© 2012 AOAC INTERNATIONAL
solution 2,
D
(
p
)(
2
), and 25.0 mL of the calibration standard stock
solution 3,
D
(
p
)(
3
). Then make up to the mark with
n
-hexane.
Dilute accordingly to the type of injector used.
Note
: This solution keeps for about 6 months if stored in the
dark at –20°C. To prevent contamination of the standard solution,
immediately distribute the solution into different vials (ready
to inject) and store at –20°C before use. Use each vial once then
discard it.
(
q
)
Commercially available calibration standard FAME
mixture solution
.—Before use, allow the ampoule to come to
room temperature (maximum 25°C) in the dark without heating.
Cut the ampoule with a glass knife and using a Pasteur pipet,
rapidly transfer the content of the ampoule into a 50 mL pre-tarred
volumetric flask, weigh, and make up to the mark with
n
-hexane.
Dilute accordingly to the type of injector used.
Note
: This solution keeps for about 6 months if stored in the
dark at –20°C. To prevent contamination of the standard solution,
immediately distribute the solution into different vials (ready to
inject) and store at –20°C before use. Use each vial once and then
discard it.
E. Preparation of Test Sample
Milk powder and infant formula
.—Mix well to ensure that
sample is homogeneous.
(
1
)
Class or group of fatty acids in 100 g fat.—
Calculate the
mass fraction of all fatty acids included in a group or in a class
of fatty acids by simple addition of individual fatty acids results
(expressed in g FA/100 g fat).
(
2
)
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 in %, is
calculated on the recovery of the tritridecanoin as a second internal
standard as follows:
ܲ
ݐ
= ݉
ଵଵ
×
ܣ
ଵଷ
× ܴ
ଵଷ
× ܵ
ଵଷ
(ܶ
)ܩܣ
ܣ
ଵଵ
× ݉
ଵଷ
× 100
where m
C11
is the mass, in milligrams, of C-11:0 internal standard
added to the solution; A
C13
is the peak area of C-13:0 internal
standard in the chromatogram; R
C13
is the response factor of C13:0
relative to C11:0, calculated according to
G
(
a
)(
2
); S
C13
is the
stoichiometric factor to convert C13:0 FAME into C13:0 TAG; A
C11
is the peak area of C-11:0 internal standard in the chromatogram;
and m
C13
is the mass of liquid milk and liquid formula. Bring sample
to room temperature and shake vigorously before use.
F. Procedure
As a check on method performance, carry out two single
determinations in accordance with
F
(
a
) and (
b
).
Note
: An alternative procedure using fat-extraction tubes with
siphon or wash-bottle fittings is given in Figures
2012.13A–C
.
(
a
)
Resolution between C18:1 trans and cis
.—Inject once the
qualitative
cis/trans
FAME isomers standard mixture solution,
D
(
o
).
(
b
)
Calibrating solution for the determination of response
factor
.—Inject three times the calibrated solution,
D
(
q
).
(
c
)
Test portion
.—Into a 25 mL centrifuge tube with a screw
cap, weigh to the nearest 0.1 mg an equivalent quantity of sample
in order to have ca 50 mg fat in the tube (
Example
: For a sample
containing 26 g fat/100 g product, the corresponding sample weight
is approximately 190 mg).
Note 1
: For fatty acid analysis on fat extracted from foods, the
same amount of fat is required. For milk powder or infant formula
powder, add 2.0 mL water using a micropipet. Close the tube, and
then dissolve gently using a vortex mixer. Wait for 15 min at room
temperature.
Note 2
: For liquid milk samples and fat extracted from foods, no
pretreatment (water addition) is required.
Pipet 5 mL of internal standard solution,
D
(
j
). Add with a pipet
5 mL of 5% (w/v) methanolic sodium methoxide solution,
D
(
d
).
The transesterification time starts with the addition of the first drop.
Close the tube hermetically and shake well for 10 s using a vortex
mixer.
After 180 s, open the tube and add 2 mL hexane. After 210 s
add 10 mL disodium hydrogen citrate and sodium chloride
aqueous solution,
D
(
f
). The transesterification time stops after the
addition of the last drop. Shake gently using a vortex mixer. The
transesterification time should not exceed 240 s.
Note 3
: To respect the total reaction time (240 s), the
transesterification reaction should be carried out with a maximum
of six tubes at the same time. Rapid delivery system (dispenser)
can be used to add reagents, but not for the addition of internal
standard solution.
Centrifuge the tube at 1750 rpm (or equivalent rpm to
g
= 375 ±
25) for 5 min.
Into a 10 mL volumetric flask, pipet 200 μL of the supernatant
and make up to the mark with
n
-hexane.
Note 4
: The dilution factor is calculated for on-column and
splitless injection only. When using split injection, adapt the
dilution to obtain the desired peak responses accordingly to split
ratio used (ensure sufficient and accurate detection level for small
peaks especially). Stored in the dark at 4°C, the sample solution
after dilution is stable for 2 days.
G. Determination
(
a
)
Quantitative determination.
(
1
)
Resolution.—
Inject into the gas chromatograph 1.0 μL of the
calibrating solution,
F
(
a
). Determine peak width at half height and
distance between the left of the chromatogram and the top of peak
for C18:1
trans
Δ13/14 and oleic acid methyl ester. The resolution
criteria (R) is calculated as follows:
ܴ = 1.18(
ݐ
ோଶ
െ
ݐ
ோଵ
)/ܹ
൫ ଵ ଶൗ ൯ଵ
+ ܹ
൫ ଵ ଶൗ ൯ଶ
where t
R1
= distance, in centimeters, between the left of the
chromatogram and the top of peak 1 (C18:1
trans
Δ13/14); t
R2
=
distance, in centimeters, between the left of the chromatogram and
the top of peak 2 (oleic acid); W
(½)1
= peak width, in centimeters, at
half height of peak 1 (C18:1
trans
Δ13/14); W
(½)2
= peak width, in
centimeters, at half height of peak 2 (oleic acid).
The resolution test is acceptable when value calculated for R is
equivalent or higher than 1.00 (±5%) (to determine with the ring test).
Note
: Example of the calculation is given in Figure
2012.13C
.
(
2
)
Calculation of response factor.—
Inject into the gas
chromatograph 1.0 μL of the calibrating solution,
F
(
b
). Determine
the area of peaks attributable to each FAME present in the
calibration standard mixture,
D
(
q
), and calculate their respective
response factors (Rf
i
) relative to the internal standard (C11:0):
ܴ݂
= ݉Ԣ
ȉ
ܣ
Ԣ
ை
݉Ԣ
ை
ȉ
ܣ
Ԣ
