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S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016 

3

(ab)

Syringe filters, PTFE 0.45 μm.

—Acrodisc 25 mm, or

equivalent. Pall Corporation, Port Washington, NY.

(ac)

50 mL self-standing, plastic centrifuge tubes.

(ad)

Autosampler vials.

—9 mm amber with screw top

12 × 32 mm presplit PTFE-silicon septa (Waters, Part No.

186000847C, or equivalent). Waters

(ae)

Teflon coated magnetic stir bars.

C. Reagents

(a)

Nicotinamide.

—USP

Reference

Standard

(U.S.

Pharmacopeial Convention, Inc. Rockville, MD) Official Lot;

Cat. No. 1462006. Store as indicated on label.

(b)

Niacin (nicotinic acid).

—USP Reference Standard

Official Lot; Cat. No. 1461003. Stored as indicated on label.

(c)

Pyridoxine hydrochloride.

—USP Reference Standard

Official Lot; Cat. No. 1587001. Store in desiccator protected

from white light. Dry according to manufacturer’s instructions

before use.

(d)

Riboflavin.

—USP Reference Standard Official Lot; Cat.

No. 1603006. Store in desiccator protected from white light.

Dry according to manufacturer’s instructions before use.

(e)

Thiamine hydrochloride.

—USP Reference Standard

Official Lot; Cat. No. 1656002. Store in desiccator protected from

white light. Measure the moisture content of the powder before use.

(f)

Pyridoxamine

dihydrochloride.

—Fluka

Analytical

Standard, Cat. No. P9380. Sigma-Aldrich, St. Louis, MO.

(g)

Pyridoxal hydrochloride.

—Sigma, Cat. No. P9130.

Sigma-Aldrich

(h)

2

H

4

-nicotinamide.

—CDN Isotopes, Pointe-Claire, Quebec,

Canada; Cat. No. D-3457.

(i)

2

H

4

-nicotinic acid.

—CDN Isotopes; Cat. No. D-4368.

( j )

1 3

C

4

- p y r i d o x i n e .

—P y r i d o x i n e : H C l

(4,5-bis(hydroxymethyl)-

13

C

4

); Cambridge Isotope Laboratory,

Tewksbury, MA; Cat. No. CLM-7563.

(k)

2

H

3

-pyridoxal.

—IsoSciences, King of Prussia, PA; Cat.

No.7098.

(l)

2

H

3

-pyridoxamine.

—IsoSciences; Cat. No. 7099.

(m)

13

C

4

-thiamine chloride.

—IsoSciences, King of Prussia,

PA; Cat. No. 9209.

(n)

13

C

4

,

15

N

2

-riboflavin.

—IsoSciences, King of Prussia, PA.,

Cat. No. 7072.

(o)

Acid phosphatase, type II from potato, 0.5–3.0 U/mg.

Sigma, Cat. No. P3752. Sigma-Aldrich

(p)

Papain from Carica papaya, ≥3 U/mg.

—Sigma, Cat. No.

76220. Sigma-Aldrich

(q)

α

-

Amylase from Aspergillus oryzae, 150 U/mg.

—Sigma,

Cat. No. A9857. Sigma-Aldrich

(r)

Hydrochloric acid concentrated (12 M).

—American

Chemical Society (ACS) grade, or equivalent.

(s)

Ammonium formate: for mass spectrometry (≥99.0%).

Fluka 70221 or equivalent. Sigma-Aldrich

(t)

Glacial acetic acid.

—Sigma ACS reagent grade, or

equivalent. Sigma-Aldrich, St. Louis, MO.

(u)

Formic acid.

—Sigma ACS reagent grade, or equivalent.

Sigma-Aldrich, St. Louis, MO.

(v)

Laboratory water.

—18.0 MΩ, <10 ppb total organic

carbon (TOC), or equivalent.

(w)

Methanol.

—Fisher Scientific (Franklin, MA) LC-MS/

MS Optima grade or EMD (EMD Millipore) Omni-Solve

LC-MS grade.

(x)

EDTA, disodium salt dihydrate.

—ACS grade (99–101%),

or equivalent.

(y)

Potassium phosphate dibasic.

—ACS grade (>98%), or

equivalent.

(z)

Metaphosphoric acid.

—ACS grade (33.5–36.5%), or

equivalent.

(aa)

 pH 4.0, 7.0, and 10.0 buffer solutions for pH meter

calibration.

(ab)

Phosphoric acid.

—85%, ACS grade, or equivalent.

(ac)

Potassium hydroxide.

—40%, ACS grade, or equivalent.

D. Standard and Solution Preparation

(a)

Mobile phase A.

—20 mM ammonium formate in water.

Using a graduated cylinder, transfer 500 mL laboratory water to

a mobile phase reservoir. Add 0.631 g of ammonium formate

and mix well. Scale as needed. Expiration: 3 days.

(b)

Mobile phase B.

—Methanol.

(c)

Weak needle wash.

—10% methanol in water, expiration

3 months.

(d)

Strong needle wash.

—Methanol.

(e)

50 mM ammonium formate.

—Using a graduated cylinder,

transfer 1400 mL laboratory water to an appropriate reservoir.

Add 4.41 g of ammonium formate and mix well. 1400 mL is

adequate for six working standards and 32 samples. Scale as

needed. Expiration: 3 days.

(f)

Enzyme cocktail.

—Using a graduated cylinder, transfer

200 mL 50 mM ammonium formate buffer to an appropriate

reservoir.Add 200 ± 10mg acid phosphatase, 80 ± 5mg α-amylase,

and 400 ± 10 mg papain. Mix for 10 min with magnetic stir plate

and stir bar. Adjust to pH 4.0–4.5 with formic acid (approximately

100 μL). 200 mL is adequate for six working standards and 32

samples. Scale as needed. Prepare fresh daily.

(g)

Stable-isotope labeled compounds.

—Individual, internal

standard stock solutions.

(

1

) 

2

H

4

-nicotinamide

stock

solution

(approximate

concentration: 560 μg/mL).

—Weigh 14.0 ± 0.1 mg into a tared

weighing vessel. Quantitatively transfer to a 25 mL volumetric

flask with laboratory water and

quantum satis

(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.

(

2

) 

2

H

4

-nicotinic acid stock solution

(approximate

concentration: 500 μg/mL)

.—Weigh 12.5 ± 0.1 mg into a tared

weighingvessel.Quantitatively transfer to a 25mLvolumetricflask

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.

(

3

) 

13

C

4

-pyridoxine

stock

solution.

—Approximate

concentration: 70 μg/mL.Weigh 7.0 ± 0.1mg into a taredweighing

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.

(

4

) 

2

H

3

-pyridoxal

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.

107