© 2012 AOAC INTERNATIONAL
8
8
12
S
w
12
12
12
12
12
2
2
18
18
w
Table 2011.10C
.
System configuration
Time, min
Valve configuration
up and concentrated, insert a
600–
900 mg SPE cartridge onto the
stopcock of the vacuum manifold and attach a 30 mL disposable
syringe barrel to the top of each cartridge.
As a general guideline,
0.00–10.5
Configuration 1
600 mg Alltech C or C cartridges have enough capacity for use
10.5–14.5
Configuration 2
14.5–30.0
Configuration 1
with most products; however, if products on an RTF basis contain
high levels of protein (>5–6%) or high levels of hydrolyzed proteins
(>4%), 900 mg Alltech C or C cartridges should be used.
Note
:
Alltech C and C
cartridges can be used interchangeably.
8
18
give a stock standard concentration of 10 000
g/L. Dissolve in and
dilute to 100 mL with 25% ethanol. Expiration 6 months.
Use the following equation to calculate the amount of vitamin B
reference standard that should be weighed:
= 10000 × 0.1 × 1/P
Condition each cartridge with at least 20 mL acetonitrile
b y
a l lowi n g a c e ton it r il e t o g rav i t y fi l t e r th rou gh th e
c a r t r id ge
and
rinse each cartridge with at least 10 mL laboratory water.
Using volumetric pipets, transfer sample filtrates to cartridges
using the guidelines in Table
2011.10B
. If necessary apply enough
vacuum so that the samples drip steadily through the cartridges.
Discard eluant.
After all of the sample filtrate has passed through the cartridge,
where S
= amount of vitamin B
standard to be weighed in
rinse each cartridge with 5 mL laboratory water and discard eluant.
Air-dry each cartridge by pulling a vacuum until no more effluent
mg; 10 000 = desired stock standard concentration in μg/L; 0.1 =
dilution volume in L; P = purity of the USP reference standard in μg
cyanocobalamin/mg of the standard.
See
standard label.
is observed. Close each stopcock.
Place a 5 or 10 mL volumetric flask under each cartridge.
(
2
)
Vitamin B
intermediate standard (1000 μg/L).—
Dilute
Add 4.4 mL
25% acetonitrile to all Alltech 600 mg cartridges
and 4.4 mL
30% acetonitrile to all Alltech 900 mg cartridges. Open
10 mL vitamin B
stock standard solution to 100 mLwith laboratory
each stopcock and elute vitamin B
into the volumetric flasks.
water. Expiration 1 week.
Final dilution
.—For samples
12
collected
in 10 mL volumetric
(
3
)
Vitamin B
working standards (2.5–25 μg/L).—
Dilute 0.5,
flasks, dilute to volume with water. For samples collected in 5 mL
1, 2, 3, 4, and 5 mL vitamin B
intermediate standard solution to
volumetric flasks, in a hood add 0.1 mL freshly prepared 0.4%
200 mL with 10% acetonitrile. Expiration 1 month.
E. Procedure
**A section describing a procedure for evaluating SPE cartridge
performance will be added here.**
Prepare all samples under UV shielded fluorescent lights. Store
prepared product samples up to 14 days after preparation (store at
2–8°C in tightly stoppered volumetric flasks). Mix or stir products
before sampling to ensure all product samples are uniform and
representative.
(
a
)
Sample preparation for infant and adult nutritional
products.—
(
1
)
Sampling.—
Mix all products thoroughly before
sampling. Reconstitute nonhomogeneous powders per label
instructions. Weigh the appropriate amount of sample (±10%)
KCN to each volumetric flask. Place prepared samples in a 95°C
oven for at least 1.5 h, but for no more than 4 h. After at least
1.5 h, remove samples from the oven and cool to room temperature.
Dilute to volume with laboratory water. Filter an aliquot of each
standard and prepared sample through a 0.45
m syringe filter into
an autosampler vial.
(
b
)
HPLC analysis.—
(
1
)
System setup and configuration.—See
Figures
2011.10A
and
B
for configurations.
(
2
)
Instrument operation conditions.—
(
a
)
Run time
.—30 min.
(
b
)
Injection volume
.—
900 uL to
2.0 mL.
(
c
)
System configuration.—See
Table
2011.10C
.
(
d
)
Isocratic pump.—
Mobile phase D: 2.5% acetonitrile.
into a 100 mL volumetric flask and record the weight to at least 4
Flow rate: Adjust so that vitamin B
elutes from the size-
significant figures. Typical weights are 20
g for adult and pediatric
and
–
3025
g for
infant
ready-to-feed
exclusion column between 10.5 and
12
14.5 min. Typical flow rates,
(RTF) liquids and reconstituted powders and 3 g for unreconstituted
powders. Add 25 mL laboratory water to all unreconstituted powder
samples and mix until all of the powder dissolves.
1.1–1.2 mL/min.
Note
: To determine an appropriate flow rate,
connect the size-exclusion column directly to the UV-Vis detector
and inject the high standard. Adjust flow rate as necessary so that
Add 1 mL of 6% taka-diastase if samples contain significant
vitamin B
elutes between 10.5 and 14.5 min.
levels of starch. Allow taka-diastase to react with samples for at
(
e
)
12
Gradient
pump
.—Mobile phase compositions: mobile
least 30 min before continuing with the extraction.
(
2
)
Extraction.—
Add 30 mL 0.
125
M sodium acetate
buffer
(pH 4.5) to each sample and swirl to mix.
In a hood, add 1 mL freshly prepared 1% KCN to each sample
and swirl to mix.
Heat samples in a 105°C oven for at least 60 min, but for no
more than 120 min. (Oven temperature will drop when the door
is opened. Start timing when oven temperature returns to 105°C.)
phase A, 0.4% TEA in laboratory water, pH 5–7; mobile phase B,
0.4% TEA and 25% acetonitrile in H O, pH 5–7; mobile phase C,
0.4% TEA and 75% acetonitrile in H O, pH 5–7. Gradient to
Table 2011.10D. C18 column gradient
Mobile phase, %
After at least 60 min, remove samples from oven and immediately
Time, min
A
B
C
cool in ice bath.
Dilute samples to volume with laboratory water. Mix well.
Filter samples through Whatman 2V filter paper (
.
com) into 125 mL Erlenmeyer flasks or equivalent glassware.
Note
:
If prepared samples are milky and contain very small insoluble
