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LC-MS/MS Analysis of Nicotinic acid
and Nicotinamide
Chemistry Method
Version 2
Page 7/15
Prepared by
S. Bhandari
Approved by
Issue Date
Implementation Date
________________________________________________________________________________
Draft Date: 3/2/16 Copyright © Merieux NutriSciences, 2016
Uncontrolled When Printed
Name
volume
Stock
(uL)
Final
Vol
(mL)
ppb
(ng/mL)
Nicotinic Acid
13
C
3
10
10
1000.0
Nicotinamide
13
C
3
40
4000.0
4.5 Standard Solutions and mixtures
c)
100 mcg/mL B-Vitamin Stock Solution.
—
Weigh 10mg of nicotinic acid as well as nicotinamide in
individual 100mL volumetric flask. Dilute to volume with Milli-Q water to make a 100 mcg/mL stock
solution. Sonicate to ensure completely dissolved. Transfer to amber storage bottle. The standard is
stable for 1 month when stored at -20
o
C. (Adjust for purity)
d) 500 ng/mL nicotinic acid and nicotinamide intermediate Solution.
—
Take 0.05mL (50uL) of nicotinic
acid as well as nicotinamide Stock solutions in a 10mL volumetric flask and dilute to volume with
Extraction Buffer pH 5.0. Make Fresh each batch.
e)
Nicotinic acid and nicotinamide Working Standard Solutions.
—
See table below for dilutions of the
working calibration curve. The 12mM ammonium formate Extraction buffer pH 5.0 is used as the
diluent. Place 1mL of each standard in an HPLC vial and add 30uL of IS Mix Std.
Std Level
Std Used
Vol. of Interm
Mix Std (mL)
Final Vol (mL)
Final Concen.
(ng/mL, ppb)
6
Interm
3.50
10.00
175
5
Interm
2.00
10.00
100
4
Interm
0.90
10.00
45
3
Std Level 6
1.00
10.00
17.5
2
Std Level 5
0.50
10.00
5.0
1
Std Level 4
0.20
10.00
0.9
4.4.3 Mixture of nicotinic acid and nicotinamide standard Solutions with Internal
Standard:
Take accurately 1 mL of all the calibration standards (levels 1 through 6) in separate
HPLC vials. To 1mL of each calibration standard solution add 0.03 mL of the internal
standard mix solution (IS-Mix). The details of the preparations of different calibration
standard mixture with mixed-internal standard solution is provided in the following
Table.
Preparation of mixture of all calibration standards with the mixed internal standard (IS-
Mix).
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE