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LC-MS/MS Analysis of Nicotinic acid
and Nicotinamide
Chemistry Method
Version 2
Page 8/15
Prepared by
S. Bhandari
Approved by
Issue Date
Implementation Date
________________________________________________________________________________
Draft Date: 3/2/16 Copyright © Merieux NutriSciences, 2016
Uncontrolled When Printed
Std Level
Final Concen.
(ng/mL, ppb)
Vol. of Std (mL)
Internal Std Mix
Solution (IS-
Mix) (mL)
Final Concen.
(ng/mL, ppb)
6
175
1.00
0.030
175
5
100
1.00
0.030
100
4
45
1.00
0.030
45
3
17.5
1.00
0.030
17.5
2
5.0
1.00
0.030
5.0
1
0.9
1.00
0.030
0.9
The net concentration of different internal standards in the calibration standards is
provided in the following Table.
The net concentration of different nicotinic acid and nicotinamide internal standards in
the calibration standards are as the following.
Name
ppb
(ng/mL)
Added to
each
Calibration
std. (mL)
Internal std.
final conc. in
each calibration
std. (ng/mL)
Nicotinic Acid
13
C
3
1000.0
0.03
30.0
Nicotinamide
13
C
3
4000.0
120.0
4.6
Reagent Solutions and mixtures
a)
Extraction Buffer 12.0 mM Ammon
ium Formate; pH 5.0
.
—
In a 1L Solvent reservoir
bottle add 0.771g Ammonium formate and 1L Milli-Q water. Stir until dissolved completely. Adjust
pH with formic acid to pH 5.0. (
1-2 drops
)
b)
Pro
Protease Solution
–
50mg/mL. Weigh 100 mg of protease in a test tube and add 2 mL of
water. Gently mix to dissolve/suspend and make sure no foaming. 0.1mL/sample needed. Make
fresh for each batch.
c)
T
akadiastase
–
weigh 3g of taka-diastase and add 30ml 10mM ammonium formate pH 5.0
extraction buffer (1mL/sample needed) Make fresh each batch.
d) Mobile Phase A 12.0 mM Ammonium Formate; pH 3.0
.
—
I
n a 1L Solvent reservoir bottle add
0.771g Ammonium formate and 1L Milli-Q water. Stir until dissolved completely. Adjust pH with
formic acid to pH 3.0
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE