© 2012 AOAC INTERNATIONAL
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Table 2011.10B. Guidelines for loading sample filtrates onto
SPE cartridges
a
diluted with 750 mL laboratory water and adjust pH to 5–7 with
concentrated formic acid.
Vitamin B
in RTF
concentration
μg/L
Volume of filtrate loaded
onto SPE cartridge, mL
Final dilution
volume, mL
(
3
)
Mobile phase C.—
4.0 mL TEA and 750 mL acetonitrile
diluted with 250 mL laboratory water and adjust pH to 5–7 with
product,
<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
concentrated formic acid.
(
4
)
2.5% Acetonitrile in H O (mobile phase D).—
50 mL
acetonitrile diluted to 2000 mL with laboratory water.
(
5
)
10% Acetonitrile in H O.—
150 mL acetonitrile diluted to
1500 mL with laboratory water.
(
6
)
25% Acetonitrile in H O SPE elution solvent.—
25 mL
Alltech C or C
cartridge.
acetonitrile diluted to 100 mL with laboratory water.
8
18
(
c
)
Drierite.—
Desiccant, anhydrous calcium sulfate, 8 mesh.
(
d
)
Ethanol.—
Denatured.
(
e
)
Formic acid.—
ACS.
(
f
)
Laboratory water.—≥
15 MΩ·cm
.
(
g
)
Potassium cyanide.—
97%ACS reagent.
(
h
)
Potassium phosphate dibasic.—
ACS.
Riboflavin - <96% ACS
(
i
)
Sodium acetate anhydrous or sodium acetate trihydrate
.—
ACS.
(
j
)
Taka-diastase.—
Accurate
Chemical
Co.
accuratechemical.com) or equivalent.
(
k
)
Triethylamine (TEA).—
HPLC grade.
(
7
)
30% Acetonitrile in H O SPE elution solvent.—
30.0 mL
acetonitrile diluted to 100 mL with laboratory water.
(
8
)
50% Acetonitrile in H O, column cleaning and storage
solution.—
500 mL acetonitrile diluted to 1000 mL in a volumetric
flask. Expiration 6 months.
(
9
)
25% Ethanol.—
50 mL ethanol diluted to 200 mL with
laboratory water.
(
10
)
0.40% Potassium cyanide.—
Dissolve 0.02 g potassium
cyanide in and dilute to 5 mL with 0.
125
M sodium acetate buffer.
Make fresh immediately before use.
(
11
)
1% Potassium cyanide.—
Dissolve 0.25 g potassium
cyanide in and dilute to 25 mL with laboratory water. Prepare fresh
immediately before use.
Resolution Test Solution:
Weigh ~0.005grams riboflavin onto a
weigh paper. Transfer to a 100 mL volumetric flask and bring to
volume with 10% acetonitrile solution. Stir to dissolve. Mix equal
amounts of solution with the highest concentration vitamin B12
working
standard.
Expiration:
1
day
(
l
)
Vitamin B
(cyanocobalamin) standard.—
USP reference,
(
12
)
0.
251
M sodium acetate buffer.—
Dissolve
16.441
g
sodium
official lot number (refer to USP catalog for current lot). Store in
desiccator protected from white light. (
Note
:
See
standard label for
purity.)
D. Solution and Standard Preparation
All solutions can be scaled up or down for convenience provided
good laboratory practices are observed. Solutions can be stored at
2–30°C in tight, inert containers unless otherwise noted.
(
a
)
Solutions.—
(
1
)
Mobile phase A.—
4.0 mL TEA diluted with
acetate anhydrous or
27.268
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 immediately before use.
(
b
)
Standards.—
Prepare all standards under UV shielded
fluorescent lights and store at 2–8°C in tightly stoppered volumetric
flasks.
1000 mLwater and adjust pH to 5–7 with concentrated formic acid.
(
1
)
Vitamin B
stock standard (10000
μ
g/L).—
Accurately weigh
(
2
)
Mobile phase B.—
4.0 mL TEA and 250 mL acetonitrile
the appropriate amount of vitamin B
USP reference standard to
