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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method BVit-04
Total Nicotinic Acid and Nicotinamide by LCMSMS
Title: Validation of a LC-MS/MS Method of Analysis for Total Nicotinic Acid and Nicotinamide in
Infant Formula and Adult Nutritional Samples
Author:
Sneh Bhandari
Summary of Method:
Dissolved (or liquid) samples are digested at pH 5.0 and 60
o
C with protease for 30 min. The enzyme is
deactivated by boiling, cooled and then incubated with Takadiastase enzyme at 45
o
C for 3 hr. The
extract is made to 50mL and centrifuged. Following filtration, 1 mL is mixed with 30µL of mixed ISTD
containing
13
C
3
15
N isomers of nicotinic acid and nicotinamide.
2µL is injected into a Dionex 3000 UPLC with Thermo C30 Acclaim column and ammonium formate
buffer pH 3.0. The analytes are eluted with acetonitrile gradient into a Sciex 5500 QTrap MS detector
with optimized voltages for nicotinic acid and nicotinamide. Quantitative transitions of 124.3 - 78.1
and 123.3 - 80.1 are made for the two analytes, each with two qualifying transitions. Corresponding
transitions for the ISTDs were made from Q1 molecular ions of 128.1 and 127.0 respectively, each to
three fragment ions.
Method Scope/Applicability:
All SPIFAN matrices and related formulations.
General comments about the method:
Sample preparation resembles the enzymatic procedure of method 2015.14 for vitamins B
1
, B
2
and B
6
,
although uses a 2-step digestion. Chromatography is also related albeit on C30 rather than C18 and
using acetonitrile in phase B rather than methanol. The stable isotopes are sourced from IsoSciences
containing
13
C
3
15
N rather than
2
H
4
isotopes in 2015.14 (from CDN Isotopes).
The new method is good although it must be questioned whether a separate procedure is required.
The need for takadiastase (amylase) digestion is questioned unless the samples contain starch. Vitamin
B
3
has no phosphorylated forms so a phosphatase is unnecessary. Many past uses of crude enzyme
extracts like takadiastase for B-vitamin extraction have been for their residual phosphatase activity.
Method Clarity: