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Butler-Thompson et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
6, 2015
local, state, and federal regulations.
See
Tables
2011.10A–C
for results of the single-laboratory
validation and interlaboratory study supporting acceptance of
the method.
A. Principle
Vitamin B
12
is extracted from samples using sodium acetate
buffer (pH 4.5) and potassium cyanide at 105°C. Extracts
are purified and concentrated with C
8
or C
18
SPE cartridges
and analyzed with size-exclusion and RP chromatography.
Determination of B
12
is made by LC with UV at 550 nm.
B. Apparatus and Materials
(a)
HPLC system
.—Gradient systemwith switching valve and
additional isocratic pump and a UV-Vis detector equipped with
a tungsten lamp (capable of monitoring at 550 nm wavelength).
Autosampler capable of injecting 900 µL to 2 mL sample.
(b)
Column
.—Analytical size-exclusion column 4 μm,
250 × 9.4 mm (Zorbax GF-250, P/N 884973-901;
www.chem. agilent.com),5 μm, 300 × 8 mm (Showa Denko America,
Inc., New York, NY) Protein KW-802.5, P/N F6989000), or
equivalent.
(c)
Column
.—Analytical C
18
column 3 μm, 100 × 4.6 mm
(Thermo Scientific Aquasil P/N 77503-104630; www.
thermoscientific.com) with C
18
drop-in guard cartridges 3 μm,
10 × 4.6 mm (Thermo Scientific Aquasil P/N 77503-014001),
Epic phenyl hexyl, 3 µm, 120 Å, 100 × 4.6 mm (ES Industries
P/N 125191-EPHX;
www.esind.com)with appropriate guard
cartridge, or equivalent.
(d)
Oven
.—Capable of maintaining temperatures of
95 ± 5°C and 105 ± 5°C.
(e)
pH meter
.—With calibration buffer.
(f)
Analytical balance
.—Capable of weighing to 0.00001 g.
(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 or equivalent glassware.
(l)
Filter paper
.—Whatman 2V
(www.whatman.com)or
equivalent.
(m)
Funnels
.—Plastic, suitable to use with filter paper.
(n)
Gloves
.
(o)
Pipettor
.—Variable volume, 100–1000 μL.
(p)
Shields
.—Yellow or clear shields with a cutoff of at least
385 nm.
(q)
SPE cartridges
.—C
8
900 mg (Alltech/Grace Davison,
Bannockburn, IL; P/N 20966), C
18
900 mg (Alltech/Grace
Davison; P/N 20942), or equivalent.
See
E
for SPE cartridge
qualification.
(r)
Syringes
.—Disposable, assorted sizes.
(s)
Syringe filters
.—0.45 μm nylon.
(t)
Vacuummanifold
.—24 ports with stopcocks or equivalent.
(u)
Volumetric pipets
.—Assorted sizes.
(v)
Volumetric flasks
.—Assorted sizes.
C. Reagents
(a)
Acetic acid
.—Glacial, ACS.
(b)
Acetonitrile
.—HPLC grade.
(c)
Drierite
.—Desiccant, anhydrous calcium sulfate, 8 mesh.
(d)
Ethanol
.—Reagent alcohol, 95%, denatured.
(e)
Formic acid
.—88%, ACS.
(f)
Laboratory water
.—≥15 MΩ·cm.
(g)
Potassium cyanide
.—≥97%, ACS.
(h)
Riboflavin
.—≥96%, ACS.
(i)
Sodium acetate anhydrous or sodium acetate
trihydrate
.—ACS.
(j)
Taka-diastase
.—Accurate
Chemical
Co.
(www.
accuratechemical.com) or equivalent.
(k)
Triethylamine (TEA)
.—HPLC grade.
(l)
Vitamin B
12
(cyanocobalamin) standard
.—USP reference,
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 1000 mL water and pH adjusted to 5–7 with about 1.25 mL
concentrated formic acid. Expiration 1 week.
(
2
)
Mobile phase B
.—4.0 mL TEA and 250 mL acetonitrile
diluted with 750 mL laboratory water and pH adjusted
to 5–7 with about 1.25 mL concentrated formic acid.
Expiration 1 week in tightly stoppered container.
(
3
)
Mobile phase C
.—4.0 mL TEA and 750 mL acetonitrile
diluted with 250 mL laboratory water and pH adjusted
to 5–7 with about 1.25 mL concentrated formic acid.
Expiration 1 week in tightly stoppered container.
(
4
)
Mobile phase D (2.5% acetonitrile in water)
.—50 mL
acetonitrile diluted to 2000 mL with laboratory water.
Expiration 1 week in tightly stoppered container.
(
5
)
10% Acetonitrile in water
.—150 mL acetonitrile diluted
to 1500 mL with laboratory water. Expiration 1 month in tightly
stoppered container.
(
6
)
30% Acetonitrile in H
2
O SPE elution solvent
.—
30.0 mL acetonitrile diluted to 100 mL with laboratory water.
Expiration 1 month in tightly stoppered container.
(
7
)
50% Acetonitrile in water, column cleaning and storage
solution
.—500 mL acetonitrile diluted to 1000 mL in a
volumetric flask. Expiration 6 months.
(
8
)
25% Ethanol
.—50 mL ethanol diluted to 200 mL
with laboratory water. Expiration 1 year in tightly stoppered
container.
(
9
)
0.40% Potassium cyanide for samples with 5 mL final
dilution volume
.—Dissolve 0.02 g potassium cyanide in and
dilute to 5 mL with 0.25 M sodium acetate buffer. Make fresh
daily before use.
(
10
)
1% Potassium cyanide
.—Dissolve 0.25 g potassium
cyanide in and dilute to 25 mL with laboratory water. Prepare
fresh daily before use.
(
11
)
Resolution test solution for determining appropriate
gradient conditions when a new analytical column is installed
.—
Weigh about 0.005 g riboflavin onto a weighing paper. Transfer
to a 100 mL volumetric flask and dilute to volume with 10%
acetonitrile solution. Stir to dissolve. Mix equal amounts of
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