© 2012 AOAC INTERNATIONAL
where m′
i
= mass fraction of FAME
i
in the calibration standard
solution,
D
(
q
); A′
O
= peak area of C11:0 in the calibration standard
solution chromatogram; m′
O
= mass of C11:0 in the calibration
standard solution,
D
(
q
); and A′
i
= peak area of FAME
i
in the
calibration standard solution chromatogram.
The variation between three injections is optimal when
coefficients of variation are less than 2.
Note
: The response factors calculated for C18:2 n-6 could be
applied for C-18:2 CLA and that calculated for C18:3 n-3
cis
could
be applied for C18:3
trans
isomers.
(
3
)
Determination of the test portion
.—Inject 1 μL of the test
portion,
F
(
b
), into the gas chromatograph applying the same
conditions as used in the calibrating solution.
(
b
)
Fatty acid identification
.—Identify the fatty acids in the
sample solution chromatogram by comparing their retention times
with those of the corresponding peaks in the calibration standard
solution,
D
(
q
), and in the qualitative standard mixture containing
TFAs and CLA,
D
(
o
).
C18:1 TFA
.—Identify and group all TFA of C18:1 (include also
the peak area of
trans
C18:1
trans
Δ16 eluted in the
cis
C18:1 region
just after the oleic acid) according to Figures
2012.13A
and
B
.
Note
: When milk fat is present, two trans isomers of C18:1 are
eluted in the
cis
C18:1 region (the C18:1
trans
Δ15 and C18:1
trans
Δ16, respectively), but only one (C18:1
trans
Δ16) is resolved with
the 100 m length capillary column. The second isomer (C18:1
trans
Δ15) is generally overlapped with the oleic acid peak (C18:1
cis
Δ9) and its area is only quantifiable using a preliminary separation
(TLC Ag
+
, HPLC Ag
+
) followed by a capillary GLC analysis.
According to recent findings, it has been demonstrated that there is
not significant difference of total C18:1
trans
amount when the area
of C18:1
trans
Δ15 (not resolved peak) is not included in the sum
in comparison to the result obtained after preliminary separation
techniques (Ag
+
) followed by a capillary GLC analysis. A part
of this phenomenon is explained by presence of some C18:1
cis
isomers (Δ6-8) which elute in the C18:1
trans
and consequently are
indirectly added to the sum of C18:1
trans
and compensate the fact
that C18:1
trans
Δ15 is not taken into account.
C18:2 TFA
.—Identify and group all TFA of linoleic (C18:2 n-6)
acids (
see
Figures
2012.13A
and
B
). For the total TFA of C18:2,
include all the
trans
isomers present in milk fat sample as shown in
Figures
2012.13A
and
B
.
C18:3 TFA
.—Identify and group all TFAof linolenic (C18:3 n-3)
acids (
see
Figures
2012.13A
and
B
).
Note
: In presence of milk fat and/or fish oil in the sample,
another isomer of C20:1 elutes just before C20:1 n-9. Depending
on the column resolution, the retention time of this fatty acid may
also correspond to a
trans
isomer of C18:3 n-3 (the c,t,c or t,c,c).
When there is only one peak in the corresponding zone of C18:3
TFA, its correct identification corresponds to a C20:1 isomer. When
two, three, or four peaks are encountered in the corresponding
zone for C18:3 TFA, each peak area should be included in the
total areas of C18:3 TFA (
see
elution order and formation rules
below). Interferences could be also observed between C18:3 TFA
isomers (C18:3 c,c,t; c,t,c; or t,c,c) and C20:1 n-9. When C20:1
n-9 elute with C18:3 c,t,c, (the minor C18:3 TFA isomer), their
contribution on the total C18:3 TFA is negligible. However, if
C20:1 n-9 is interfered with C18:3 c,c,t or with C18:3 t,c,c the
chromatography conditions should be slightly modified to obtain
sufficient separation. Interference is also visible when wrong ratio
between C18:3 n-3 c,c,t and C18:3 n-3 t,c,c is observed (normal
ratio is always 5:4).
The kinetics of C18:3
trans
isomers formation in refined and
deodorized oils have been analyzed using highly polar capillary
column and described in the literature. They could be used as a
Figure 2012.13A. Example of GC chromatogram (enlarged view of C18:1 TFA, C18:2 TFA, and CLA) using split injection.
