μg/μL) was analyzed five times on the same day and five times on different days.

Fatty Acid Hexyl Ester Preparation of Nutritional CLA Supplements

The S1–S5 CLA supplements (2 mg) were subjected to sulfuric acid esterification by the reaction with n-hexanol (200 μL) and sulfuric acid (10 μL). The reaction was carried out in pentane (1 mL) at 65 °C for 15 min, as reported in the previous section.

The S6 CLA supplement was transesterified with hexanolic KOH under the same experimental conditions reported in a previous paper [21].

An aliquot of the final products, FA hexyl esters obtained from S1 to S6 supplements, was analyzed by Ag+-HPLC as reported in the previous paragraph and an aliquot was further derivatized and analyzed by HRGC-MS (mass spectrometry) as reported in the following section.

HRGC-MS Analysis of Hexyl Ester MTAD Adducts of Nutritional CLA Supplements

The preparation of hexyl ester MTAD adducts of nutritional CLA supplements was carried out following the procedure reported by Reaney et al. [24], with minor changes. In brief, FA hexyl esters (40 μg) were mixed with MTAD (65 μg) in dichloromethane (100 μL) at 0 °C under magnetic stirring for 40 s. Then the reactions were immediately stopped by addition of 1,3-hexadiene (0.5 μL), followed by agitation for a few seconds. The mixture was dried under nitrogen and dissolved in dichloromethane. The MTAD adducts were analyzed by HRGC-MS as reported in a previous paper [25].

Statistical Analysis

Three capsules of each supplement were opened and the contents mixed. The results of HRGC and Ag+-HPLC analyses are the means of three parallel solutions of each sample with the respective standard deviation.

The values of the repeatability assay (precision of the analytical method) are expressed as RSDr %.

Results and Discussion

Analysis of CLA Supplements by TLC

It is extremely important that the composition label, reported on supplement packaging, is detailed in order to provide nutritional information to consumers, who, consequently, are then in a position to make the right purchasing decision. Among the ingredients written on the packaging, only some supplement labels (S2, S3 and S5) reported the presence of anti-oxidants, which are very important compounds taking into account the presence of polyunsaturated FA. Moreover, only the S6 supplement label reported the following caption “Might contain traces of soy, gluten, milk and eggs”, information needed to avoid allergic reactions.

Since the ingredients reported on the label of the considered CLA supplements were not clearly stressed, the first step of this research was a TLC analysis to evaluate if the CLA isomers were present as free FA, simple esters or TAG. This is valuable information considering the absorption processes of the different chemical forms [26]. The TLC analysis showed that only the S6 supplement contained TAG, while all the other supplements (S1, S2, S3, S4 and S5) contained CLA as free FA. In some cases, this information was not in agreement with that on the nutritional label, in particular some supplements (S1 and S3) contained free FA, while vegetable oils were indicated as ingredients.

Fatty Acid Analysis of Six Nutritional Supplements by HRGC-FID

The most commonly used method for FA analysis is the derivatization to methyl esters and the successive HRGC analysis. Unfortunately, this procedure permits only the partial separation of the CLA isomers, even if it is used for the quality control of these compounds.

In order to evaluate the FA % composition, in this research six nutritional supplements, after derivatization to the relative FAME, were analyzed by HRGC. The S1–S5 supplements were esterified, the S6 supplement was transesterified. Table 1 shows the total (% mol) acidic compositions of the six nutritional supplements, as well as the results of CLA main isomer % contents. The CLA % content (mg/100 mg total FA) ranged from 79.4 % (S6 supplement) to 94.4 % (S4 supplement). In all considered supplements, t10,c12 and c9,t11 were the main CLA isomers. The t,t isomer contents ranged from 1.4 % (S6 supplement) to 7.3 % (S3 supplement). The differences can be due to the starting vegetable oil and to the production method of the supplements.
Table 1

Fatty acid composition (% mol) of six nutritional CLA supplements analyzed by HRGC-FID analysis










1.4 ± 0.3

3.6 ± 0.1

3.4 ± 0.2

0.2 ± 0.0

4.2 ± 0.5

4.1 ± 0.3


2.1 ± 0.1

2.1 ± 0.0

2.2 ± 0.1


2.3 ± 0.1

2.6 ± 0.0


14.4 ± 0.1

11.4 ± 0.0

11.4 ± 0.1

4.9 ± 0.3

11.7 ± 0.1

12.4 ± 0.2


0.4 ± 0.1

1.1 ± 0.0

0.9 ± 0.1

0.6 ± 0.0

1.1 ± 0.1

1.1 ± 0.0


0.1 ± 0.1

0.4 ± 0.1

0.4 ± 0.1


0.3 ± 0.1

0.4 ± 0.0


38.7 ± 0.3 (47.4)

37.1 ± 0.1 (45.6)

34.3 ± 0.2 (41.9)

44.1 ± 0.1 (46.7)

37.0 ± 0.0 (46.0)

36.5 ± 0.1 (46.0)


39.1 ± 0.5 (47.9)

39.9 ± 0.3 (49.0)

37.3 ± 0.0 (45.5)

46.5 ± 0.4 (49.3)

39.6 ± 0.1 (49.2)

40.5 ± 1.7 (51.0)


2.0 ± 0.3

2.2 ± 0.2

3.0 ± 0.3

2.2 ± 0.1

2.1 ± 0.3

1.0 ± 1.4


1.9 ± 0.5

2.2 ± 0.0

7.3 ± 0.3

1.6 ± 0.0

1.8 ± 0.3

1.4 ± 0.1

Total CLA







The results were obtained using a CP-select CB for the FAME column (50 m × 0.25 mm i.d., 0.25 μm film thickness); oven temperature program, 180 °C (6 min), 3 °C/min to 250 °C (10 min); He flow rate, 1.0 mL/min

16:0, palmitic acid; 18:0, stearic acid; 18:1n-9, oleic acid; 18:2n-6, linoleic acid; 18:3n-3, α-linolenic acid; c9,t11-CLA, cis9,trans11-CLA isomer; t10,c12-CLA, trans10,cis12-CLA isomer. Total CLA: sum of c9,t11-CLA, t10,c12-CLA, c,c-CLA and t,t-CLA isomers

The results are the means of three parallel solutions of each sample with the respective standard deviation

The values reported in brackets were obtained by normalization to total CLA

nd not detected

In addition to the two main CLA isomers (c9,t11 and t10,c12), oleic acid (C18:1n-9) was the most abundant FA, with percentages ranging from 4.9 % (S4 supplement) to 14.4 % (S1 supplement). The S4 nutritional supplement showed the highest content of CLA isomers (94.4 %) and the smallest percentages of the other acids, while stearic (C18:0) and linolenic (C18:3n-3) acids were not detected.

As regards the CLA isomer % content, the results of FAME HRGC analysis were confirmed by the Ag+-HPLC analytical technique necessary to evaluate the complete CLA isomer profile.

Optimizing the Separation of CLA Alkyl Esters by Ag+-HPLC

Before analyzing the nutritional supplements, a CLA standard mixture containing four c,t positional isomers (c11,t13, t10,c12, c9,t11 and t8,c10) was used to optimize the resolution of the cited CLA isomers [25]. The HRGC critical pairs (c11,t13: t10,c12c and c9,t11: t8,c10) have been generally resolved by Ag+-HPLC analysis using at least three columns in series [15]. In this research, the CLA standard mixture was derivatized using six alcohols, with different length chain (methanol, ethanol, n-propanol, n-butanol, n-pentanol and n-hexanol), and the derivatized compounds were analyzed by using an Ag+-HPLC single column and a UV detection system, set at 232 nm.

Figure 1 shows the Ag+-HPLC profiles of the different alkyl (methyl, ethyl, butyl, propyl, pentyl and hexyl) esters of the commercial CLA standard. The analytes were eluted for 32 min, with a slight decrease of retention times from methyl to hexyl esters. Moreover, the separation degree of the four CLA isomers increased from methyl to hexyl ester derivatives. The resolution values (R) between adjacent peaks of alkyl esters, synthesised from CLA standard mixture, are reported in Table 2. The values were calculated applying the following formula [27]:
Fig. 1

Ag+-HPLC profiles of methyl CLA (a), ethyl CLA (b), propyl CLA (c), butyl CLA (d), pentyl CLA (e) and hexyl CLA (f) esters. 1c11,t13-CLA, 2t10,c12-CLA, 3c9,t11-CLA, 4t8,c10-CLA. Experimental conditions: the separation was carried out on a silver-impregnated ChromSpher 5 Lipids column (5 μm, 250 mm × 4.6 mm i.d.); the mobile phase was 0.1 % acetonitrile in n-hexane (0.6 mL/min); the UV detector was set at 232 nm

Table 2

Resolution of CLA alkyl esters analyzed by Ag+-HPLC



c11,t13 : t10,c12

t10,c12 : c9,t11

c9,t11 : t8,c10

Methyl CLA

0.9 ± 0.3

0.3 ± 0.1

0.8 ± 0.1

Ethyl CLA

0.8 ± 0.0

0.5 ± 0.0

0.9 ± 0.0

Propyl CLA

0.8 ± 0.1

0.5 ± 0.0

0.8 ± 0.1

Butyl CLA

0.9 ± 0.0

0.8 ± 0.1

1.1 ± 0.1

Pentyl CLA

1.0 ± 0.0

0.7 ± 0.1

1.1 ± 0.1

Hexyl CLA

1.0 ± 0.1

0.8 ± 0.0

1.2 ± 0.1

The results are the means of three repetitions with the respective standard deviations

Ag+-HPLC analyses have been performed in the same experimental conditions reported in Fig. 1 legend

$$ R = \frac{{2\left( {{\text{tr}}_{2} - {\text{tr}}_{1} } \right)}}{{W_{ 1} + \, W_{ 2} }} $$
where: tr1 and tr2 are the retention times of the analytes, 1 and 2, respectively, W1 and W2 are the peak widths of peaks in time units, 1 and 2, respectively.

As concern the resolution of the isomer pairs (c11,t13: t10,c12; t10,c12: c9,t11; c9,t11: t8,c10), it was possible also to observe that it increased when the chain length of the alcohol, used for the esterification, increased. The c11,t13: t10,c12 and c9,t11: t8,c10 isomer pairs showed acceptable resolution values taking also into account the CLA methyl ester derivatives; these values had a tendency to increase up to reach the maximum values with the CLA hexyl esters. The critical pair t10,c12: c9,t11 showed a very low resolution value, equal to 0.3 for the methyl esters. The most interesting result was the increasing of the resolution value for the cited critical pair, from methyl to hexyl esters (0.8).

In order to validate the procedure, the parameters of the response linearity for CLA standard derivatized to hexyl esters were calculated and are listed in Table 3, together with the detection (LOD) and the quantification (LOQ) limits. An adequate linearity was obtained with R2-values higher than 0.9983. The LOD and LOQ ranges were 0.03–2.08 and 0.10–6.29 ng/μL; the lowest values were obtained for t10,c12 isomer. The precision of the Ag+-HPLC method has been evaluated using the RSDr % values, both for intra-day and for inter-day reproducibility. The intra-day % values ranged from 3.0 (c9,t11) to 6.2 (c11,t13), while inter-day % values ranged from 4.1 (t8,c10) to 11.6 (c11,t13).
Table 3

Estimated regression parameters, LOD, LOQ for CLA hexyl ester standard analyzed by Ag+-HPLC

CLA hexyl esters

Calibration curve equations


LOD (ng/μL)

LOQ (ng/μL)


Y = 5293.81X−4.39





Y = 7287.29X−2.88





Y = 8466.72X−9.73





Y = 5235.55X−7.00




Ag+-HPLC analyses were performed under the same experimental conditions reported in the Fig. 1 caption

R2 correlation coefficient, LOD limit of detection, LOQ limit of quantification

The improvement of the Ag+-HPLC separation of CLA isomers as hexyl esters gave very interesting results, also considering that only a column was used, and therefore less time, less solvents were necessary with respect to the procedures reported in literature [28].

CLA Isomer Analysis of Six Nutritional Supplements by Ag+-HPLC

To evaluate the profile of CLA isomers of the six nutritional supplements, the Ag+-HPLC method optimized was used as described in the previous section; in particular, the CLA isomers were derivatized as FA hexyl esters. Figure 2 shows the Ag+-HPLC profiles of two nutritional supplements: S1 with the highest content of the two CLA main isomers (c9,t11 and t10,c12) and S3 with the highest content of t,t isomers. In Table 4 the CLA isomer composition of the supplements has been reported. It can be observed that t10,c12 and c9,t11 were in similar amounts and that low percentages of the other minor isomers (t10,t12; t9,t11; c10,c12; c9,c11) were present. The t,t isomer % contents ranged from 0.9 of the S1 supplement to 9.3 of the S3 supplement; the c,c isomer % contents ranged from 0.1 of S5 supplement to 1.2 of S2 supplement, similar to the S4 supplement, while they were not detected in the S6 supplement. However, in all the analyzed supplements, the c,c isomers were in lower amounts with respect to the t,t isomers.
Fig. 2

Ag+-HPLC profiles of hexyl CLA esters of S1 and S3 nutritional supplements. The experimental conditions have been reported in Fig. 1 caption

Table 4

CLA isomer composition of nutritional CLA supplements analyzed by Ag+-HPLC as hexyl esters

Nutritional supplements









0.5 ± 0.2

1.3 ± 0.1

4.8 ± 0.0

0.7 ± 0.0

1.3 ± 0.1

3.8 ± 5.3


0.4 ± 0.2

1.2 ± 0.1

4.5 ± 0.1

0.6 ± 0.0

1.3 ± 0.2



49.0 ± 0.1

49.7 ± 0.4

46.7 ± 0.4

48.7 ± 1.3

50.9 ± 0.3

49.6 ± 4.2


49.8 ± 0.1

46.6 ± 0.3

43.4 ± 0.4

48.7 ± 1.0

46.5 ± 0.5

46.6 ± 1.1


0.2 ± 0.2

0.5 ± 0.0

0.6 ± 0.8

0.4 ± 0.1

0.1 ± 0.1



0.1 ± 0.2

0.7 ± 0.0


0.8 ± 0.2



The results are the means of three parallel solutions of each sample with the respective standard deviations

Ag+-HPLC analyses were performed under the same experimental conditions reported in the Fig. 1 caption

nd not detected

The results obtained by Ag+-HPLC of CLA hexyl esters (Table 4) were comparable to those obtained by HRGC of FAME derivatives (Table 1, values in brackets), as no different CLA isomers were present. Obviously, the comparison between these results can be made only after the normalization of HRGC data to the total % CLA content. It is interesting to note that all the considered nutritional supplements did not contain the 8,10 and 11,13 isomers, that have been found in CLA commercial samples [16, 20].

Analysis of CLA Supplements by HRGC-MS

To verify the identity of the CLA isomers, the supplements were subjected to a double-step derivatization process in order to obtain the Diels–Alder adducts with the dienic conjugated system of CLA isomers esterified as FA hexyl esters; MTAD adducts were analyzed by HRGC-MS. It was possible to identify the c9,t11 and t10,c12 isomers by means of the characteristic MS fragments, reported in a previous paper [25]. The HRGC-MS analysis of MTAD adducts confirmed the results obtained by Ag+-HPLC that is the presence of c9,t11 and t10,c12 CLA isomers, as main components in nutritional supplements; it was also possible to confirm that the 8,10 and 11,13 CLA isomers were not present in the considered samples.


In this research an optimized single-column Ag+-HPLC method for CLA isomer analysis has been developed and validated. CLA isomers have been analyzed as hexyl esters instead of the most commonly used methyl esters and good resolutions between isomers have been obtained with shorter times and lower costs of analysis. In this research, the developed method has been used to analyze some nutritional supplements; however, it could be useful for analyzing food products with more complex CLA compositions.

All the analyzed commercial supplements contained t10,c12-CLA and c9,t11-CLA in similar amounts, while c,c and t,t CLA isomers were present in low % contents. Only the S3 supplement showed a higher amount of t,t isomers, which have lesser known effects.

It can be concluded that the quality control of the nutritional CLA supplements must be carried out with analytical methodologies that give not only a good resolution of the isomers, but also allow their sure identification. The evaluation of the qualitative and quantitative profile of the CLA isomers is important because the CLA biological effects are isomer-specific, and therefore it is useful to provide consumers with beneficial information.

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