Identification of Conjugated Linoleic Acid Isomers in Cheese
by Gas Chromatography, Silver Ion High Performance Liquid
Chromatography and Mass Spectral Reconstructed Ion Profiles.
Comparison of Chromatographic Elution Sequences
Najibullah Sehata, John K.G. Kramerb,1, Magdi M. Mossobaa,
Martin P. Yurawecza,*, John A.G. Roacha, Klaus Eulitza, Kim M. Morehousea, and Youh Kua
a
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC 20204, and bSouthern
Crop Protection, Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario N1G2W1, Canada
ABSTRACT: Commercial cheese products were analyzed for
their composition and content of conjugated linoleic acid (CLA)
isomers. The total lipids were extracted from cheese using pe-
troleum ether/diethyl ether and methylated using NaOCH3. The
fatty acid methyl esters (FAME) were separated by gas chro-
matography (GC), using a 100-m polar capillary column, into
nine minor peaks besides that of the major rumenic acid,
9c,11t-octadecadienoic acid (18:2), and were attributed to 19
CLA isomers. By using silver ion–high performance liquid chro-
matography (Ag+–HPLC), CLA isomers were resolved into seven
trans,trans (5–9%), three cis/trans (10–13%), and five cis,cis
(<1%) peaks, totaling 15, in addition to that of the 9c,11t-18:2
(78–84%). The FAME of total cheese lipids were fractionated by
semipreparative Ag+–HPLC and converted to their 4,4-dimethyl-
oxazoline derivatives after hydrolysis to free fatty acids. The
geometrical configuration of the CLA isomers was confirmed by
GC–direct deposition–Fourier transform infrared, and their dou-
ble bond positions were established by GC-electron ionization
mass spectrometry. Reconstructed mass spectral ion profiles of
the m + 2 allylic ion and the m + 3 ion (where m is the position
of the second double bond in the parent conjugated fatty acid)
were used to identify the minor CLA isomers in cheese. Cheese
contained 7t,9c-18:2 and the previously unreported 11t,13c-
18:2 and 12c,14t-18:2, and their trans,trans and cis,cis geomet-
ric isomers. Minor amounts of 8,10-, and 10,12-18:2 were also
found. The predicted elution orders of the different CLA isomers
on long polar capillary GC and Ag+–HPLC columns are also
presented.
Lipids 33, 963–971 (1998).
1
On sabbatical leave at the U.S. Food and Drug Administration, Washington,
DC.
*To whom correspondence should be addressed at Office of Food Labeling
(HFS-175), U.S. Food and Drug Administration, 200 C St., S.W., Washing-
ton DC 20204. E-mail: mpy@cfsan.fda.gov
Abbreviations: c; cis; cis/trans, refers to the same positional isomers that
have either a cis,trans or a trans,cis configuration; CLA, conjugated linoleic
acid; DMOX, 4,4-dimethyloxazoline; FAME, fatty acid methyl esters;
GC–DD–FTIR, gas chromatography–direct deposition–Fourier transform in-
frared; GC–EIMS, gas chromatography–electron ionization mass spectrome-
try; HPLC, high performance liquid chromatograph(y); t, trans.
Copyright © 1998 by AOCS Press 963
Conjugated linoleic acid (CLA) has been reported to lead to
reduced carcinogenesis (1–5) and atherosclerosis (6,7), in-
creased bone mass (8) and muscle mass (9–11), and to have
antidiabetic properties (12) in laboratory animals. The term
CLA refers to a mixture of positional and geometric conju-
gated octadecadienoic acid (18:2) isomers. The active isomer
has been assumed to be 9c,11t-18:2 (also called rumenic acid)
because it is the major CLA isomer present in milk (13–20),
cheese (15,21–27), and meat (14,15,28) from ruminant ani-
mals. It is formed as an intermediate in the biohydrogenation
of linoleic acid in the rumen (29–31), and possibly by ∆9 de-
saturation of vaccenic acid (11t-18:1) (32).
Besides the major 9c,11t-18:2 established by Parodi (13),
there are a number of minor CLA isomers in cheese and milk.
To date, their reported identities and levels remain question-
able, because of inadequate chromatographic separations and
confirmatory methods used to establish the double bond posi-
tions and configurations. Ha et al. (21) were the first to report
six additional CLA peaks in cheese separated on a 60-m Su-
pelcowax-10 gas chromatographic capillary column. Based
on comparisons of gas chromatographic equivalent chain
lengths with published data (33), they reported the presence
of 10c,12t-18:2, 10t,12c-18:2, 11c,13c-18:2, 9c,11c-18:2,
10c,12c-18:2, 9t,11t-18:2, and 10t,12t-18:2 as fatty acid
methyl esters (FAME) after BF3 methylation. They deter-
mined the molecular weight of the CLA isomers by gas chro-
matography (GC)–chemical ionization mass spectrometry
(MS), but were unable to identify CLA positional isomers by
GC–MS using 4-phenyl-1,2,4-triazoline-3,5-dione deriva-
tives. Nevertheless, their BF3 methylation procedure, GC sep-
aration conditions, and equivalent chain length comparisons
were subsequently used by many investigators (17,23,24,34),
with variation in the methylation catalyst, i.e., HCl (15,22),
tetramethylguanidine (25), or NaOCH3 (35). Acid (BF3 or
HCl)-catalyzed methylations (8,15,17,21–24,34) were shown
to lead to isomerization of cis/trans to trans,trans CLA iso-
mers and the formation of methoxy artifacts (19), as well as
CLA artifacts from allylic hydroxy fatty acids (36). Acid fat
extraction procedures (22,25) may also lead to CLA isomer-
Lipids, Vol. 33, no. 10 (1998)
964 N. SEHAT ET AL.
ization. On the other hand, base-catalyzed methylation did not
isomerize CLA isomers nor produce methoxy artifacts (19).
The GC resolution of CLA isomers was improved by using
100-m polar capillary columns under optimal conditions,
which resulted in the separation of 10 GC peaks attributed to
CLA isomers (19,37).
Recently, Lavillonnière et al. (26) reported the identity of
five additional minor CLA isomers in cheese (8c,10t-18:2,
8c,10c-18:2, 8t,10t-18:2, 11t,13t-18:2, and 11?,13?-18:2).
They methylated cheese lipids with NaOCH3, then partially
reduced the isolated CLA mixture with hydrazine, separated
the resultant monounsaturated (18:1) FAME by silver nitrate
thin-layer chromatography, and analyzed them by GC. The
positional 18:1 isomers were identified as 4,4-dimethylox-
azoline (DMOX) derivatives by GC–electron ionization mass
spectrometry (GC–EIMS). These authors also evaluated the
total CLA DMOX mixture by GC–EIMS.
We have just reported the separation of 12 CLA isomers
as their FAME from a commercial CLA mixture by using sil-
ver ion high-performance liquid chromatography (Ag+–HPLC)
based on their double-bond geometry (trans,trans, cis/trans,
and cis,cis) and position (8,10-, 9,11-, 10,12-, and 11,13-18:2)
(27). The Ag+–HPLC method, together with reconstructed
GC–EIMS ion profiles of isomer-specific ions for CLA
DMOX derivatives, permitted us to identify the previously
unrecognized and abundant minor isomer, 7t,9c-18:2, in milk,
cheese, beef, and human milk and adipose tissue (38).
In the present communication, we report the accurate iden-
tification and composition of CLA isomers in cheese, includ-
ing for the first time the previously unreported 11t,13c- and
12c,14t-18:2 isomers. Cheese lipids were fractionated by
semipreparative Ag+–HPLC, and the minor CLA isomers
were identified by GC–EIMS and GC–direct deposition-
Fourier transform infrared (GC–DD–FTIR) spectrometry.
MATERIALS AND METHODS
A mixture of CLA FAME was purchased from Nu-Chek-
Prep, Inc. (Elysian, MN). Pure CLA isomers (9c,11t-18:2,
10t,12c-18:2, 9c,11c-18:2, and 9t,11t-18:2) were obtained as
free fatty acids from Matreya Inc. (Pleasant Gap, PA). Ace-
tonitrile and hexane were ultraviolet grade. Other solvents
were distilled-in-glass quality. 2-Amino-2-methyl-1-propanol
(95%) was purchased from Aldrich Chemical Company, Inc.
(Milwaukee, WI). A 10% solution of trimethylsilyldi-
azomethane in hexane was obtained from TCI America (Port-
land, OR). The cheeses were purchased locally.
Lipid extraction. Cheese was extracted using a published
procedure (39). Briefly, 20 g cheese, 2 g potassium oxalate,
and 100 mL ethyl alcohol were placed in a blender jar and ho-
mogenized for 3 min. The jar contents were poured into a
250-mL centrifuge tube, to which 50 mL diethyl ether and 50
mL petroleum ether were added. The contents were mixed
well for 0.5 min after each addition of solvent. The mixture
was centrifuged at 1700 rpm for 7 min at room temperature.
The lower phase was reextracted two more times with 50 mL
Lipids, Vol. 33, no. 10 (1998)
petroleum ether and diethyl ether (1:1, vol/vol). The com-
bined organic phases were transferred into a 1000-mL sepa-
ratory funnel containing 500 mL distilled water and 30 mL
saturated sodium chloride. The combined organic extract was
washed two more times with 100 mL distilled water. Any
emulsion formed was broken up by the addition of saturated
sodium chloride (2–5 mL), and allowed to stand for 30 min.
The combined organic layer was poured through a glass col-
umn (2.5 cm × 50 cm) containing a bed of anhydrous sodium
sulfate (15 cm). The organic solvent was removed using a ro-
tary evaporator at 37°C and the total lipids were determined
gravimetrically.
Preparation of FAME. Free fatty acids of CLA were dis-
solved in 1 mL of 20% methanol/benzene, placed in a reac-
tion tube fitted with a Teflon-lined screw cap, and reacted
with trimethylsilyldiazomethane for 30 min at room tempera-
ture (40). Unreacted trimethylsilyldiazomethane was de-
stroyed by adding acetic acid with gentle swirling until the
yellow color disappeared. After addition of H2O (5 mL), 1
mL isooctane was added to extract the FAME which were
subsequently dried over Na2SO4.
Portions of total lipids from cheese (20–70 mg) were
placed into 15-mL reaction tubes fitted with a Teflon-lined
screw cap. One milliliter of benzene was added to dissolve
the lipids, followed by 4 mL anhydrous NaOCH3. The tubes
were flushed with nitrogen, then heated at 50°C for 10 min
with occasional shaking. After completion of methylation, 0.2
mL of water was added, and the FAME were extracted with
hexane, dried over Na2SO4, and analyzed directly by GC and
HPLC.
HPLC. The HPLC (Waters 510 solvent delivery system;
Waters Associates, Milford, MA) was equipped with an au-
tosampler and 2-mL injection loop for the semipreparatory
column and a 200-µL injection loop for the analytical column
(Waters 717), an ultraviolet detector (Waters 486 tunable ab-
sorbance), a fraction collector (Waters LR 76413), and an op-
erating system (Waters MillenniumTM version 2.15.01). Both
ChromSpher 5 Lipids semipreparative (10 mm i.d. × 250 mm
stainless steel; 5 µm particle size) and an analytical (4.6 mm
i.d. × 250 mm stainless steel; 5 µm particle size) silver-im-
pregnated column were used (Chrompack, Bridgewater, NJ).
CLA isomers were measured at 233 nm. The mobile phase
was 0.1% acetonitrile in hexane and operated isocratically.
The solvent flows were 4.0 and 1.0 mL/min, for the semi-
preparative and analytical columns, respectively. The flow
was commenced for 0.5 h prior to sample injection. For solu-
tion containing about 20 mg/mL, typical injection volumes
were 150–200 µL for the semipreparative column and 5–15
µL for the analytical column. Whenever necessary, the col-
umn was restored by flushing with 1% acetonitrile in hexane
for 4 h followed by 0.5 h with 0.1% acetonitrile in hexane.
DMOX derivatives. In a reaction tube, 10–20 mg of methyl
heptadecanoate (17:0) and ca. 1–2 mg of CLA FAME were
added, and the mixture was hydrolyzed with 1 N KOH/95%
ethanol at 50°C for 30 min. The free fatty acids were ex-
tracted with petroleum ether after the reaction was neutral-
 IDENTIFICATION OF CLA ISOMERS IN CHEESE 965

ized with HCl. The free fatty acids were added to a screw-cap
reaction tube (1 mL) and a threefold excess of 2-amino-2-
methyl-1-propanol (w/w) was added. The tube was purged
with argon, capped, and heated at 170°C for 0.5 h in an oven.
The reaction mixture was transferred into a 250-mL separa-
tory funnel containing 40 mL of petroleum ether and 50 mL
of water and shaken vigorously. Saturated NaCl was added to
break the emulsion. The aqueous layer was removed, and the
petroleum ether was rewashed with water, dried over anhy-
drous Na2SO4 and concentrated to the desired volume under
a stream of argon (36).
GC. The cheese FAME were analyzed by GC (model
5890; Hewlett-Packard, Palo Alto, CA) using a fused-silica
capillary column (CP-Sil 88; 100 m × 0.25 mm i.d. × 0.2 µm
film thickness; Chrompack Inc.). The column was held at
70°C for 4 min after injection, temperature-programmed at
13°C/min to 175°C, held there for 27 min, then temperature-
programmed at 4°C/min to 215°C, and held there for 31 min.
Hydrogen was the carrier gas, at a split ratio of 20:1.
GC–EIMS. The GC–EIMS was performed using a GC
(Hewlett-Packard 5890, series II) coupled to a mass spec-
trometer (Autospec Q mass spectrometer) and a data system
(OPUS 4000; Micromass, Manchester, United Kingdom).
The GC–EIMS system utilized version 2.1 BX software. This
system was used with a 50-m CP-Sil 88 capillary column de-
scribed previously (41). The GC–EIMS conditions were:
splitless injection with helium sweep restored 1 min after in-
jection; injector and transfer lines’ temperature 220°C; oven
temperature was 75°C for 1 min after injection, then temper-
ature-programmed 20°C/min to 185°C, held there for 15 min,
then temperature-programmed 4°C/min to 220°C, and held
there for 45 min.
GC–DD–FTIR. A Bio-Rad (Cambridge, MA) TracerTM
GC–FTIR 60A spectrometer system was used. This system
was used with a 50 m CP-Sil 88 capillary column (42).
RESULTS AND DISCUSSION
Fat extraction from cheese. The total fat was extracted from
cheese (39). In this method, potassium oxalate served as a
grinding agent and formed calcium oxalate, which prevented
calcium ions from causing emulsions. The total fat content of
11 commercial cheese products ranged from 14 to 37% (Table
1). For the extraction of fats and oils, acid conditions were
avoided because they may lead to isomerization of the
cis/trans to trans,trans CLA isomers. In a previous study (22)
preliminary acid digestion may have contributed to the low
content of 9c,11t-18:2, 48–68% (22), instead of 78–84% re-
ported for selected cheese products (Table 1).
Methylation. The total lipids from cheese were methylated
using NaOCH3 to avoid any isomerization of conjugated di-
enes and formation of methoxy and CLA artifacts. Acid-cat-
alyzed methylation using either BF3, HCl or H2SO4 would lead
to conversion of cis/trans to trans,trans CLA isomers. Reduc-
ing the temperature during methylation reduced the extent of
this conversion (17,23,43), but the methylation, particularly of
phospholipids, may not be complete (19). Therefore, to avoid
isomerization and incomplete methylation of CLA, NaOCH3
is recommended. If test samples contained significant amounts
of free fatty acids, alkali hydrolysis followed by subsequent
methylation using tetramethylsilyldiazomethane (40) was car-
ried out and checked by thin-layer chromatography (19), and
these steps are recommended. However, neither of these two
methods will methylate sphingomyelin, since the N-acyl bond
is resistant to alkali. In the present cheese study, we ignored the
small contribution of CLA from sphingomyelin because its
level in cheese was less than 0.1%, and the occurrence of CLA
in sphingomyelin is generally the lowest compared to all the
other phospholipids (37).
GC. The CLA region of the FAME GC trace for cheese
(Fig. 1) was a complex mixture of 10 peaks attributed to many

TABLE 1
Lipid Content and Composition of Conjugated Linoleic Acid (CLA) Isomers in Commercial Cheesesa
Lipid Total CLA isomers (%)d
contentb CLAc 7t,9c
Types of cheese (%) (%) 9c,11t 8t,10c 11t,13ce 12c,14t Total t,t
American processed cheese 16.5 0.46 79.35 10.17 1.71 0.67 8.11
Cheddar, sharp 29.5 0.54 82.58 8.31 2.14 0.61 6.36
Cheddar, extra sharp 23.4 0.52 77.72 12.13 0.90 0.66 8.6
Colby 29.7 0.40 82.01 9.89 0.98 0.63 6.48
Cream cheese 36.7 0.77 83.53 4.61 4.23 1.57 6.06
Feta 24.7 0.49 80.76 11.05 1.51 0.90 5.78
Monterey Jack 26.9 0.47 80.01 10.97 1.32 0.93 6.77
Mozzarella 16.5 0.47 78.07 11.11 0.88 0.77 9.16
Parmesan 25.1 0.38 80.83 10.01 1.09 0.63 7.44
Prepared cheese product 15.3 0.56 81.74 8.63 1.80 0.99 6.84
Processed, Cheddar 13.5 0.50 84.15 8.57 1.26 0.61 5.41
a
As determined by silver ion–high-performance liquid chromatography (Ag+–HPLC).
b
Determined gravimetrically after total lipid extraction; see the Materials and Methods section.
c
Total CLA fatty acid methyl ester (FAME) content (% of total FAME) in extracted lipids determined by gas chro-
matography (GC).
d
CLA isomeric composition (% of total CLA FAME) determined by Ag+–HPLC.
e
See text regarding discussion about double-bond configuration.

