105 / 258
S
alvati
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
3, 2016
1
Simultaneous Determination of Total Vitamins B
1
, B
2
, B
3
, and
B
6
in Infant Formula and Related Nutritionals by Enzymatic
Digestion and LC-MS/MS: Single-Laboratory Validation, First
Action 2015.14
L
ouis
M. S
alvati
, S
ean
C. M
c
C
lure
, T
odime
M. R
eddy
,
and
N
icholas
A. C
ellar
1
Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219
This method provides simultaneous determination
of total vitamins B
1
, B
2
, B
3
, and B
6
in infant formula
and related nutritionals (adult and infant). The method
was given First Action for vitamins B
1
, B
2
, and B
6
,
but not B
3
, during the AOAC Annual Meeting in
September 2015. The method uses acid phosphatase
to dephosphorylate the phosphorylated vitamin
forms. It then measures thiamine (vitamin B
1
);
riboflavin (vitamin B
2
); nicotinamide and nicotinic
acid (vitamin B
3
); and pyridoxine, pyridoxal, and
pyridoxamine (vitamin B
6
) from digested sample
extract by liquid chromatography-tandem mass
spectrometry. A single-laboratory validation was
performed on 14 matrixes provided by the AOAC
Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) to demonstrate method
effectiveness. The method met requirements of
the AOAC SPIFAN
Standard Method Performance
Requirement
for each of the three vitamins, including
average over-spike recovery of 99.6 ± 3.5%, average
repeatability of 1.5 ± 0.8% relative standard deviation,
and average intermediate precision of 3.9 ± 1.3%
relative standard deviation.
A
OAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) released a call for methods for total
vitamins B
1
, B
2
, B
3
, and B
6
in infant formula and related
nutritionals. In the European Union and other countries, label
claim is regulated based upon total vitamin content and not just
the fortified form. Historically, microbiological methods were used
to estimate total vitamin. However, these methods are challenged
with newer, more diverse nutritional products and are no longer
considered the gold standard. Newer, chromatographic methods,
especially with mass spectral detection, are quickly becoming
the new standard because their specificity enables accurate
quantitation across more complex and diverse matrixes. However,
that specificity then requires explicit definition of the vitamin forms
necessary for a “total” vitamin determination. SPIFAN gathered
experts in industry, government, and academia to provide these
definitions. Total vitamin B
1
is defined as the sum of thiamine,
thiamine monophosphate, thiamine pyrophosphate, and thiamine
triphosphate in the
Standard Method Performance Requirement
(SMPR
®
; 1). Total B
2
is defined as riboflavin, riboflavin-5′-
phosphate, and flavin adenine dinucleotide (2). Total B
3
is defined
as the sum of nicotinic acid and nicotinamide (3). Finally, total B
6
includes five forms: pyridoxine, pyridoxal, pyridoxal-5′-phosphate,
pyridoxamine, and pyridoxamine-5′-phosphate (4).
SPIFAN defined forms of vitamins B
1
, B
2
, B
3
, and B
6
that
represent the major contributors to total vitamin concentration in
formulas and enter the formulation through both fortification and
from ingredient sources. For example, depending on the protein
fraction of milk and the degree of processing, the contribution of
inherent (unfortified) vitamin can be virtually 0 to >45% of the
total value (5). But, regardless of source, the concentration of
each vitamin must be verified to meet label claim. Remarkably,
the necessary sample treatments and separation as described
later are similar for the intended vitamins and thus lend
themselves to simultaneous determination, saving both time
and cost. Further, the availability of modern mass spectrometry
(MS) instrumentation with electrospray ionization (ESI)
facilitates simultaneous determination by removing remaining
hurdles associated with detection. Suppression of ionization is
problematic for quantitation with ESI, but is overcome with the
use of stable-isotope labeled internal standards. The associated
cost of isotopically labeled standards, although perceived as
great, only adds a few cents to the cost of a sample because
of the small amount necessary. By contrast, the syringe filter
required to prevent clogging the liquid chromatography (LC)
column adds about $1 (USD) to the cost of a sample.
The combined method was developed to measure thiamine,
riboflavin, pyridoxamine, pyridoxal, pyridoxamine, nicotinic acid,
and nicotinamide directly. Separation was achieved with 20 mM
ammonium formate mobile phase without ion pairing agent.
Thiamine is not well retained in reverse phase at low pH without
an ion pairing agent (6). However, ion pairing agents bring
additional challenges to LC-tandem MS (MS/MS) determination.
Improved retention of thiamine has been previously demonstrated
by increasing the mobile phase pH (6). In fact, there is a striking
improvement in retention for many water-soluble vitamin under
reverse-phase conditions at moderate pH (5–7). This improvement
in retention was harnessed to achieve good method performance
for a subset of the targeted vitamin forms; however, elution
of phosphorylated compounds is notoriously difficult (8). The
phosphate moiety complexes with Fe
3+
and thus phosphate
INFANT FORMULA AND ADULT NUTRITIONALS
Submitted for publication February 18, 2016.
The method was approved by the AOAC Expert Review Panel for
SPIFAN Nutrient Methods as First Action.
The expert review panel invites method users to provide feedback
on the First Action methods.
Feedback from method users will help verify that the methods
are fit-for-purpose and are critical for gaining global recognition and
acceptance of the methods. Comments can be sent directly to the
corresponding author or
methodfeedback@aoac.org.
1
Corresponding author’s e-mail:
nick.cellar@abbott.comDOI: 10.5740/jaoacint.15-0315
105