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Butler-Thompson et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
6, 2015
1655
INFANT FORMULA AND ADULT NUTRITIONALS
Received on April 30, 2015. Accepted by SG July 23, 2015.
The method was approved by the AOAC
Official Methods Board
as Final Action.
See
“Standards News,” (2014)
Inside Laboratory
Management
, November/December issue.
The AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) invites method users to provide feedback on the
Final Action methods. Feedback from method users will help verify
that the methods are fit for purpose and are critical to gaining global
recognition and acceptance of the methods. Comments can be sent
directly to the corresponding author.
1
Corresponding author’s e-mail:
Karen.schimpf@abbott.comDOI: 10.5740/jaoacint.15-108
Determination of Vitamin B
12
in Infant, Adult, and Pediatric
Formulas by HPLC-UV and Column Switching: Collaborative
Study, Final Action 2011.10
Linda D. Butler-Thompson, Wesley A. Jacobs, and Karen J. Schimpf
1
Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH, 43219
Collaborators: J. Austad, B. Chen, S. Christiansen, C. Domer, G. Hostetler, D. Labitan, B. Liu, I. Malaviole, B. Marosanović,
F. Martin, A. Mittal, J.M. Oberson, A. Ruosch, Y. Shen, G. Wang
AOAC First Action Method 2011.10, Vitamin B
12
in
Infant and Pediatric Formulas and Adult Nutritionals,
was collaboratively studied. This method uses a
pH 4.5 sodium acetate buffer and potassium cyanide
at 105°C to extract and convert all biologically active
forms of vitamin B
12
present to cyanocobalamin;
octylsilyl (C
8
) or C
18
SPE cartridges to purify and
concentrate cyanocobalamin; a combination of
size-exclusion and RPLC to isolate cyanocobalamin;
and visible absorbance at 550 nm to detect and
quantitate cyanocobalamin in infant, pediatric, and
adult nutritionals with vitamin B
12
concentrations
greater than 0.025 µg/100 g ready-to-feed (RTF)
liquid. During this collaborative study, nine to
11 laboratories from eight different countries
analyzed blind duplicates of 12 infant, pediatric,
and adult nutritional formulas. Per the AOAC
Expert Review Panel (ERP) on Stakeholder Panel
on Infant Formula and Adult Nutritionals (SPIFAN)
Nutrient Methods the method demonstrated
acceptable repeatability and reproducibility
and met SPIFAN
Standard Method Performance
Requirements
(SMPRs
®
) for the majority of product
matrixes analyzed. Vitamin B
12
SPIFAN SMPRs
for repeatability were ≤15% RSD at vitamin B
12
concentrations of 0.01 µg/100 g RTF liquid and
≤7% RSD at vitamin B
12
concentrations of
0.2–5.0 µg/100 g RTF liquid. Vitamin B
12
SPIFAN
SMPRs for reproducibility were ≤11% RSD in
products with vitamin B
12
concentrations ranging
from 0.3 to 5.0 µg/100 g RTF liquid. During this
collaborative study, the RSD
r
ranged from
2.98 to 9.77%, and the RSD
R
ranged from 3.54 to
19.5%. During previous single-laboratory validation
studies, the method LOQ was estimated to
be 0.025 µg/100 g RTF liquid.
V
itamin B
12
is a generic term used to describe
all cobalamins that demonstrate antipernicious
anemia activity. These compounds are corrinoids,
tetrapyrrole structures where the cobalt ion is chelated by
four pyrrole nitrogens. The fifth coordinate covalent bond
to cobalt is with a nitrogen of the dimethylbenzimidazole
moiety, while the sixth position may be occupied by a
cyanide, 5'-deoxyadenosyl, methyl, water, hydroxyl, nitrite,
ammonia, or sulfite ligand (1).
The AOAC Stakeholder Panel on Infant Formula
and Adult Nutritionals (SPIFAN) has defined vitamin
B
12
as any cobalt-containing corrinoid with the biological
activity of cyanocobalamin, including cyanocobalamin,
aquocobalamin, hydroxycobalamin, methylcobalamin, and
adenosylcobalamin. Most nutritional products are fortified with
cyanocobalamin, a synthetic form of vitamin B
12
, because of
its stability and commercial availability. Methylcobalamin and
5'-deoxyadenosylcobalamin occur naturally and are known
to exhibit metabolic activity in humans. Naturally occurring
vitamin B
12
originates solely from synthesis by bacteria and
other microorganisms growing in soil, water, or sewage and in
the rumen and intestinal tract of animals (2).
A new chromatographic method capable of determining
vitamin B
12
in infant, adult, and pediatric formula powders,
ready-to-feed (RTF) liquids, and liquid concentrates was needed
after SPIFAN determined that traditional microbiological
vitamin B
12
methods were not precise and accurate enough to
meet the nutrient specification requirements of infant, pediatric,
and adult nutritionals. The SPIFAN vitamin B
12
working
group developed
Standard Method Performance Requirements
(SMPRs
®
) that were then approved by SPIFAN (3).
In June 2011, the AOAC Expert Review Panel (ERP)
on SPIFAN nutrient methods granted First Action status to
multiple vitamin B
12
methods, AOAC
2011.08
(4),
2011.09
(5),
AOAC
2011.10
(6), and
2011.16
(7). Single-laboratory
validations (SLVs) were completed for AOAC
2011.08
and
AOAC
2011.10
(8), and in March 2013, AOAC
2011.10
was
selected by the ERP for further evaluation in a multilaboratory
collaborative study to determine method reproducibility.
133