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and 400 ± 10 mg papain. Mix for ten minutes with magnetic stir plate and stir bar. Adjust to
pH 4.0-4.5 with formic acid (~ 100 μL). 200 mL is adequate for 6 working standards and 32
samples. Scale as needed. Prepare fresh daily.
5.
Stable-Isotope Labeled Compounds – Individual, Internal Standard Stock Solutions
a)
2
H
4
-Nicotinamide Stock Solution (Approximate Concentration: 560
µ
g/mL) - Weigh 14.0
±
0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask
with laboratory water and QS with laboratory water. Mix well and transfer to a 50 mL
amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of
contamination.
b)
2
H
4
-Nicotinic Acid Stock Solution (Approximate Concentration: 500
µ
g/mL) - Weigh 12.5
±
0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask
with laboratory water and QS with laboratory water. Mix well and transfer to a 50 mL
amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of
contamination.
c)
13
C
4
-Pyridoxine Stock Solution (Approximate Concentration: 70
µ
g/mL) - Weigh 7.0
±
0.1
mg into a tarred 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.
d)
2
H
3
-Pyridoxal Stock Solution (Approximate Concentration: 40
µ
g/mL) - Weigh 4.0
±
0.1 mg
into a tarred 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.
e)
2
H
3
-Pyridoxamine Stock Solution (Approximate Concentration: 40
µ
g/mL) - Weigh 4.0
±
0.1 mg into a tarred 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.
e)
13
C
4
-Thiamine Chloride Stock Solution (Approximate Concentration: 100
µ
g/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 tarred 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.
f)
13
C
4
,
15
N
2
-Riboflavin Stock Solution (Approximate Concentration: 73
µ
g/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.
Candidates for 2016 Method of the Year
191