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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