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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.
4
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
f)
Extraction Buffer 12.0 mM Ammonium 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
)
g)
Protease Solution ( 50mg/mL) – Weigh 100 mg 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.
h)
Takadiaistase – weigh 3gram of taka-diastase and add 30ml 12mM ammonium formate
pH 5.0 extraction buffer (1mL/sample needed) Make fresh each batch.
i)
Mobile Phase A 12.0 mM Ammonium Formate; pH 3.0
. — 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 3.0.
j)
Mobile Phase B
— 100% Acetonitrile
Sample Preparation
Samples should be completely homogenous and well blended .
NIST 1849a can be prepared as a slurry before the analysis 25 g and 200 mL
of water and mixed to uniform mixture.
Extraction of Niacin (Vitamin B3) in samples:
Weigh approximately 0.2g -2.3 g in 50mL Centrifuge tube
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE