201 / 363
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.
Candidates for 2016 Method of the Year
200