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© 2012 AOAC INTERNATIONAL
AOAC Official Method 2012.13
Determination of Labeled Fatty Acids Content
in Milk Products and Infant Formula
Capillary Gas Chromatography
First Action 2012
A. Scope
The method involves the quantification of all labeled fatty acids
that includes groups of fatty acids [i.e.,
trans
fatty acids (TFA),
conjugated linoleic acids (CLA), saturated fatty acids (SFA),
monounsaturated fatty acids (MUFA), polyunsaturated fatty acids
(PUFA), omega-3, omega-6, omega-9] and/or individual fatty acids
[i.e., linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid
(ARA), ecosapentaenoic acid (EPA), docosahexaenoic acid (DHA)]
(
see
also
J
) in milk products and infant formula, containing milk
fat and/or vegetable oils, supplemented or not supplemented with
long-chain polyunsaturated fatty acids (LC-PUFA).
The determination is performed by direct transesterification of
food matrixes, without prior fat extraction, and consequently it is
applicable to liquid samples or reconstituted powders.
Products containing less than 1.5% fat can be analyzed after
preliminary fat extraction using methods described.
In the case of products supplemented/enriched with PUFA
having fish oil or algae origins, a cold fat extraction procedure is
recommended (evaporation of solvents at lower temperature).
B. Principle
Addition of the internal standard solution to the sample,
preparation of fatty acid methyl esters (FAMEs) by direct
transesterification with methanolic sodium methoxide for liquid
samples; dissolution in water and direct transesterification with
methanolic sodium methoxide for powdered samples. Separation of
FAMEs using capillary gas-liquid chromatography. Identification
by comparison with the retention time of pure standards and
quantification as fatty acids by reference to an internal standard.
Verification of the transesterification performance using a second
internal standard.
C. Apparatus
Common laboratory equipment and, in particular, the following:
(
a
)
Analytical balance
.—Capable of weighing to the nearest
1 mg, with a readability of 0.1 mg.
(
b
)
One-mark volumetric flasks
.—50, 100, 250, 300, and
500 mL.
(
c
)
One-mark volumetric pipets
.—2, 5, 10, 25, and 50 mL; ISO
class AS (ISO).
(
d
)
Two-marks pipet, volumetric, with two marks.—
2 and 5 mL;
class AS (ISO).
(
e
)
Micropipet
.—200 μL.
(
f
)
Dispensers
.—2, 5, and 10 mL.
(
g
)
Test tube
.—26 mm (diameter) × 100 mm (length), fitted with
PTFE-lined screw cap.
(
h
)
Test tube mixer
.—Vortex Genie, or equivalent.
(
i
)
Laboratory centrifuge
.—Equipped with adapters for test
tubes with external diameter of 26 mm.
(
j
)
Gas–liquid chromatograph
.—Equipped with flame
ionization detector and capillary split injection system or on-
column.
Note
: Use of the cleanest possible glassware and caps is required
to avoid impurities in the FAME chromatogram.
(
1
)
Carrier gas.—
Hydrogen or helium. Purity ≥99.9997%.
Note
: The use of hydrogen or helium will affect the
chromatography duration especially, and lessen the resolution.
(
2
)
Other gases
.—Free from organic impurities (CnHm of
below 1 ppm), nitrogen and hydrogen, purity at least ≥99.995%,
and synthetic air.
(
3
)
Capillary column
.—With a stationary phase which has
been successfully employed to perform FAMEs separation. It
is suggested to use cyanopropyl-polysiloxane phase capillary
columns (100 m length × 0.25 mm id, 0.25 μm film thickness) that
elute the FAMEs, primarily by carbon chain length and secondarily
by the number of double bonds.
Note
: Traces of oxygen and humidity will damage the polar
phase of the column. If pure gas is not available, use a gas purifying
filter device.
D. Chemicals and Reagents
Use only reagents of recognized analytical grade, unless
otherwise specified, and distilled or demineralized water or water
of equivalent purity.
(
a
)
Water
.—HPLC grade.
(
b
)
Sodium methoxide solution (CH
3
ONa)
.—Dissolved in
methanol 30% (w/v) (ca 5.4 M).
(
c
)
Transesterification solution
.—Sodium methoxide solution
5% in methanol.
Into a 300 mL volumetric flask, pipet 50 mL sodium methoxide
solution,
D
(
b
), and complete gently with 250 mL methanol using
a magnetic stirrer. Remove the magnetic stirrer, then cool to room
temperature, and make up to the mark with methanol.
Stored in the dark at 4°C, this solution is stable for 1 week. Allow
the solution to come to room temperature before use. This solution
volume is sufficient to analyze ca 40 samples. In case of smaller
number of analysis, reagent volume can be adapted accordingly.
Perform the transesterification reaction at ambient temperature
(between 20 to 25°C).
(
d
)
Disodium hydrogen citrate sesquihydrate [HOC(COOH)
(CH
2
COONa)
2
·1.5H
2
O]
.
(
e
)
Neutralization solution
.—Disodium hydrogen citrate
sesquihydrate 10%, sodium chloride 15% in water.
Weigh 50.0 g disodium hydrogen citrate sesquihydrate and 75.0 g
sodium chloride in a 500 mL volumetric flask,
C
(
b
). Dissolve
in 450 mL water using a magnetic stirrer. Remove the magnetic
stirrer, then make up to the mark with water.
Stored in the dark at 4°C, this solution is stable for 1 month.
Presence of salt crystals may appear in the solution during storage,
but disappear after shaking.
Allow the solution to come to room temperature before use. This
solution volume is sufficient to analyze ca 40 samples or more. In
case of a smaller number of analysis (or single analysis), weights
and volume of solution can be adapted accordingly.
(
f
)
tert-Butyl methyl ether (MTBE).
(
g
)
Methyl undecanoate (C11:0 FAME)
.—Purity ≥99% mass
fraction.
(
h
)
Tritridecanoin (C13:0 TAG)
.—Purity ≥99 % mass fraction.
(
i
)
C11:0 FAME/C13:0 TAG standard solution.—
Into a 250 mL
volumetric flask, weigh to the nearest 0.1 mg about 500 mg
tritridecanoin and 500 mg methyl undecanoate. Dissolve and make
up to the mark with MTBE.
Stored in the dark at 4°C, this solution is stable for 1 week. Allow
the solution to come to room temperature before use.
Candidates for 2016 Method of the Year
362