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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.
2
Principle
Free as well as bound nicotinic acid and nicotinamide are extracted using a slightly
acidic buffer employing protease and takadiastase treatments. The sample extracts are
analyzed using liquid chromatography (LC) with tandem mass spectrometric detection.
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
Standards
a)
Nicotinic acid (B3-acid) -- 99% reagent grade or better (USP P/N #1461003)
b)
Nicotinamide (B3-amide) -- 99% reagent grade or better (USP P/N #1462006)
Internal Standards
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
Instrumentation
a)
Chromatographic system.— ABSciex 5500 QTrap / ABSciex 6500 QTrap
b)
Dionex Ultimate 3000 RS Pump, Autosampler, Column Compartment &Degasser
Thermo Fisher / Agilent 1290 Infinity Pump, Autosampler, column compartment &
Degasser
c)
Agilent 1290 Infinity Pump, Autosample, column compartment & Degasser
d)
Software.—AbSciex Analyst and MultiQuant chromatography data system
e)
Analytical column.— Thermo Acclaim C30 3um 2.1 x 150mm; (P/N 075725)
Labwares
a)
Autosampler vials.—Agilent 2.0ml amber vials, Fisher P/N 50-929-364)
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE