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Butler-Thompson et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
6, 2015
1659
riboflavin solution with the highest concentration vitamin B
12
working standard. Expiration: 1 week.
(
12
)
0.25 M Sodium acetate buffer
.—Dissolve 41 g
sodium acetate anhydrous or 68 g sodium acetate trihydrate
in approximately 1800 mL laboratory water. Adjust pH to
4.50 with concentrated acetic acid. Dilute to 2000 mL with
laboratory water. Expiration 3 months.
(
13
)
6% Taka-diastase
.—Dissolve 0.6 g taka-diastase in
10 mL water. Prepare fresh daily before use.
(b)
Standards
.—Prepare all standards in volumetric
glassware under UV shielded fluorescent lights and store at
2–8°C in tightly stoppered volumetric flasks.
(
1
)
Vitamin B
12
stock standard (10 000
μ
g/L)
.—Accurately
weigh, to 0.00001 g, the appropriate amount of vitamin B
12
USP reference standard to give a stock standard concentration
of 10 000 μg/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
12
reference standard that should be weighed:
S
w
= 10 000 × 0.1 × 1/P
where S
w
= amount of vitamin B
12
standard to be weighed
in mg; 10000 = 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.
(
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
) and (
2
) of
the sample preparation procedure described below. (
3
) Combine
all extracted sample filtrates. Accurately transfer 1 mL solution
prepared in step (
1
) to 80 or 100 mL of sample filtrate (spiked
sample), and accurately transfer 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 vitaminB
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, pediatric, 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 four significant figures. Typical weights
are 20 g for adult and pediatric 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 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 an
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 to which 0.5 g milk protein has
been added, 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 pass by gravity 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 the vitamin B
12
concentration is unknown, use guidelines for RTF products
containing 1
–
10 µg/L. 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 eluent. After all of the sample filtrate
has passed through the cartridge, rinse each cartridge with 5 mL
Table 2011.10D. Guidelines for loading sample filtrates
onto SPE cartridges
a
Vitamin B
12
concentration
in RTF product, µg/kg
Volume of filtrate loaded
onto SPE cartridge, mL
Final dilution
volume, mL
<1
80
5
1–10
70–80
10
11–20
50–60
10
21–50
20–40
10
a
Do not load more than 60 mL adult and pediatric nutritionals onto an
Alltech C
8
or C
18
cartridge.
137