© 2012 AOAC INTERNATIONAL
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AOAC Official Method 2011.10
Scientific Aquasi1 P/N 77503-104630;
.
Vitamin B
in Infant Formula
com) with C drop-in guard cartridges 3 μ, 10 × 4.6 mm (Thermo
and Adult Nutritionals
High-Performance Liquid Chromatography
First Action 2011
Scientific Aquasil P/N 77503-014001)
, Epic Phenyl Hexyl, 3u,
120A, 100 x 4.6 mm (ES Industries 125191-EPHX) with
appropriate guard cartridge;
or equivalent.
(
d
)
Oven.—
Capable of maintaining temperatures of 95 ± 5°C
and 105 ± 5°C.
(Applicable to the determination of vitamin B
and adult nutritionals.)
in infant formula
(
e
)
pH meter.—
With calibration buffer
.
(
f
)
Analytical balance.—
Capable of weighing 0.00001 g.
Caution:
Refer to Material Safety Data Sheets (MSDS) of
chemicals prior to use and follow the suggested personal
protective equipment.
NOTE: Potassium cyanide is
highly toxic. When handling this chemical, wear gloves
and appropriate personal protective equipment. Weigh
chemical and dispense solutions in a 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
Table
2011.10A
for infant formula and adult nutritional
matrixes for which the method was validated.
A. Principle
(
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.
(
l
)
Filter paper.—
Whatman 2V or equivalent.
(
m
)
Funnels.—
Plastic, suitable to use with filter paper.
Vitamin B
is extracted from the sample using sodium acetate
(
n
)
Gloves.
buffer (pH 4.5) and potassium cyanide at 105°C. Extracts are
purified and concentrated with C8 or C18 solid-phase extraction
(SPE) cartridges and analyzed with size-exclusion and reversed-
(
o
)
Pipettor.—
Variable volume, 100–1000 μL.
(
p
)
Shields.—
Yellow or clear shields with a cutoff of 385 nm.
(
q
)
SPE cartridges.—
C 600 mg (Alltech/Grace Davidson P/N
phase chromatography. Determination of B
is made by liquid
20958;
),
C 900 mg (Alltech/Grace
chromatography with visible detection at 550 nm.
B. Apparatus and Materials
(
a
)
HPLC system.—
Gradient system with switching valve
and isocratic pump on side and a UV-Vis detector equipped with
a tungsten lamp (capable of monitoring at 550 nm wavelength).
Autosampler capable of injecting
900 uL or
2 mL sample.
(
b
)
Column.—
Analytical size-exclusion column 4 μ, 250 ×
9.4 mm (Zorbax GF-250, P/N 884973-901;
com), 5 μ, 300 × 8 mm (Shodex Protein KW-802.5, P/N F6989000)
or equivalent.
Davidson P/N 20966),
C 600 mg (Alltech/Grace Davidson P/N
20934),
C 900 mg (Alltech/Grace Davidson P/N 20942), or
equivalent.
(
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.—
ACS.
(
c
)
Column.—
Analytical C
column 3 μ, 100 × 4.6 mm (Thermo
(
b
)
Acetonitrile.—
HPLC grade.
Table 2011.10A. Infant formula and adult nutritional matrixes for which method was validated
Product type
HPLC method, μg/kg (RSD, %) Microbiological method, μg/kg
HPLC/micro, %
Hypoallergenic infant formula powder
51.4 (4.00)
n
= 12
Soy-based infant formula powder
47.8 (5.30)
n
= 15
High-calorie milk-based infant formula powder
59.5 (5.19)
n
= 12
Milk-based infant formula liquid
4.30 (4.42)
n
= 9
Lactose-free infant formula powder
42.9 (4.70)
n
= 12
Child nutritional powder
7.04 (5.27)
n
= 9
Adult nutritional powder
51.7 (5.66)
n
= 12
Adult nutritional beverage
11.2 (4.85)
n
= 21
Metabolic child/adult nutritional powder
48.0 (8.88)
n
= 9
Adult diabetic nutritional beverage
2
n
= 9
Metabolic child/adult nutritional powder
8
n
= 14
Disease-specific adult nutritional beverage
1
n
= 9
