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AOAC
2011.10
uses a pH 4.5 sodium acetate buffer and
potassium cyanide at 105°C to extract and convert all forms of
vitamin B
12
present to cyanocobalamin, 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.
This method is applicable to the determination of vitamin B
12
,
including cyanocobalamin, aquocobalamin, hydroxycobalamin,
methylcobalamin, and adenosylcobalamin, in all forms of
infant, adult, and pediatric formula (powders, RTF liquids, and
liquid concentrates.)
Multilaboratory Collaborative Study
Initially 17 laboratories expressed interest in participating
in the AOAC
2011.10
vitamin B
12
collaborative study, but
only 11 laboratories were able to complete the study. The
11 participating laboratories were located in eight different
countries. The remaining six laboratories were not able to
participate because of time and resource constraints and issues
with the importation of samples into their countries. Two of the
participating laboratories only received partial sample shipments.
Before actual multilaboratory collaborative study samples
were analyzed, each participating laboratory was asked to
analyze two practice samples in duplicate to identify and resolve
any testing issues that the laboratories may have had executing
the method. The practice samples included National Institute of
Standards and Technology (NIST; Gaithersburg, MD) standard
reference material (SRM) 1849a and a high protein adult
nutritional RTF product. After approval of the practice sample
results by the study directors, laboratories began testing the
study samples.
Blind duplicates of the 12 SPIFAN matrixes were shipped
to each participating laboratory. The matrixes included SRM
1849a, an adult nutritional milk protein-based powder, an
infant formula partially hydrolyzed milk-based powder, an
infant formula partially hydrolyzed soy-based powder, an adult
nutritional low-fat powder, a child formula powder, an infant
elemental powder, an infant formula milk-based powder, an
infant formula soy-based powder, an infant formula milk-based
RTF liquid, an adult high-fat nutritional RTF liquid, and an
adult high protein nutritional RTF liquid.
Participants were asked to reconstitute all powders prior
to analysis. SRM 1849a was reconstituted by dissolving the
entire contents of the sachet (10 g) in 90 mL water. All other
powders were reconstituted by dissolving 25 g powder in
200 mL laboratory water. Participants were asked to split the
24 test samples into two groups of 12 according to the data
reporting sheets included in the protocol and to test each
group on a separate day. Although the original AOAC
2011.10
First Action method allowed for the use of different size SPE
cartridges, depending on the sample type being analyzed,
collaborative study participants were required to test all samples
with cartridges containing at least 900 mg resin since the study
samples were blinded. Most laboratories used the 900 mg
octylsilyl (C
8
) or C
18
cartridges referenced in the method, but
one participating laboratory used 1 g C
18
cartridges.
Upon completion of the sample analyses, participating
laboratories were asked to send all of their data to the study
directors. This included all standard and sample chromatograms
for the instrument check, practice sample analyses, test
sample analyses, standard curve information, calculations, and
completed reporting of analysis forms with dilution and sample
weights. Participants were also asked to report any deviations
to the method and any relevant comments based on their
experiences with the method.
All data were statistically analyzed using AOAC
INTERNATIONAL guidelines to determine overall mean,
repeatability SD (s
r
), RSD
r
, reproducibility SD (s
R
), RSD
R
, and
Horwitz ratio (HorRat; 9). Cochran (
P
= 0.025, one-tail) and
Grubbs (single and double,
P
= 0.025, two-tail) tests were used
to determine statistical outliers.
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. Requirements for reproducibility
were ≤11% RSD in products with vitamin B
12
concentrations
ranging from 0.3 to 5.0 µg/100 g RTF liquid.
Method
A few minor modifications were made to AOAC Official
First Action method
2011.10
before it was sent to the study
participants in the collaborative study protocol. These changes
included increasing the concentration of the sodium acetate
buffer from 0.1 to 0.25 M, adding more RP column options,
providing guidance for the preparation of samples containing
free amino acids or no intact protein, and providing guidance
for choosing appropriate size SPE cartridges.
After completion of the study, the modifications noted
above were incorporated in the Final Action method along
with a few additional modifications based on study results and
feedback from study participants and the ERP. In addition to
the modifications listed above, procedures for safely handling
potassium cyanide, qualifying SPE cartridges, and establishing
appropriate elution gradients to adequately resolve vitamin B
12
on the RP column were added to the Final Action method.
The option for using SPE cartridges smaller than 900 mg and
a guidance for choosing appropriate size SPE cartridges were
removed from the method since many laboratories may not
have enough information about the samples that they are testing
to use smaller size SPE cartridges.
AOAC Official Method 2011.10
Vitamin B
12
in Infant and Pediatric Formulas
and Adult Nutritionals
HPLC
First Action 2011
Final Action 2014
ISO-AOAC Method
(Applicable to the determination of vitamin B
12
in infant and
pediatric formulas and adult nutritionals.)
Caution
: Refer to Material Safety Data Sheets of chemicals
prior to use. Use the suggested personal protective
equipment and follow good laboratory practices.
Note
: Potassium cyanide is highly toxic. When handling
this chemical, wear gloves and appropriate personal protective
equipment. Weigh chemical and dispense solutions in a fume
hood. Perform test in a well-ventilated area. Treat sample waste
with sodium hypochlorite and dispose of waste according to
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