196 / 258
212
G
olay
&
M
oulin
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
omega-6, and omega-9] in milk products, infant formula, and
adult/pediatric nutritional formula containing milk fat and/or
vegetable oils, supplemented or not supplemented with oils rich
in long-chain PUFAs.
The determination is performed by direct transesterification
of food samples without prior fat extraction and, consequently,
it is applicable to liquid samples or reconstituted powder.
Products containing <1.5% fat can be analyzed after
preliminary fat extraction using a suitable fat-extraction
reference method (i.e., ISO/IDF-AOAC).
In the case of products supplemented or enriched with PUFAs
having fish-oil or algae origins, the extraction solvents must be
evaporated at a maximum of 40°C.
B. Principle
Addition of the internal standard solution to the sample,
preparation of FAMEs by direct transesterification with
methanolic sodium methoxide for liquid samples and fat
extracted from food; dissolution (i.e., reconstitution) in water
for powder sample and direct transesterification with methanolic
sodium methoxide. Separation of FAMEs using capillary GLC.
Identification of peak by comparison with the retention time of
pure standards and quantification as fatty acids by reference to
an internal standard (C11:0 FAME) and instrument response
factors. Verification of the transesterification performance using
a second internal standard [C13:0 triacylglycerols (TAG)].
C. Apparatus and Materials
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;
class AS (ISO).
(d)
Two-mark pipet, volumetric.—
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 Scientific Industries,
Inc., Bohemia, NY, 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. Autosampler and integration system preferably
computerized.
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 affects principally the
chromatography duration but does not have any significant
impact on the chromatographic resolution.
(
2
)
Other gases
.—Free from organic impurities (C
n
H
m
<1 ppm), nitrogen and hydrogen, purity at least ≥99.995%, and
compressed pure air.
(
3
)
Capillary column
.—Cyanopropyl-polysiloxane phase
(or equivalent polarity) capillary columns with 100 m length ×
0.25 mm id, 0.2 μm film thickness.
Note
: Traces of oxygen and humidity will damage the polar
phase of the column. When 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.
(a)
Water
.—HPLC grade or equivalent quality.
(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 dilute 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. Perform the transesterification reaction
at ambient temperature (20–25°C).
(d)
Disodium hydrogen citrate sesquihydrate [HOC(COOH)
(CH
2
COONa)
2
·1.5H
2
O].
(e)
Sodium chloride (NaCl).
—Puriss.
(f)
Neutralization
solution
.—Disodium hydrogen citrate
sesquihydrate 10%, sodium chloride 15% in water. Weigh
50.0 g disodium hydrogen citrate sesquihydrate,
D
(
d
), and
75.0 g sodium chloride,
D
(
e
), in a 500 mL volumetric flask,
C
(
b
). Dissolve in 450 mL water using a magnetic stirrer.
Remove the magnetic stirrer, and dilute to volume with water.
Stored in the dark at 4°C, this solution is stable for 1 month. Salt
crystals may appear in the solution during storage but disappear
after shaking. Allow the solution to come to room temperature
before use.
(g)
tert-Butyl methyl ether (MTBE)
.
(h)
Methyl undecanoate (C11:0 FAME)
.—Purity ≥99% mass
fraction.
(i)
Tritridecanoin (C13:0 TAG)
.—Purity ≥99% mass
fraction.
(j)
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,
D
(
i
), and 500 mg methyl undecanoate,
D
(
h
). Dissolve and dilute to the mark with MTBE. Stored in the
dark at 4°C, this solution is stable for 1 wk. Allow the solution
to come to room temperature before use.
(k)
Octadecenoic acid methyl ester
.—
Cis/trans
isomer mixture
of C18:1 with
trans
-4 to
trans
-16 (all isomers) and principal
cis
isomers. Concentration 2.5 mg/mL in methylene chloride.
Note
: This standard is commercially available from the
Supelco brand of Sigma-Aldrich St. Louis, MO (Cat. No.
40495-U).
(l)
Linoleic acid methyl ester
.—
Cis/trans
isomer mixture
of C18:2 with
trans
-9,
trans
-12-octadecadienoic acid (50%);
cis
-9,
trans
-12-octadecadienoic acid (20%);
trans
-9,
cis
-12-
octadecadienoic acid (20%); and
cis
-9,
cis
-12-octadecadienoic
acid (10%). Concentration 10 mg/mL in methylene chloride.
Note
: This standard is commercially available from the
Supelco brand of Sigma-Aldrich (Cat. No. 47791).
196