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4
S
alvati
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
3, 2016
(
5
)
2
H
3
-pyridoxamine
stock
solution.
—Approximate
concentration: 40 μg/mL. Weigh 4.0 ± 0.1 mg into a tared
weighing vessel. Quantitatively transfer to a 100 mL volumetric
flask with laboratory water and QS with laboratory water.
Mix well and transfer to a 100 mL amber bottle and store
refrigerated (2°–8°C). Expiration: Until exhausted or evidence
of contamination.
(
6
)
13
C
4
-thiamine chloride stock solution.
—Approximate
concentration: 100 μg/mL.
(
i
)
0.12 M HCl.
—Add approximately 300 mL water to a
500 mL graduated cylinder. Add 5.0 ± 0.1 mL conc. HCl and
swirl to mix. Bring to 500 mL with laboratory water and mix
well.
(
ii
) Weigh 5.0 ± 0.1 mg
13
C
4
-thiamine into a tared weighing
vessel. Quantitatively transfer to a 50 mL volumetric flask with
0.12 N HCl and QS with 0.12 N HCl. Mix well and transfer to a
100 mL amber bottle and store refrigerated (2–8°C). Expiration:
Until exhausted or evidence of contamination.
(
7
)
13
C
4
,
15
N
2
-riboflavin stock solution.
—Approximate
concentration: 73 μg/mL.
(
i
)
1.0% Acetic acid in water.
—Add approximately 30 mL
water to a 500 mL graduate cylinder. Add 5.0 ± 0.1 mL glacial
acetic acid and swirl to mix. Bring to 500 mL with laboratory
water and mix well.
(
ii
) Weigh 7.3 ± 0.1 mg
13
C
4
,
15
N
2
-riboflavin into a tared
weighing vessel. Quantitatively transfer to a 100 mL volumetric
flask with 1.0% acetic acid and QS with 1.0% acetic acid.
Mix well and transfer to a 100 mL amber bottle and store
refrigerated (2–8°C). Expiration: Until exhausted or evidence
of contamination.
(h)
Internal standard stock mixture (ISSM).
—Combine
2500 μL of 50 mM ammonium formate with 250 μL
2
H
4
-
nicotinamide stock, 250 μL
2
H
4
-nicotinic acid stock, 250 μL
13
C
4
-pyridoxine stock, 200 μL
2
H
3
-pyridoxal stock, 50 μL
2
H
3
-
pyridoxamine stock, 250 μL
13
C
4
-thiamine stock, and 250 μL
13
C
4
,
15
N
2
-riboflavin acid stock. Volume provides sufficient
ISSM for six working standards and 32 samples. Scale as
needed. Prepare fresh daily.
(i)
Phosphate buffer solution, pH 5.0.
—0.10 M potassium
phosphate dibasic, 1% EDTA, 2% metaphosphoric acid.
(
1
) Weigh 20.0 ± 0.2 g EDTA into a tared weighing vessel
and quantitatively transfer to a 2000 mL beaker containing
approximately 1800 mL laboratory water and add a magnetic
stir bar.
(
2
) Weigh 34.8 ± 0.1 g potassium phosphate dibasic into a
tared weighing vessel and quantitatively transfer to the 2000 mL
beaker already containing approximately 1800 mL laboratory
water and EDTA. Mix by stirring on a magnetic stir plate until
both the EDTA and potassium phosphate dibasic is completely
dissolved.
(
3
) Weigh 40.0 ± 0.2 g metaphosphoric acid into a tared
weighing vessel and quantitatively transfer to the 2000 mL beaker
containing approximately 1800 mL laboratory water, EDTA, and
potassium phosphate dibasic. Mix by stirring on a magnetic stir
plate until the metaphosphoric acid is completely dissolved.
Adjust the pH of the solution to pH 5.00 ± 0.02 using 40%
potassium hydroxide or 85% phosphoric acid. Quantitatively
transfer the solution to a 2000 mL volumetric flask and dilute to
volume with laboratory water. Expiration: 48 h.
(j)
Native compounds.
—Stock standard solutions.
(
1
)
Vitamin standard stock mixture (VSSM).
—Accurately
weigh the indicated amounts for the following standards using
separate weighing funnels or other appropriate weighing vessel
and quantitatively transfer to a 100 mL volumetric flask using
phosphate buffer (pH 5):
(
i
)
Niacinamide.
—70.5 ± 0.5 mg.
(
ii
)
Thiamine hydrochloride.
—10.5 ± 0.2 mg.
Determine the moisture of the USP thiamine hydrochloride
reference standard as directed on the container immediately
before weighing. The percent moisture determined for the
reference standard is used to calculate the concentration of
thiamine in the VSSM.
(
iii
)
Riboflavin.
—7.0 ± 0.2 mg. Dry an appropriate amount
of the USP riboflavin reference standard at 105 (±1)°C for 2 h
(±10 min) before weighing.
(
iv
)
Pyridoxine hydrochloride.
—10.8 ± 0.2 mg. Dry an
appropriate amount of the USP pyridoxine hydrochloride
reference standard over indicating absorbent in vacuo for 4 h
before weighing.
QS to volume with phosphate buffer (pH 5) solution. Heat
and slowly stir until the standards have completely dissolved
(riboflavin dissolves more slowly) and the solution is clear. Do
not heat the solution for more than 40 min and do not exceed
90°C. Store refrigerated (2–8°C). Expiration: 3 months.
(
2
)
Nicotinic acid stock solution (550 µg/mL).
—Accurately
weigh 13.7 ± 0.1 mg USP niacin (nicotinic acid, Cat. No.
1461003) reference standard. Quantitatively transfer the
nicotinic acid to a 25 mL volumetric flask. Add laboratory water
to a total volume of about 20 mL and swirl until completely
dissolved. Bring to volume with laboratory water. Mix well.
Expiration: 3 months.
(
3
)
Pyridoxal stock solution.
—140 µg/mL. Accurately
weigh 17.0 ± 0.5 mg pyridoxal dihydrochloride standard.
Quantitatively transfer to a 100 mL volumetric flask. Add
laboratory water to a total volume of about 70 mL and swirl
until completely dissolved. Bring to volume with laboratory
water. Mix well. Expiration: 3 months.
(
4
)
Pyridoxamine stock solution.
—160 µg/mL. Accurately
weigh 23.0 ± 0.5 mg pyridoxamine hydrochloride standard.
Quantitatively transfer to a 100 mL volumetric flask. Add
laboratory water to a total volume of about 70 mL and swirl
until completely dissolved. Bring to volume with laboratory
water. Mix well. Expiration: 3 months.
(k)
MWS.
—Combine 500 μL VSSM, 25 μL pyridoxamine
stock, 25 μL pyridoxal stock, and 65 μL nicotinic acid stock
solutions in a 10 mL volumetric flask containing approximately
5 mL of 50 mM ammonium formate. Bring to volume with
50 mM ammonium formate and mix well. Prepare fresh daily.
(l)
Working standard solution preparation.
—(
1
)
WS1.
—
Add 20 μL MWS and 980 μL of 50 mM ammonium formate
to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to
mix. Prepare fresh daily.
(
2
)
WS2.
—Add 50 μL MWS and 950 μL of 50 mM
ammonium formate to a 50 mL centrifuge tube. Add 100 μL of
ISSM, and vortex to mix. Prepare fresh daily.
(
3
)
WS3.
—Add 100 μL MWS and 900 μL of 50 mM
ammonium formate to a 50 mL centrifuge tube. Add 100 μL of
ISSM, and vortex to mix. Prepare fresh daily.
(
4
)
WS4.
—Add 200 μL MWS and 800 μL of 50 mM
ammonium formate to a 50 mL centrifuge tube. Add 100 μL of
ISSM, and vortex to mix. Prepare fresh daily.
108