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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.
5
Add 10mL Extraction Buffer (12mM Ammonium Formate pH 5.0)
Sonicate 30min (quick vortex every 10 min)
Add 0.1mL Protease Solution (50mg/mL)
Incubate in 60
o
C shaking water bath 30min
Boil 10min ; cool to R.T
Add 1mL Taka Diastase Solution (1% in Extraction Buffer)
Incubate 3Hrs @ 45
o
C shaking water
Boil 5min; Cool to R.T
Bring to Final Volume 50mL with Extraction Buffer
Centrifuge 5min 8500rpm
PVDF filter into 16x25 disposable glass culture tube
Pipette 1mL into HPLC vial and add 30u: of IS Mix
Inject 2uL on LCMS/MS
Instrument HPLC Conditions
Dionex Ultimate 3000 / Agilent 1290 System
(
a
)
Columns.—
Thermo Acclaim C30 3um 2.1 x 150mm
(
b
)
Mobile Phase. —
M.P.A: 12 mM ammonium formate (Optima) in water pH = 3.0
M.P.B: 100% ACN
(
c
)
Gradient. —
#
Time (min)
Flow Rate
(uL/min)
A%
B%
0
0.0
300
100
0
1
4.0
600
100
0
2
11.5
600
64
36
3
12.0
600
0
100
4
15.5
600
0
100
5
16.0
600
100
0
6
21.0
600
100
0
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE