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Validation of a LC-MS/MS method of Analysis of Nicotinic acid and Nicotinamide in infant formula and adult
nutritional samples
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Copyright © 2012 Silliker, Inc.
3
b)
Storage bottles.—Thermo Scientific Nalgene™ narrow-mouth packaging bottles; natural
HPDE with PP closures, 250 mL, 24-415 closure, bulk pack (P/N 312089-0008, or
equivalent).
c)
Centrifuge tubes. — BD Falcon™ 50 mL conical, polypropylene; 17 (od) x 115 (length)
mm; 25/bag (Fisher Scientific; P/N 14-432-22).
d)
Disposable glass culture tubes 15x125
e)
Filters. —Fisher PVDF auto vials (P/N 09-916A, or equivalent).
f)
Analytical balance. —Mettler Toledo
g)
10, 100 and 1000mL volumetric flasks
Solutions and Reagents Preparation
a)
Internal standard (IS) Stock Solution – Add 1mL water to each individual vials of nicotinic
acid and nicotinamide 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.
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
c)
100 mcg/mL Stock Solutions.
— 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 3 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.
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE