AOAC Official Method 2011.10 Scientific Aquasi1 P/N 77503-104630; www.thermoscientific.

Vitamin B12 in Infant Formula com) with C18 drop-in guard cartridges 3 μ, 10 × 4.6 mm (Thermo
and Adult Nutritionals
High-Performance Liquid Chromatography
First Action 2011
(Applicable to the determination of vitamin B12 in infant formula
and adult nutritionals.)
Caution: Refer to Material Safety Data Sheets (MSDS) of
chemicals prior to use and follow the suggested personal
protective equipment.
See Table 2011.10A for infant formula and adult nutritional
matrixes for which the method was validated.
A. Principle
Vitamin B12 is extracted from the sample using sodium acetate
buffer (pH 4.5) and potassium cyanide at 105°C. Extracts are
purified and concentrated with C8 or C18 solid-phase extraction
(SPE) cartridges and analyzed with size-exclusion and reversed-
phase chromatography. Determination of B12 is made by liquid
chromatography with visible detection at 550 nm.
B. Apparatus and Materials
(a) HPLC system.—Gradient system with switching valve
and isocratic pump on side and a UV-Vis detector equipped with
a tungsten lamp (capable of monitoring at 550 nm wavelength).
Autosampler capable of injecting 2 mL sample.
(b) Column.—Analytical size-exclusion column 4 μ, 250 ×
9.4 mm (Zorbax GF-250, P/N 884973-901; www.chem.agilent.
com), 5 μ, 300 × 8 mm (Shodex Protein KW-802.5, P/N F6989000)
or equivalent.
(c) Column.—Analytical C18 column 3 μ, 100 × 4.6 mm (Thermo
Scientific Aquasil P/N 77503-014001) or equivalent.
(d) Oven.—Capable of maintaining temperatures of 95 ± 5°C
and 105 ± 5°C.
(e) pH meter.—With calibration buffer.
(f) Analytical balance.—Capable of weighing 0.00001 g.
(g) Beakers.—Glass, assorted sizes.
(h) Bottle top dispenser.—Capable of dispensing 30 mL.
(i) Cylinders.—Graduated glass, assorted sizes.
(j) Desiccator.
(k) Erlenmeyer flasks.—125 mL.
(l) Filter paper.—Whatman 2V or equivalent.
(m) Funnels.—Plastic, suitable to use with filter paper.
(n) Gloves.
(o) Pipettor.—Variable volume, 100–1000 μL.
(p) Shields.—Yellow or clear shields with a cutoff of 385 nm.
(q) SPE cartridges.—C8 600 mg (Alltech/Grace Davidson P/N
20958; www.discoverysciences.com), C8 900 mg (Alltech/Grace
Davidson P/N 20966), C18 600 mg (Alltech/Grace Davidson P/N
20934), C18 900 mg (Alltech/Grace Davidson P/N 20942), or
equivalent.
(r) Syringes.—Disposable, assorted sizes.
(s) Syringe filters.—0.45 μm nylon.
(t) Vacuum manifold —24 ports with stopcocks or equivalent.
(u) Volumetric pipets.—Assorted sizes.
(v) Volumetric flasks.—Assorted sizes.
C. Reagents
(a) Acetic acid.—ACS.
(b) Acetonitrile.—HPLC grade.

Table 2011.10A. Infant formula and adult nutritional matrixes for which method was validated
Product type HPLC method, μg/kg (RSD, %) Microbiological method, μg/kg HPLC/micro, %
Hypoallergenic infant formula powder 51.4 (4.00) 53.6 95.9
n = 12
Soy-based infant formula powder 47.8 (5.30) 48.5 98.6
n = 15
High-calorie milk-based infant formula powder 59.5 (5.19) 61.2 97.2
n = 12
Milk-based infant formula liquid 4.30 (4.42) 4.20 102
n=9
Lactose-free infant formula powder 42.9 (4.70) 45.6 94.1
n = 12
Child nutritional powder 7.04 (5.27) 7.80 90.2
n=9
Adult nutritional powder 51.7 (5.66) 47.6 109
n = 12
Adult nutritional beverage 11.2 (4.85) 10.8 104
n = 21
Metabolic child/adult nutritional powder 48.0 (8.88) 49.5 97.0
n=9
Adult diabetic nutritional beverage 21.7 (0.76) 23.2 93.5
n=9
Metabolic child/adult nutritional powder 89.8 (4.85) 87.7 102
n = 14
Disease-specific adult nutritional beverage 16.6 (5.10) 18.3 90.7
n=9

© 2012 AOAC INTERNATIONAL

Table 2011.10B. Guidelines for loading sample filtrates onto diluted with 750 mL laboratory water and adjust pH to 5–7 with
SPE cartridgesa concentrated formic acid.
Vitamin B12 concentration Volume of filtrate loaded Final dilution
(3) Mobile phase C.—4.0 mL TEA and 750 mL acetonitrile
in RTF product, μg/L onto SPE cartridge, mL volume, mL diluted with 250 mL laboratory water and adjust pH to 5–7 with
concentrated formic acid.
<1 80 5
(4) 2.5% Acetonitrile in H2O (mobile phase D).—50 mL
1–10 70–80 10 acetonitrile diluted to 2000 mL with laboratory water.
11–20 50–60 10 (5) 10% Acetonitrile in H2O.—150 mL acetonitrile diluted to
21–50 20–40 10 1500 mL with laboratory water.
a
Do not load more than 60 mL adult and pediatric nutritionals onto an
(6) 25% Acetonitrile in H2O SPE elution solvent.—25 mL
Alltech C8 or C18 cartridge. acetonitrile diluted to 100 mL with laboratory water.
(7) 30% Acetonitrile in H2O SPE elution solvent.—30.0 mL
acetonitrile diluted to 100 mL with laboratory water.
(c) Drierite.—Desiccant, anhydrous calcium sulfate, 8 mesh. (8) 50% Acetonitrile in H2O, column cleaning and storage
(d) Ethanol.—Denatured. solution.—500 mL acetonitrile diluted to 1000 mL in a volumetric
(e) Formic acid.—ACS. flask. Expiration 6 months.
(f) Laboratory water.—≥15 MΩ·cm. (9) 25% Ethanol.—50 mL ethanol diluted to 200 mL with
(g) Potassium cyanide.—97% ACS reagent. laboratory water.
(h) Potassium phosphate dibasic.—ACS. (10) 0.40% Potassium cyanide.—Dissolve 0.02 g potassium
(i) Sodium acetate anhydrous or sodium acetate trihydrate.— cyanide in and dilute to 5 mL with 0.1 M sodium acetate buffer.
ACS. Make fresh immediately before use.
(j) Taka-diastase.—Accurate Chemical Co. (www. (11) 1% Potassium cyanide.—Dissolve 0.25 g potassium
accuratechemical.com) or equivalent. cyanide in and dilute to 25 mL with laboratory water. Prepare fresh
(k) Triethylamine (TEA).—HPLC grade. immediately before use.
(l) Vitamin B12 (cyanocobalamin) standard.—USP reference, (12) 0.1 M sodium acetate buffer.—Dissolve 16.4 g sodium
official lot number (refer to USP catalog for current lot). Store in acetate anhydrous or 27.2 g sodium acetate trihydrate in
desiccator protected from white light. (Note: See standard label for approximately 1800 mL laboratory water. Adjust pH to 4.50 with
purity.) concentrated acetic acid. Dilute to 2000 mL with laboratory water.
D. Solution and Standard Preparation
Expiration 3 months.
(13) 6% Taka-diastase.—Dissolve 0.6 g taka-diastase in 10 mL
All solutions can be scaled up or down for convenience provided water. Prepare fresh immediately before use.
good laboratory practices are observed. Solutions can be stored at (b) Standards.—Prepare all standards under UV shielded
2–30°C in tight, inert containers unless otherwise noted. fluorescent lights and store at 2–8°C in tightly stoppered volumetric
(a) Solutions.—(1) Mobile phase A.—4.0 mL TEA diluted with flasks.
1000 mL water and adjust pH to 5–7 with concentrated formic acid. (1) Vitamin B12 stock standard (10 000 μg/L).—Accurately weigh
(2) Mobile phase B.—4.0 mL TEA and 250 mL acetonitrile the appropriate amount of vitamin B12 USP reference standard to

Figure 2011.10A. System setup and configuration: Figure 2011.10B. System setup and configuration:
Configuration 1. Configuration 2.

© 2012 AOAC INTERNATIONAL

Table 2011.10C. System configuration
Time, min Valve configuration
0.00–10.5 Configuration 1
10.5–14.5 Configuration 2
14.5–30.0 Configuration 1
give a stock standard concentration of 10 000 g/L. Dissolve in and
dilute to 100 mL with 25% ethanol. Expiration 6 months.
Use the following equation to calculate the amount of vitamin B12
reference standard that should be weighed:
Sw = 10 000 × 0.1 × 1/P
where Sw = amount of vitamin B12 standard to be weighed in
mg; 10 000 = desired stock standard concentration in μg/L; 0.1 =
dilution volume in L; P = purity of the USP reference standard in μg
cyanocobalamin/mg of the standard. See standard label.
(2) Vitamin B12 intermediate standard (1000 μg/L).—Dilute
10 mL vitamin B12 stock standard solution to 100 mL with laboratory
water. Expiration 1 week.
(3) Vitamin B12 working standards (2.5–25 μg/L).—Dilute 0.5,
1, 2, 3, 4, and 5 mL vitamin B12 intermediate standard solution to
200 mL with 10% acetonitrile. Expiration 1 month.
E. Procedure
Prepare all samples under UV shielded fluorescent lights. Store
prepared product samples up to 14 days after preparation (store at
2–8°C in tightly stoppered volumetric flasks). Mix or stir products
before sampling to ensure all product samples are uniform and
representative.
(a) Sample preparation for infant and adult nutritional
products.—(1) Sampling.—Mix all products thoroughly before
sampling. Reconstitute nonhomogeneous powders per label
instructions. Weigh the appropriate amount of sample (±10%)
into a 100 mL volumetric flask and record the weight to at least 4
significant figures. Typical weights are 20–30 g for ready-to-feed
(RTF) liquids and reconstituted powders and 3 g for unreconstituted
powders. Add 25 mL laboratory water to all unreconstituted powder
samples and mix until all of the powder dissolves.
Add 1 mL of 6% taka-diastase if samples contain significant
levels of starch. Allow taka-diastase to react with samples for at
least 30 min before continuing with the extraction.
(2) Extraction.—Add 30 mL 0.1 M sodium acetate buffer
(pH 4.5) to each sample and swirl to mix.
In a hood, add 1 mL freshly prepared 1% KCN to each sample
and swirl to mix.
Heat samples in a 105°C oven for at least 60 min, but for no
more than 120 min. (Oven temperature will drop when the door
is opened. Start timing when oven temperature returns to 105°C.)
After at least 60 min, remove samples from oven and immediately
cool in ice bath.
Dilute samples to volume with laboratory water. Mix well.
Filter samples through Whatman 2V filter paper (www.whatman.
com) into 125 mL Erlenmeyer flasks or equivalent glassware. Note:
If prepared samples are milky and contain very small insoluble
particles, centrifuge samples and transfer liquid layer to funnels
lined with filter paper.
(3) Sample concentration.—For each sample that will be cleaned
up and concentrated, insert a 600–900 mg SPE cartridge onto the
stopcock of the vacuum manifold and attach a 30 mL disposable
syringe barrel to the top of each cartridge. As a general guideline,
600 mg Alltech C8 or C18 cartridges have enough capacity for use
with most products; however, if products on an RTF basis contain
high levels of protein (>5–6%) or high levels of hydrolyzed proteins
(>4%), 900 mg Alltech C8 or C18 cartridges should be used. Note:
Alltech C8 and C18 cartridges can be used interchangeably.
Condition each cartridge with at least 20 mL acetonitrile and
rinse each cartridge with at least 10 mL laboratory water.
Using volumetric pipets, transfer sample filtrates to cartridges
using the guidelines in Table 2011.10B. If necessary apply enough
vacuum so that the samples drip steadily through the cartridges.
Discard eluant.
After all of the sample filtrate has passed through the cartridge,
rinse each cartridge with 5 mL laboratory water and discard eluant.
Air-dry each cartridge by pulling a vacuum until no more effluent
is observed. Close each stopcock.
Place a 5 or 10 mL volumetric flask under each cartridge.
Add 4.4 mL 25% acetonitrile to all Alltech 600 mg cartridges
and 4.4 mL 30% acetonitrile to all Alltech 900 mg cartridges. Open
each stopcock and elute vitamin B12 into the volumetric flasks.
Final dilution.—For samples collected in 10 mL volumetric
flasks, dilute to volume with water. For samples collected in 5 mL
volumetric flasks, in a hood add 0.1 mL freshly prepared 0.4%
KCN to each volumetric flask. Place prepared samples in a 95°C
oven for at least 1.5 h, but for no more than 4 h. After at least
1.5 h, remove samples from the oven and cool to room temperature.
Dilute to volume with laboratory water. Filter an aliquot of each
standard and prepared sample through a 0.45 m syringe filter into
an autosampler vial.
(b) HPLC analysis.—(1) System setup and configuration.—See
Figures 2011.10A and B for configurations.
(2) Instrument operation conditions.—(a) Run time.—30 min.
(b) Injection volume.—2.0 mL.
(c) System configuration.—See Table 2011.10C.
(d) Isocratic pump.—Mobile phase D: 2.5% acetonitrile.
Flow rate: Adjust so that vitamin B12 elutes from the size-
exclusion column between 10.5 and 14.5 min. Typical flow rates,
1.1–1.2 mL/min. Note: To determine an appropriate flow rate,
connect the size-exclusion column directly to the UV-Vis detector
and inject the high standard. Adjust flow rate as necessary so that
vitamin B12 elutes between 10.5 and 14.5 min.
(e) Gradient pump.—Mobile phase compositions: mobile
phase A, 0.4% TEA in laboratory water, pH 5–7; mobile phase B,
0.4% TEA and 25% acetonitrile in H2O, pH 5–7; mobile phase C,
0.4% TEA and 75% acetonitrile in H2O, pH 5–7. Gradient to
Table 2011.10D. C18 column gradient
Mobile phase, %
Time, min A B C
0.00 90 10 0
14.5 90 10 0
14.6 41 59 0
26.0 41 59 0
26.1 0 10 90
28.0 0 10 90
© 2012 AOAC INTERNATIONAL
 170.00