Lipids, Vol. 33, no. 10 (1998)

966
FIG. 1. The conjugated linoleic acid (CLA) region of a gas chromatogra-
phy (GC) trace for cheese.
more unresolved CLA isomers, even with a highly polar 100-m
capillary column. The CLA isomers in the gas chromatogram
were labeled based on an elution order that was verified re-
cently (19,37,38) as well as in this study; see the predicted
elution order in Table 2. Besides the major 9c,11t-18:2, there
were minor peaks in the cis/trans region corresponding to
9t,11c- plus 10c,12t-18:2; 11t,13c-18:2; and a mixture of
10t,12c- plus 12c,14t-18:2. The 7t,9c,10c,12c- and 8t,10c-
18:2 isomers eluted on the leading and the tailing edges of
9c,11t-18:2, respectively. In the cis,cis region (Fig. 1) there
was one major peak consisting of a mixture of 7c,9c-, 8c,10c-,
and 9c,11c-18:2, followed by a smaller peak due to 10c,12c-,
11c,13c-, plus 12c,14c-18:2. The trans,trans region showed
four responses attributed to the FAME of 12t,14t-18:2;
11t,13t-18:2; a mixture of 10t,12t-, 9t,11t plus 8t,10t-18:2;
and 7t,9t-18:2 (Fig. 1). To separate and confirm the identity
of CLA isomers, complementary techniques, such as
Ag+–HPLC, GC–EIMS and GC–DD–FTIR were used.
Ag+–HPLC. The separation of cheese FAME by Ag+–HPLC
is shown in Figure 2. The elution order observed by Ag+-
HPLC for a commercial mixture of 12 CLA isomers (27),
plus those found in natural products (38, and the present
study), are listed in Table 3. The main CLA isomer in cheese
was 9c,11t-18:2. To determine the structure of the many
minor CLA isomers, FAME were prepared from 1 g of cheese
lipids and were fractionated using a semipreparative
Ag+–HPLC column. Five fractions were collected and those
containing minor CLA isomers, Fractions 1, 2, 4 and 5, were
shown after expanding the y-scale in Figure 2. The fractions
were subsequently derivatized to DMOX and analyzed by
GC–EIMS and GC–DD–FTIR.
Lipids, Vol. 33, no. 10 (1998)
N. SEHAT ET AL.
TABLE 2
Expected Gas Chromatographic Elution Order of Positional
and Geometric CLA Fatty Acid Methyl Ester, or Dimethyloxazoline,
Isomers on a 100 m CP-Sil 88 Capillary Columna
cis/trans-18:2b cis,cis-18:2c trans,trans-18:2d
7c,9t (7c,9c)e 12t,14t
(6t,8c) 8c,10c 11t,13t
(8c,10t) 9c,11c 10t,12t
7t,9c 10c,12c 9t,11t
9c,11t 11c,13c 8t,10t
8t,10c 12c,14c 7t,9t
10c,12t
9t,11c
11c,13t
10t,12c
12c,14t
a
The elution order was: all the cis/trans, followed by all the cis,cis, followed
by all the trans,trans CLA positional isomers. Many CLA isomers overlapped
within each geometric group.
b
The observed elution time of cis/trans CLA isomers increased as the ∆ value
of the cis double bond increased in the molecule. For a pair of cis/trans iso-
mers in which the cis double bond has the same ∆ value, the isomer with
the lower ∆ trans value eluted first. Therefore, it followed that for the same
positional isomer, the cis,trans eluted before the trans,cis geometric isomer.
c
The observed elution time of cis,cis CLA isomers increased with increased
∆ values.
d
The observed elution time of trans,trans CLA isomers increased with de-
creased ∆ values.
e
CLA isomers shown in parentheses were predicted. For abbreviations see
Table 1.
GC–EIMS reconstructed ion profiles for DMOX deriva-
tives of conjugated dienes. The fatty acid DMOX mass spec-
tra consisted of a series of even-mass ions separated by 14
mass units due to successive losses of methylene units. A gap
of 12 mass units between ions containing n−1 and n carbon
atoms indicated the presence of a double bond between car-
bons n and n + 1 of the parent fatty acid (44). For monounsat-
urated fatty acids, more abundant peaks occurred due to al-
lylic cleavages on either side of the gap of 12 mass units for
ions containing n − 2 and n + 2 carbons (44). In addition, the
n + 3 carbons fragment ion occurred in equal abundance to
the one containing n + 2 carbons; the mechanism of forma-
tion of this ion remains unknown (45). Therefore, for conju-
gated dienes, prominent ions occurred at n − 2, m + 2 and m
+ 3, where n is the position of the first double bond and m is
the position of the second double bond along the parent con-
jugated fatty acid chain. These three prominent ions were
clearly recognizable in previous published spectra of CLA
DMOX (27,46,47), and in the series of mass spectra of 12,14-
18:2 to 8,10-18:2 isomers found in cheese (Fig. 3).
The characteristic abundant ions with m + 2 and m + 3 car-
bons in the GC–EIMS DMOX spectra of CLA isomers were
used in this study to identify trace amounts of isomers, be-
cause the gap of 12 mass units was often too weak to observe.
For example, all the minor CLA isomers in Figure 3, except
for the major 9c,11t-18:2 isomer, were missing one or more
of the expected ions of the conjugated system, but the allylic
m + 2 and the m + 3 ions were clearly visible. The prominent
ion profiles for the m + 2 and m + 3 pair of ions due to a spe-
 IDENTIFICATION OF CLA ISOMERS IN CHEESE
FIG. 2. Silver ion–high performance liquid chromatography (Ag+–HPLC)
profile for a commercial cheese product. Expanded traces are shown
for Fractions 1 (A), 2 (B), 4 (C), and 5 (D). These fractions were collected
by Ag+–HPLC for further analysis.
967
TABLE 3
Elution Order of Positional and Geometric CLA FAME Isomers
by (Ag+–HPLC)a
trans,trans-18:2b cis/trans-18:2b,c cis,cis-18:2b
(13t,15t)d (13,15 c/t)
12t,14t 12,14 c/t 12c,14c
11t,13t 11,13 c/t 11c,13c
10t,12t 10,12 c/t 10c,12c
9t,11t 9,11 c/t 9c,11c
8t,10t 8,10 c/t 8c,10c
7t,9t 7,9 c/t 7c,9c
a
The elution order was: all the trans,trans, followed by all the cis/trans, fol-
lowed by all the cis,cis CLA positional isomers.
b
The observed elution time of each group of geometric CLA isomers in-
creased as the ∆ values decreased.
c
For the same positional isomer, the cis,trans and the trans,cis geometric iso-
mers were not clearly separated under our experimental conditions.
d
CLA isomers shown in parentheses were not confirmed. For abbreviations
see Table 1.
cific CLA isomer were normalized and displayed. If these two
ion profiles superimposed, then this GC peak was due to this
specific CLA isomer. For nearly coeluting (or coeluting) CLA
isomers, the reconstructed ion chromatograms showed a
small (or lack of) difference in retention times. This recon-
structed ion profile GC-EIMS method allowed the identifica-
tion of most minor CLA isomers in cheese.
Identification of Ag+–HPLC fractions. Fraction 1 consisted
of a mixture of seven trans,trans CLA peaks (Fig. 2), as evi-
denced by the elution order of geometric CLA isomers previ-
ously found by Ag+–HPLC (27) and verified by GC–DD–FTIR
with characteristic =C-H stretching (3016 cm−1) and deforma-
tion (990 cm−1) vibrations for trans,trans CLA isomers. The
GC–EIMS reconstructed ion profile for the molecular ion m/z
333 of the DMOX derivative of all CLA isomers is shown in
Figure 4A. The total trans,trans CLA DMOX mixture exhib-
ited three GC–EIMS peaks using a 50-m CP-Sil 88 capillary
column. In using the reconstructed ion profile technique de-
scribed above, the identify of the first small peak (Fig. 4C) was
established as 12t,14t-18:2 (Fig. 3). The second CLA peak
(Fig. 4B) was due to 11t,13t-18:2 (Fig. 3), and the third one
(Fig. 4D) to 9t,11t-18:2 (Fig. 3). The CLA isomers 8t,10t- and
10t,12t-18:2 coeluted with 9t,11t-18:2 (reconstructed ion pro-
file not shown), while the 7t,9t-18:2 eluted on the tail of the
third peak [see reference (38), Fig. 1 bottom]. The first small
peak in the Ag+–HPLC chromatogram (Fig. 2, Fraction 1)
would appear to be 13t,15t-18:2 based on the Ag+–HPLC elu-
tion order, although this was not confirmed by GC–EIMS.
In Figure 3, GC–EI mass spectra were obtained from dif-
ferent cheese fractions, except the one for 10,12-18:2, be-
cause this trans,trans isomer coeluted by GC–EIMS with
9t,11t-18:2 (Fig. 4D), and the corresponding cis/trans and
cis,cis isomers were very weak. However, its identity was
confirmed from the reconstructed ion profiles for m/z 276 (m
+ 2) and 290 (m + 3) in each case. A mass spectrum of
10t,12c-18:2, shown for comparison in Figure 3, was selected
from a previous study on the distribution of CLA isomers in
pig lipid classes (37).
Lipids, Vol. 33, no. 10 (1998)
968 N. SEHAT ET AL.

FIG. 3. GC–electron ionization mass spectra of the 4,4-dimethyloxazoline (DMOX) derivatives of 12t,14t-18:2 (Fraction 1), 11t,13t-18:2 (Fraction
1), 10t,12c-18:2 [reference (37), see text], 9c,11t-18:2 (Fraction 2), and 8c,10c-18:2 (Fraction 5). The allylic ions n − 2 and m + 2, and the m + 3
ion are labeled for each of the positional CLA DMOX isomers; the molecular ion was m/z 333. For other abbreviations see Figure 1.

Fraction 2 (Fig. 2) showed two minor cis/trans CLA peaks
eluting before the major 9c,11t-18:2, whose cis/trans geomet-
ric configuration was consistent with Ag+–HPLC and
GC–DD–FTIR data. In using the same reconstructed ion pro-
file techniques described for the trans,trans CLA isomers, the
peaks were shown to be due to 12,14- and 11,13-18:2. The EI
mass spectra of the DMOX derivative of the 12,14- and
11,13-18:2 isomer were similar to the corresponding
trans,trans CLA isomers shown in Figure 3. The geometric
configuration of 12,14-18:2 was likely 12c,14t-18:2 based on
the GC elution sequence and because it was possibly derived
from biohydrogenation and isomerization of α-linolenic acid
in the rumen. The geometric configuration of the 11,13-18:2
isomer in cheese was probably 11t,13c-18:2 based on the fol-
lowing considerations. On a 100-m CP Sil 88 column (Fig.
1), the 11,13-18:2 isomer in cheese eluted after the 11c,13t-
18:2 isomer previously found in a Nu-Chek-Prep commercial
mixture (27,37). In using two Ag+–HPLC columns in series,
the 11,13-18:2 isomer in cheese eluted before the 11c,13t-
18:2 present in the Nu-Chek-Prep commercial mixture when
these two lipid mixtures were coinjected (Sehat, N., unpub-
lished data). Therefore, the relative elution sequences of the
two c/t 11,13-18:2 isomers observed by both Ag+–HPLC and
GC are consistent with the presence of 11t,13c-18:2 in cheese.
On the other hand, the fact that 11t,13c-18:2 eluted before
10t,12c-18:2 by GC cannot be reconciled with the expected
GC elution sequence (Table 2), namely, 10t,12c-18:2 is ex-
pected to elute first, followed by 12c,14t-18:2 and then by
11t,13c-18:2. Therefore, the identification of the c/t 11,13-
18:2 peak in cheese is only tentatively attributed to the
11t,13c-18:2 isomer. The double-bond configuration of these
two c/t 11,13-18:2 geometric isomers (after isolation) still
needs to be definitively confirmed by such methods as hy-
drazine reduction and GC separation of the resulting 18:1
fatty acids (26, and references cited therein). Trace amounts
of 10t,12c-18:2 were generally not resolved chromatographi-
cally from the major 9c,11t-18:2 by Ag+–HPLC because of
the large differences in concentration between these two iso-
mers.
Fraction 3 contained the major 9c,11t-18:2 (Fig. 2), which
was not further investigated. Fraction 4 (Fig. 2) contained one
cis/trans CLA peak. The main isomer under this minor peak

Lipids, Vol. 33, no. 10 (1998)

 IDENTIFICATION OF CLA ISOMERS IN CHEESE
FIG. 4. Reconstructed ion chromatograms for DMOX derivatives of
trans,trans CLA isomers in Fraction 1 isolated from cheese using
Ag+–HPLC showing the profiles for (A) the molecular ion m/z 333; (B)
the allylic ion (m + 2) and the m + 3 ion for 11,13-18:2, (C) 12,14-18:2,
and (D) 9,11-18:2. In each case these two ions were normalized for the
CLA isomer indicated. For abbreviations see Figures 1–3.
was identified as 7t,9c-18:2 by GC–EIMS, GC–DD–FTIR
and by comparison with partial synthesis, as reported recently
(38). In addition to the 7t,9c-18:2, small amounts of 8t,10c-
18:2 were evident under this peak by GC–EIMS.
Fraction 5 (Fig. 2) contained a mixture of five cis,cis CLA
peaks as evidenced by their elution on Ag+–HPLC (Table 3).
The more predominant CLA isomers were identified by
GC–EIMS as 9c,11c-18:2 and 8c,10c-18:2, with minor
amounts of 12c,14c-, 11c,13c-, and 7c,9c-18:2. The EI mass
spectrum of the DMOX derivative of the 8c,10c-18:2 isomer
is shown in Figure 3. The peak expected for 10c,12c-18:2 was
not observed, probably because it was too weak.
Composition of CLA isomers in cheese. The total CLA con-
tent of cheese fats was determined by GC and ranged from 0.4
to 0.8% (Table 1). Based on the separation of CLA isomers by
969
Ag+–HPLC, the compositions of the various cheese products
are reported in Table 1. The concentration of the major 9c,11t-
18:2 ranged from 78 to 84% (as percentage of total CLA) in
the cheese products investigated. Among the minor CLA iso-
mers, 7t,9c-18:2 was the most predominant at about 5 to 12%
(containing undetermined small amounts of 8t,10c-18:2), fol-
lowed by 11t,13c-18:2 at about 2%. The presence of 7t,9c-18:2
in the rumen is expected from ∆9 desaturation of 7t-18:1, and
to a smaller extent from biohydrogenation and isomerization
of γ-linolenic acid, while 12c,14t-18:2 is from biohydrogena-
tion and isomerization of α-linolenic acid. The remainder was
a mixture of total trans,trans (5 to 9%) and cis,cis (<1%) CLA
isomers produced in the complex rumenal fluids.
Alternative methods used to determine CLA isomers in
cheese by partial reduction with hydrazine, followed by GC sep-
aration of the resultant mixtures of octadecenoic acids (18:1)
(26), did not lead to the identification of the 12,14- and 7,9-18:2
isomers. A possible reason for the latter is that 14t-18:1 is only
partially resolved on polar capillary GC columns from 13t-18:1
as DMOX, and even less resolved as FAME (48). Similarly, the
7t-18:1 FAME coelutes with 6t- and 8t-18:1 FAME, while 7t-
18:1 DMOX coelutes with 9t-18:1 DMOX (48).
The 11t,13c-18:2 isomer was found as a minor CLA com-
ponent in cheese lipids. On the other hand, the 11c,13t-18:2
isomer was present at about 22% of total CLA in a commer-
cial CLA mixture (27). The latter isomer, when included in
the diet of pigs at the level found in commercial CLA mix-
tures, preferentially accumulated in heart lipids, particularly
in heart and liver diphosphatidylglycerol (37). It would be of
interest to investigate if 11t,13c-18:2 also accumulated in
heart lipids and diphosphatidylglycerol, of pigs fed a cheese
product containing 11t,13c-18:2 at about 1% of total CLA.
The availability of Ag+–HPLC, GC–EIMS, GC–DD–FTIR,
and very long polar capillary GC columns has provided the
necessary tools to investigate the minor isomers of CLA in
cheese products. Combinations of these methods plus re-
versed-phase HPLC will be required to identify the metabo-
lites of CLA. In addition to 9c,11t-18:2 and the isomers re-
ported previously by Ha et al. (21) and Lavillonniere et al.
(26), the previously unrecognized isomers 11t,13c-18:2,
12c,14t-18:2, the prominent 7t,9c-18:2 (38), and their
trans,trans, and cis,cis geometric isomers were found in this
investigation. On the basis of our observed results in this and
previous studies (19,37,38), the elution orders of CLA iso-
mers on Ag+–HPLC and long polar GC columns were deter-
mined (Tables 2 and 3).
ACKNOWLEDGMENT
Contribution number S009 from Southern Crop Protection, Food
Research Center, Guelph, Ontario, Canada.
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[Received June 4, 1998, and in final revised form and accepted
September 24, 1998]
Lipids, Vol. 33, no. 10 (1998)