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ii) Weigh 7.3
±
0.1 mg
13
C
4
,
15
N
2
-riboflavin into a tarred 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.
6.
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 6 working standards and
32 samples. Scale as needed. Prepare fresh daily.
7.
Phosphate Buffer Solution, pH 5.0 (0.10M Potassium Phosphate Dibasic, 1% EDTA, 2%
Metaphosphoric Acid)
.
a)
Weigh 20.0 ± 0.2 g of EDTA into a tarred weighing vessel and quantitatively transfer to a
2000 mL beaker containing approximately 1800 mL laboratory water and add a magnetic
stir bar.
b)
Weigh 34.8 ± 0.1 g of potassium phosphate dibasic into a tarred 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.
c)
Weigh 40.0 ± 0.2 g grams of metaphosphoric acid into a tarred 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 hours.
8.
Native Compounds – Stock Standard Solutions
a)
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):
(a)
Niacinamide: 70.5 ± 0.5 mg
(b)
Thiamine hydrochloride: 10.5 ± 0.2 mg
Determine the moisture of the USP thiamine hydrochloride reference standard as
directed on the container immediately prior to weighing. The percent moisture
determined for the reference standard is used to calculate the concentration of
thiamine in the VSSM.
(c)
Riboflavin: 7.0 ± 0.2 mg - Dry an appropriate amount of the USP riboflavin reference
standard at 105 (±1)
°C
for 2 hours (± 10 minutes) prior to weighing.
(d)
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
hours prior to weighing.
Candidates for 2016 Method of the Year
192