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cyanocobalamin/mg of the standard.
See
standard label.
(
2
)
Vitamin B
12
intermediate standard (1000 μg/L).—
Dilute
10 mL vitamin B
12
stock standard solution to 100 mL with
laboratory water. Expiration 1 week.
(
3
)
Vitamin B
12
working standards (2.5–25 μg/L).—
Dilute 0.5,
1, 2, 3, 4, and 5 mL vitamin B
12
intermediate standard solution to
200 mL with 10% acetonitrile. Expiration 1 month.
E. Procedure
Prepare all samples under UV shielded fluorescent lights. Mix
or stir products before sampling to ensure all product samples are
uniform and representative. Store prepared product samples up to
14 days after preparation in tightly stoppered volumetric flasks at
2–8°C.
(
a
)
SPE cartridge qualification
.—To establish SPE cartridge
equivalency or to verify the suitability of new lots of cartridges:
(
1
) Prepare a solution containing 160 µg/L vitamin B
12
in water.
(
2
) Prepare three samples from one representative product that
contains the highest amount of protein of any product that will
be analyzed with this method following steps
E
(
b
)(
1
)–(
2
) of the
sample preparation procedure described below.
(
3
) Combine all extracted sample filtrates. Add 1 mL of the
solution prepared in step (
1
) to 80 or 100 mL of sample filtrate
(spiked sample), and add 1 mL water to 80 or 100 mL of sample
filtrate (unspiked sample).
(
4
) Continue preparing the spiked and unspiked sample using
the sample cleanup and concentration,
E
(
b
)(
3
), and final dilution,
E
(
b
)(
4
), procedures described in the sample preparation procedure
below.
(
5
) Analyze the two samples chromatographically.
(
6
) Calculate the vitamin B
12
concentration of the spiked and
unspiked samples and calculate the spike recovery.
(
7
) In order for the cartridges to be considered acceptable, spike
recoveries should be
≥
90%.
(
b
)
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 product (±10%)
into a 100 mL volumetric flask and record the weight to at least
4 significant figures. Typical weights are 20 g for adult and
pediatric ready-to-feed (RTF) liquids and reconstituted powders,
25 g for infant RTF liquids and reconstituted powders, and 3 g for
unreconstituted powders. Add 25 mL laboratory water to flasks
containing unreconstituted powders and mix until all of the powder
dissolves.
Add 1 mL of 6% taka-diastase to products containing starch.
Allow taka-diastase to react with samples for at least 30 min before
continuing with the extraction.
Note
: Add 0.5 g of a milk protein, such as calcium caseinate,
to nutritional products that do not contain any intact protein (i.e.,
infant elemental powders) and reconstitute or add water to the
powder immediately before the extraction step.
(
2
)
Extraction.—
Add 30 mL 0.25 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.) Remove samples from
the oven and immediately cool in ice bath. Dilute samples to volume
with laboratory water. Mix well. Filter samples through Whatman
2V filter paper
(www.whatman.com)into 125 mL Erlenmeyer
flasks or equivalent glassware.
Note
: If prepared samples are milky
and contain very small insoluble particles, centrifuge samples and
then transfer liquid layer to funnels lined with Whatman 2V filter
paper.
Note
: Do not heat samples that 0.5 g of milk protein have been
added to, but continue with the dilution and filtration steps.
(
3
)
Sample cleanup and concentration.—
For each sample,
insert a 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.
Note
: Alltech C
8
and C
18
cartridges can be used
interchangeably. Condition each cartridge with at least 20 mL
acetonitrile by allowing acetonitrile to gravity filter through the
cartridge and rinse each cartridge with at least 10 mL laboratory
water.
Using volumetric pipets, transfer sample filtrates to cartridges
using the guidelines in Table
2011.10D
. If necessary apply enough
vacuum so that the samples drip steadily through the cartridges.
Sample filtrates should pass through the cartridges at a rate of no
more than 120 drops/min. Discard eluant. After all of the sample
filtrate has passed through the cartridge, rinse each cartridge with
5 mL laboratory water and discard eluant. Air-dry each cartridge
by pulling a vacuum until no more effluent is observed. Close each
stopcock. Place a 5 or 10 mL volumetric flask under each cartridge.
Add 4.4 mL 30% acetonitrile to each cartridge. Open each stopcock
and elute vitamin B
12
into the volumetric flasks.
(
4
)
Final dilution
.—For samples collected in 10 mL volumetric
flasks, dilute to volume with water. For samples collected in 5 mL
volumetric flasks, in a hood add 0.1 mL freshly prepared 0.4%
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.
Table 2011.10F. Reversed-phase column gradient
Time, min
Mobile phase, %
A
B
C
0.00
90
10
0
14.5
90
10
0
14.6
40–60
a
60–40
a
0
27.0–30.0
40–60
a
60–40
a
0
27.1–30.1
0
10
90
29.90–33.00
0
10
90
a
Appropriate gradient conditions must be established with each column
to adequately resolve vitamin B
12
and riboflavin and to elute vitamin B
12
between approximately 24 and 30 min. To establish appropriate gradient
conditions with a new column, set the gradient composition at 14.6 and
27.0–30.0 min to the midpoint of the allowable range from the table.
Inject the resolution test solution and calculate the resolution (R) between
vitamin B
12
and riboflavin. Adjust the mobile phase composition between
14.6 and 27.0–30.0 min until R is > 1.5. After vitamin B
12
elutes from the
C
18
or phenyl column, rinse the column with 90% mobile phase C for at
least 2.8 min.
Table 2011.10E
.
System configuration
Time, min
Valve configuration
0.00–10.5
Configuration 1
10.5–14.5
Configuration 2
14.5–30.0 to 33
Configuration 1
Candidates for 2016 Method of the Year
342