203 / 258
G
olay
&
M
oulin
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
219
(
5
)
Performance of the transesterification.
—Record the
areas of the two internal standard peaks (methyl undecanoate
and tritridecanoin) in the analyzed samples. The performance of
transesterification (Pt) expressed as a percentage, is calculated
on the recovery of the tritridecanoin as a second internal
standard as follows:
=
× × ×
×
×
(
)
100
11
13
13 13
11
13
Pt
m A R S TAG
A m
c
c
c
c
c
c
where
m
c11
is the mass in milligrams of the C11:0 internal
standard added to the solution;
A
c13
is the peak area of the C13:0
internal standard in the chromatogram;
R
c13
is the response
factor of C13:0 relative to C11:0, calculated according to
G
(
1
);
S
c13
is the stoichiometric factor to convert C13:0 FAME into
C13:0 TAG;
A
c11
is the peak area of the C11:0 internal standard
in the chromatogram; and
m
c13
is the mass in milligrams of the
C13:0 internal standard added to the solution.
The performance of the transesterification method should
be always 100.0±2.0%. When the performance of the
transesterification is >102.0 or <98.0%, the origin of the
problem could be the following: incomplete transesterification,
partial degradation of internal standard(s), or a matrix effect
problem. The evaluation of transesterification performance in
a blank sample can be used to monitor the stability of reagents
and chemicals.
Results and Discussion
Forty-six analytes (Table 2) were recorded in 12 selected
samples (Table 1) analyzed as double blinds (i.e., 24 analyses)
by 18 participants; in total, 19 872 results were collected from
the study. The results were carefully evaluated and summarized
in an Excel template used for statistical evaluation. Single values
reported for one double-blind sample were removed from the
evaluation, and a list of fatty acids was selected according to
the composition (dairy versus nondairy) of analyzed samples
and their abundance. Outlier values were removed based on
Cochran and/or Grubbs tests following the ISO 5725 guideline.
All statistical decisions regarding the evaluation of data were
carefully recorded and provided to the ERP to assist in their
decision to accord Final Action status in July 2014.
Results corresponding to Samples 1–6 were expressed in
grams per 100 g finished product, except for Sample 5 (cheese),
which are expressed in grams per 100 g extracted fat from cheese.
Results corresponding to SPIFAN Samples 7–9 in powder form
were expressed in grams per 100 g reconstituted product (25 g
powder with 200 g water), and Samples 10–12 were expressed
in grams per 100 g liquid products. Results for Samples 7–12
were reported using criteria defined in AOAC SMPR 2012.011.
As previously discussed with the ERP during SLV data
evaluation, the requirements for repeatability and reproducibility
are not fully consistent with the whole range of fatty acids
concentration found in the samples. Values were given for fatty
acid concentrations of <0.5, ≥0.5 to <3.0, and ≥3.0 g/100 g;
however, the quantification limit is (≤0.001) is 500 times lower
than the lower fatty acid concentration indicated in SMPR (0.5).
The performance requirement at the level of 0.5 cannot be the
same for a fatty acid in a concentration at 0.499 in a sample and
at 0.001 in another sample (500 times lower). As a consequence,
additional limits for repeatability and reproducibility values
also need to be fixed in the SMPR for concentrations below
0.5. Proposed repeatability and reproducibility limits are shown
in Table 3.
The transesterification performance (i.e., recovery between
C11:0 FAME and C13:0 TAG) was monitored in all samples and
ranged between 98.9 and 100.0% with an RSD value between
0.9 and 1.6%, except for a cheese sample (2.7%).
A questionnaire was sent to all participants, along with an
invitation to give comments about the performance of the
method in their laboratory. Feedback was requested with respect
Table 2. Analytes
Fatty acid
Abrev.
Fatty acids analyzed
Individual fatty acid
C18:2
n
-6
LA
C18:2
n
-6
C18:3
n
-3
ALA
C18:3
n
-3
C20:4
n
-6
ARA
C20:4
n
-6
C20:5
n
-3
EPA
C20:5
n
-3
C22:6
n
-3
DHA
C22:6
n
-3
Group of fatty acid
Saturated fatty acids
SFAs
C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C22:0, and C24:0
Monounsaturated fatty acids
MUFAs
C14:1
n
-5, C15:1
n
-5, C16:1
n
-7, C17:1
n
-7, C18:1
n
-9 (and other
cis
isomers),
C20:1
n
-9, C22:1
n
-9, and C24:1
n
-9
Polyunsaturated fatty acids
PUFAs
C18:2
n
-6, C18:3
n
-6, C18:3
n
-3, C20:2
n
-6, C20:3
n
-3, C20:3
n
-6, C20:4
n
-6,
C20:5
n
-3, C22:2
n
-6, and C22:6
n
-3
Trans
fatty acids
TFAs
C18:1
trans
, C18:2
trans
, and C18:3
trans
Omega-3
ω-3
C18:3
n
-3, C20:3
n
-3, C20:5
n
-3, and C22:6
n
-3
Omega-6
ω-6
C18:2
n
-6, C18:3
n
-6, C20:2
n
-6, C20:3
n
-6, C20:4
n
-6, and C22:2
n
-6
Omega-9
ω-9
C18:1
n
-9, C20:1
n
-9, C22:1
n
-9, and C24:1
n
-9
Sum of all fatty acids
Total fatty acids
All fatty acids (including CLAs and omega fatty acids)
Transesterification performance
TP
C11:0 and C13:0 (internal standards)
203