30 / 220
LC-MS/MS Analysis of Nicotinic acid
and Nicotinamide
Chemistry Method
Version 2
Page 5/15
Prepared by
S. Bhandari
Approved by
Issue Date
Implementation Date
________________________________________________________________________________
Draft Date: 3/2/16 Copyright © Merieux NutriSciences, 2016
Uncontrolled When Printed
3. QUICK REFERENCE
3.1 Weigh about 0.2g -2.3 g of sample in a 50 mL centrifuge tube.
3.2 Add 10 mL Extraction Buffer & sonicate for 30 min.
3.
4 Add 0.1 mL protease and incubate at 60C for 30 min.
3.5 Boil for 10 min, cool to room temperature (RT)
. 3.6 Add 1 mL takadiastase solution and incubate at 45 C for 3 hours.
3.7 Boil for 5min and cool to RT and make volume to 40 mL with extraction buffer.
3.8 Filter a portion of extract through 0.45 micron PVDF syringe filtrer.
3.9 Pipet 1 mL of sample extract in a HPLC vial and add 30uL of mixed internal std. solution
and mix.internal standard.
4.0 Analyze by LC-MS/MS
.
4. FULL METHOD
4.1 Apparatus
(Apparatus listed was used in method validation. Any necessary substitutions should be
made based on technical equivalence, and verified with CRM’s or sample spikes.)
1. Chromatographic system
2. Dionex Ultimate 3000 RS Pump, Autosampler, Column Compartment &Degasser Thermo Fisher
/ Agilent 1290 Infinity Pump, Autosampler, column compartment & Degasser
3. Agilent 1290 Infinity Pump, Autosample, column compartment & Degasser
4. The Detection (triple quard mass Spec) System: .
—
ABSciex 5500 QTrap / ABSciex 6500 QTrap
5. Software.
—
AbSciex Analyst and MultiQuant chromatography data system
6. Analytical column.
—
Thermo Acclaim C30 3um 2.1 x 150mm; (P/N 075725)
4.2 Labwares:
1. Autosampler vials.
—
Agilent 2.0ml amber vials, Fisher P/N 50-929-364)
2. 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).
3. Centrifuge tubes.
—BD Falcon™ 50 mL conical,
polypropylene; 17 (od) x 115 (length) mm;
25/bag (Fisher Scientific; P/N 14-432-22).
4. Filters.
—
Fisher PVDF auto vials (P/N 09-916A, or equivalent).
5. Analytical balance.
—
Mettler Toledo
6. 1000mL volumetric flasks
7.
10, and 100 mL volumetric flasks (Class A)
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE