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© 2015 AOAC INTERNATIONAL
AOAC Official Method 2011.10
Vitamin B
12
in Infant and Pediatric Formulas
and Adult Nutritionals
High-Performance Liquid Chromatography
First Action 2011
Final Action 2014
(Applicable to the determination of vitamin B
12
in infant and
pediatric formulas and adult nutritionals.)
Caution:
Refer to Material Safety Data Sheets (MSDS) of
chemicals prior to use and follow the suggested personal
protective equipment.
Potassium cyanide is highly toxic. When handling this
chemical, wear gloves and appropriate personal protective
equipment. Weigh chemical and dispense solutions in a
fume hood. Perform test in a well-ventilated area. Treat
sample waste with sodium hypochlorite and dispose of
waste according to local, state, and federal regulations.
See
Tables
2011.10A–C
for results of the single-laboratory
validation and multilaboratory study supporting acceptance of the
method.
A. Principle
Vitamin B
12
is extracted from samples using sodium acetate
buffer (pH 4.5) and potassium cyanide at 105°C. Extracts are
purified and concentrated with C
8
or C
18
solid-phase extraction
(SPE) cartridges and analyzed with size-exclusion and reversed-
phase chromatography. Determination of B
12
is made by liquid
chromatography with visible detection at 550 nm.
B. Apparatus and Materials
(
a
)
HPLC system.—
Gradient system with switching valve and
additional isocratic pump and a UV-Vis detector equipped with
a tungsten lamp (capable of monitoring at 550 nm wavelength).
Autosampler capable of injecting 900 μL to 2 mL sample.
(
b
)
Column.—
Analytical size-exclusion column 4 μ, 250 ×
9.4 mm (Zorbax GF-250, P/N 884973-901; www.chem.agilent.
com), 5 μ, 300 × 8 mm (Shodex Protein KW-802.5, P/N F6989000)
or equivalent.
(
c
)
Column.—
Analytical C
18
column 3 μ, 100 × 4.6 mm (Thermo
Scientific Aquasi1 P/N 77503-104630; www.thermoscientific.
com) with C
18
drop-in guard cartridges 3 µ, 10 × 4.6 mm (Thermo
Scientific Aquasil P/N 77503-014001), Epic Phenyl Hexyl, 3 µ,
120A, 100 × 4.6 mm (ES Industries P/N 125191-EPHX; www.
esind.com)with appropriate guard cartridge; or equivalent.
(
d
)
Oven.—
Capable of maintaining temperatures of 95 ± 5°C
and 105 ± 5°C.
(
e
)
pH meter.—
With calibration buffer
.
(
f
)
Analytical balance.—
Capable of weighing 0.00001 g.
(
g
)
Beakers.—
Glass, assorted sizes.
(
h
)
Bottle top dispenser.—
Capable of dispensing 30 mL.
(
i
)
Cylinders.—
Graduated glass, assorted sizes.
(
j
)
Desiccator.
(
k
)
Erlenmeyer flasks.—
125 mL or equivalent glassware.
(
l
)
Filter paper.—
Whatman 2V or equivalent.
(
m
)
Funnels.—
Plastic, suitable to use with filter paper.
(
n
)
Gloves.
(
o
)
Pipettor.—
Variable volume, 100–1000 μL.
(
p
)
Shields.—
Yellow or clear shields with a cutoff of at least
385 nm.
(
q
)
SPE cartridges.—
C
8
900 mg (Alltech/Grace Davison
P/N 20966), C
18
900 mg (Alltech/Grace Davison P/N 20942), or
equivalent.
See
E
, procedure for SPE cartridge qualification.
(
r
)
Syringes.—
Disposable, assorted sizes.
(
s
)
Syringe filters.—
0.45 μm nylon.
(
t
)
Vacuum manifold —
24 ports with stopcocks or equivalent.
(
u
)
Volumetric pipets.—
Assorted sizes.
(
v
)
Volumetric flasks.—
Assorted sizes.
C. Reagents
(
a
)
Acetic acid.—
Glacial, ACS.
(
b
)
Acetonitrile.—
HPLC grade.
(
c
)
Drierite.—
Desiccant, anhydrous calcium sulfate, 8 mesh.
(
d
)
Ethanol.—
Reagent alcohol, 95%, denatured.
Table 2011.10A. Single-laboratory validation: Repeatability precision data for vitamin B
12
Sample type
No. of replicates
(duplicates on multiple days)
Mean (µg/100 g RTF)
SD
r
RSD
r
, %
Infant formula (NIST SRM 1849a)
18
42.4
a
1.37
3.27
Infant formula powder soy-based
12
0.415
0.0150
3.62
Infant formula powder milk-based
12
0.236
0.0083
3.52
Infant formula RTF milk-based
12
0.355
0.0083
2.34
Infant formula powder partial
hydrolyzed milk-based
12
0.377
0.0132
3.50
Infant formula powder partial
hydrolyzed soy-based
12
0.257
0.0090
3.51
Adult nutritional powder milk-based
12
0.299
0.0141
4.72
Adult nutritional RTF high protein
12
1.13
0.0250
2.21
Child formula powder
12
0.955
0.0225
2.36
Adult nutritional RTF high fat
12
1.65
0.0463
2.81
Infant elemental powder
12
0.540
0.0223
4.13
Adult nutritional powder low fat
12
0.666
0.0187
2.81
a
Results in µg/kg powder.
Candidates for 2016 Method of the Year
339