165.00

160.00

155.00

150.00

vitamin B12 - 23.655

145.00

140.00
AU

135.00

130.00

125.00

120.00

115.00

110.00

105.00

100.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00
Minutes

Figure 2011.10C. Typical standard chromatogram.

elute vitamin B12 in 20–25 min: see Table 2011.10D. Flow rate: with the vitamin B12 peak areas of the samples and verify that the
1.0 mL/min. vitamin B12 peak areas of the samples are within the range of the
(f) Detector settings.—Detection wavelengths and bandwidth, vitamin B12 peak areas of the standards.
550 and 10 nm, respectively. (c) Calculation of standard concentrations.
(3) HPLC of standards and samples.—Make 3–4 injections of
a working standard and verify the precision of those injections is WS = Sw × P × A/200
≤3%.
If the system is working properly, inject a set of 3–6 working where WS = working standard concentration in g/L; Sw = amount
standards once, followed by a control sample, a set of 1–14 of vitamin B12 standard weighed in mg; P = purity of USP reference
samples, and another set of 3–6 working standards. Every set of standard in g cyanocobalamin (vitamin B12)/mg of the standard; A
1–14 samples should be bracketed by standards of appropriate = aliquot of vitamin B12 internal standard used (0.5, 1, 2, 3, 4, 5, or
concentration. 10) in mL; and 200 = dilution volume in mL.
(d) Preparation of standard curves.—(1) At each standard
F. Calculations
concentration, average the peak area of the standard injected at the
(a) Chromatography.—Visually inspect each standard and beginning of a set of samples with the peak area of the standard
sample chromatogram and verify that vitamin B12 is resolved from injected at the end of the set of samples. Prepare a standard curve
all other peaks in the chromatograms (Figures 2011.10C and D). by performing linear least squares (regression) on concentration
(b) Measurement of peak area.—Peak areas are measured with versus the average peak areas of the working standards. A standard
a data system. Before calculating the vitamin B12 concentrations curve must have a correlation of at least 0.999 to be considered
of samples, compare the vitamin B12 peak areas of the standards acceptable for sample calculations.

132.00

130.00

128.00

126.00

124.00

122.00
23.613

120.00

118.00

116.00
AU

114.00

112.00

110.00

108.00

106.00

104.00

102.00

100.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00
Minutes

Figure 2011.10D. Typical standard chromatogram.

© 2012 AOAC INTERNATIONAL

 (2) At each working standard concentration, the peak areas of
standards injected at the beginning and end of a set of samples
should not increase or decrease by more than 10%.
(e) Calculation of vitamin B12 concentrations in samples.—The
vitamin B12 concentration in each injected sample preparation is
extrapolated from the vitamin B12 standard curve prepared above.
The concentration of vitamin B12 in each product can then be
calculated.

Cp = Ci × D1  ss × D2 V

where Cp = product concentration in g/kg; Ci = vitamin B12

concentration of the injected sample preparation extrapolated from
standard curve in g/L; D1= volume of the first dilution in mL
(100 mL); ss = sample size in g; D2 = volume of the second (final)
dilution in mL; V = volume of filtrate loaded onto the cartridge in
mL.
For each set of samples, the control result must be within 3
standard deviations of the control mean.
References: J. AOAC Int. 95, 313(2012); AOAC SMPR 2011.005
J. AOAC Int. 95, 293(2012)

© 2012 AOAC INTERNATIONAL