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LC-MS/MS Analysis of Nicotinic acid
and Nicotinamide
Chemistry Method
Version 2
Page 6/15
Prepared by
S. Bhandari
Approved by
Issue Date
Implementation Date
________________________________________________________________________________
Draft Date: 3/2/16 Copyright © Merieux NutriSciences, 2016
Uncontrolled When Printed
4.3 Reagents and Standards
4.3.1 Reagents
a) Deionized (DI) water.
—Type I reagent grade, 18 MΩ
-cm resistance or better.
b) Ammonium Hydroxide.
—
Fisher Scientific; P/N A669-500, or equivalent
c) Extraction Buffer - 12 mM Ammonium Formate in water; pH = 5.0
d) Ammonium Formate
–
Optima LC/MS Fisher Scientific A115-50g
e) Formic Acid
–
Optima LC/MS Fisher Scientific A117-50mL
f) Acetonitrile
–
Optima LCMS grade A955-4
g) Protease
–
Sigma P3910
h) Takadiastase
–
Pfaltz & Bauer P/N:T00040
4.3. 2 Calibration Standard Chemicals
Standards:
a) B3: Nicotinic acid -- 99% reagent grade or better (USP P/N #1461003)
b) B3: Nicotinamide -- 99% reagent grade or better (USP P/N #1462006)
4.3. 3 Calibration Internal Standard Chemicals
a) Internal Standard (IS) Nicotinic Acid-[13C3, 15N]
–
IsoSciences P/N 13257; 1mg
b) Internal Standard (IS) Nicotinamide-[13C3, 15N]
–
IsoSciences P/N 14327; 1mg
4.4 Internal standard Solutions and mixtures
a) IS Stock Solution
–
Add 1mL water to each individual vials of niacin and niacinamide IS Chemicals.
Stock solution Concentrations are as follows:
Name
B
Vitamin
mass
(mg) of
IS
volume
H
2
O (mL)
ppm
(
g/mL)
B
Vitamin
Nicotinic Acid
13
C
3
B3-acid
1
1.00
1000.0
B3-acid
Nicotinamide
13
C
3
B3-NH
1
1.00
1000.0
B3-NH
b) Internal Standard Mix(IS Mix)
–
Take various volumes of each stock as shown in table below and
make up to 10mL with Extraction buffer.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE