50.1.26
—10–1000 and
AOAC Official Method 2004.07
Vitamin B6 in Reconstituted Infant Formula
Liquid Chromatographic Method
First Action 2004
(Applicable to the determination of vitamin B6 in milk- and
soy-based liquid infant formula at 0–1 mg/100 g.)
See Table 2004.07 for the results of the interlaboratory study
supporting acceptance of the method.
Caution: Conduct borohydride reduction in fume hood. Consult
Material Safety Data Sheet (MSDS) for hazard
information regarding the use of all reagents. Wear
protective gloves and protective eyewear. Sodium
borohydride is a highly hydroscopic and reactive
reagent. Store in a freshly charged desiccator. Avoid
contact with water, which may react violently,
liberating extremely flammable hydrogen gas. Do not
breathe dust and avoid contact with eyes, skin, and
clothing. Wear gloves and eye protection. Use under
fume hood. There is limited evidence of carcinogenic
effect from glyoxylic acid; possibly mutagenic. Handle
carefully, avoiding contact with skin.
A. Principle
Vitamin B6 is dephosphorylated by enzy matic hy droly sis.
Simultaneously, by reaction with glyoxylic acid in presence of Fe+2
catalyst, pyridoxamine is transformed into pyridoxal, which is
subsequently reduced to pyridoxine by the action of sodium
borohydride in the alkaline medium. The total vitamin B6 is
quantified by ion-pair liquid chromatography (LC).
Notes: Glassware must be free from detergent or any other
substance that can fluoresce, giving high reagent blanks. Rinse
glassware with nitric acid and alcohol.
Cal i brate micropipets by dis pens ing a known vol ume of
deionized water into a small tared beaker and checking the weight of
water dispensed.
Use deionized water (resistivity >18MW) to prepare the mobile
phase and the aqueous reagent solution.
B. Apparatus
(a) Liquid chromatograph.—LC system with pump maintaining
constant pulseless flow of mobile phase at 1.0 mL/min, autosampler
(or manual injector), and fluorescence detector set at excitation
wavelength of 290 nm and emission wavelength of 395 nm. LC
system is preferably computer controlled with electronic data
collection.
(b) Column.—Luna 5 mm phenyl-hexyl column, 250 ´ 4.6 mm id
(Part No. 00G-4257-RO, Phenomenex Inc., Torrance, CA
90501-1456, USA; www.phenomenex.com). Other reversed-phase
col umns, includ ing C8 and C18, can be used if equiv alent
performance is demonstrated.
(c) Constant temperature incubator shaker.—Oven or water
bath continuously shaking and incubating vessels at 37°C overnight
(Controller Environment Incubator Shaker, New Brunswick
Scientific Co., Edison, NJ 08818-4005, USA; www.nbsc.com, is
suitable).
(d) Ultrasonic cleaner.—1 gal capacity or larger.
(e) Laboratory balance.—Readability, 0.1 mg.
(f) Cal i brated pre ci sion microliter pipets.
500–5000 mL ca pac ity [Gilson (Middleton, WI, USA;
www.gilson.com) Pipetman ® Pipet P-1000 and P-5000 are
suitable].
(g) Disposable syringe filters.—Nylon or cellulose acetate,
25 mm diameter, 0.45 mm pore size.
C. Reagents
(a) Glyoxylic acid monohydrate (C2H2O3×H2O).—Cat. No.
G10601 (Aldrich Chem i cal, Mil wau kee, WI 53233, USA;
www.sigmaaldrich.com).
(b) Pyridoxal hydrochloride.—Sigma Cat. No. P 9130.
(c) Pyridoxine hydrochloride, purity >98%.—Sigma Cat. No.
P9755, or equivalent.
(d) Sodium borohydride.—Sigma Cat. No. H2766.
(e) Sodium 1-heptanesulfonic acid.—Sigma Cat. No. H2766.
(f) Acid phosphatase from potato.—Grade II, 2 U/mg (Cat. No.
0108227, Roche Mo lec u lar Biochemicals, In di a nap o lis, IN
46250-0414, USA; www.roche-applied-science.com).
D. Preparation of Solutions
(a) Mobile phase.—Methanol–0.01M phosphoric acid (26 + 74).
Weigh 2.28 g ortho-phosphoric acid (85%, w/w) and 1.0 g
1-heptanesulfonic acid, sodium salt, into a 2 L volumetric flask
containing ca 1 L deionized water. Add 520 mL methanol. Swirl to
mix. Dilute to volume with deionized water, and mix thoroughly.
Filter through 0.45 mm disposable syringe filter. Degas. Check pH of
mobile phase and adjust if necessary with ortho-phosphoric acid or
sodium hydroxide solution to pH 2.50–2.60.
(b) Sodium acetate solution.—0.05M. Dissolve 6.8 g sodium
acetate·3H2O in ca 900 mL deionized water. Adjust to pH 4.5 with
glacial acetic acid and dilute to 1 L with deionized water.
(c) Acid phosphatase solution.—20 mg/mL. Prepare fresh daily.
Dissolve 0.50 g acid phosphatase in 25.0 mL 0.05M sodium acetate
solution.
(d) Sodium acetate solution.—140 g/L. Dissolve 70 g sodium
acetate·3H2O in 500 mL deionized water.
(e) Glyoxylic acid solution.—1M. Prepare fresh daily. Dissolve
4.70 g glyoxylic acid monohydrate in ca 30 mL 140 g/L sodium
acetate solution. Adjust pH to 4.5 with 6M potassium hydroxide
solution. Dilute to 50 mL with deionized water.
(f) Ferrous sulfate solution.—10 g/L. Prepare fresh daily.
Dissolve 0.50 g ferrous sulfate·7H2O in 50 mL 0.05M sodium
acetate solution, pH 4.5.
(g) Sodium acetate solution.—0.625M. Dissolve 85 g sodium
acetate·3H2O in ca 900 mL deionized water. Adjust to pH 4.5 with
glacial acetic acid and dilute to 1 L with deionized water.
(h) Sodium hydroxide solution.—0.2M. Dissolve 0.80 g sodium
hydroxide in 100 mL deionized water.
(i) Sodium borohydride solution.—0.1M. Prepare fresh daily.
Dissolve 0.378 g sodium borohydride in 100 mL 0.2M sodium
hydroxide solution. Keep solid sodium borohydride reagent in
desiccator with freshly recharged desiccant. If reagent becomes
moist, discard.
(j) Phos pho ric acid so lu tion.—1.14 g/L. Di lute 1.14 g
ortho-phosphoric acid (85%) to 1 L with deionized water.
E. Preparation of Stock Standard Solutions
(a) Stock pyridoxine hydrochloride standard.—Accurately weigh
0.385 g pyridoxine hydrochloride into a 1 L volumetric flask and
© 2005 AOAC INTERNATIONAL
Table 2004.07. Interlaboratory study results for vitamin B6 in reconstituted infant formula
Milk-based infant formula
a b
Sample No. No. labs Mean, mg/g sr RSDr, % 2.8 ´ sr sR RSDR, % 2.8 ´ sR HORRAT
1 9 0.130 0.01 10 0.04 0.02 17.5 0.06 0.81
2 9 0.36 0.01 3.3 0.03 0.03 8.2 0.08 0.44
3 8 0.57 0.01 2.0 0.03 0.05 8.4 0.13 0.48
4 9 1.01 0.04 4.0 0.090 0.060 8.4 0.24 0.53
Soy-based infant formula
5 7 0.05 0.01 16.4 0.02 0.02 52.1 0.07 2.06
6 9 0.28 0.02 5.9 0.05 0.03 11.2 0.09 0.58
7 9 0.56 0.03 4.5 0.07 0.04 7.4 0.12 0.42
8 9 1.06 0.04 3.5 0.10 0.07 6.7 0.20 0.43
a
Sample 1 is nonfortified milk-based infant formula; Samples 2–4 are fortified milk-based infant formula; Sample 5 is nonfortified soy-based infant formula;
Samples 6–8 are fortified soy-based infant formula.
b
Outliers excluded.

dilute to volume with deionized water; mix thoroughly. If standard is
not anhydrous, correct weight for water content.
(b) Stock pyridoxal hydrochloride standard.—Accurately weigh
0.385 g pyridoxal hydrochloride into a 1 L volumetric flask and
dilute to volume with deionized water; mix thoroughly.
(c) Preparation of standard for systems suitability test.—(1)
Intermediate pyridoxine hydrochloride standard solution.—Pipet
20.0 mL stock pyridoxine hydrochloride standard into 200 mL
volumetric flask and dilute to volume with deionized water; mix
well. (2) Intermediate pyridoxal standard.—Pipet 20.0 mL stock
pyridoxal hydrochloride standard solution into 200 mL volumetric
flask, and dilute to volume with deionized water. (3) Working system
suitability standard solution.—0.385 mg of each analyte/mL. Pipet
1.0 mL of each intermediate standard, E(c)(1) and E(c)(2), into
100 mL volumetric flask and dilute to volume with the aqueous
solution containing 1.14 g/L ortho-phosphoric acid (85%) solution.
Prepare fresh daily.
(d) Working standards.—Pipet 0, 0.1, 0.2, 0.4, 0.8, and 1.2 mL
prepared intermediate pyridoxine hydrochloride standard solution,
E(c)(1), into sep a rate 50 mL Erlenmeyer flasks. Stan dard
concentrations will be 0 (reagent blank), 0.038, 0.077, 0.154, 0.309,
and 0.463 mg/mL, respectively. Add 25 mL deionized water to each
flask and process similarly to test samples, G(b).
F. System Suitability
Allow LC system to warm up and equilibrate for at least 30 min
with mo bile phase flow ing at 1.0 mL/min. Vi ta min B 6 is
light-sensitive; avoid exposure to bright light.
(a) Resolution test.—Inject 10 mL working suitability standard,
E(c)(3). Pyridoxal is eluted first at ca 9 min followed by pyridoxine
at ca 10 min at flow rate of 1.0 mL/min. The pyridoxal–pyridoxine
pair must have base line resolution (R > 1).
R = T1 – T2/2(W1 + W2)
where T1 and T2 are the retention times and W1 and W2 are the width
of the peaks at baseline of the 2 components. The peaks should not
exhibit excessive tailing. The tailing factor (T) is calculated as
follows:
T = W0.05/2 f
where W0.05 is the width of peak at 5 % height; f is the distance from
peak maximum to leading edge of peak, measured at 5 % height. For
a symmetrical peak, the tailing factor T is unity and the value of T
increases as tailing becomes more pronounced. Include the standard
injection at the beginning of each set to verify proper column
performance.
(b) Retention test.—Pyridoxine peak should elute between 7 and
12 min at 1.0 mL/min. To correct, adjust the solvent ratio of the
mobile phase. An increase in methanol ratio will decrease the
retention times, and vice versa.
(c) Detector sensitivity.—The peak area for the lowest standard
should be >5 times the baseline noise. To improve sensitivity, the
injection volumes can be increased from 10 to 100 mL or the gain on
the detector can be increased.
(d) System linearity.—Inject each working standard. Prepare a
5-point calibration over the range of 0.038 to 0.463 mg/mL. Plot the
standard concentration vs peak area for each standard, using a least
square linear regression. The r2 should be >0.995.
(e) System precision.—Make 5 rep licate injec tions of the
suitability test mixture saved from the system resolution test. The
relative standard deviation (RSD) of the pyridoxine peak should be
<2%.
G. Determination
(a) Preparation of test sample.—Weigh 25 g well-mixed,
ready-to-feed infant formula into 50 mL Erlenmeyer flask. For
powdered infant formulas, dilute accordingly to manufacturer's
instruction, typically 12–15 g/100 mL.
(b) Derivatization.—Add the following reagents to the test
solutions and working standards in the following order: 2.0 mL
0.625M sodium acetate solution; 2.5 mL 1M glyoxylic acid reagent;
0.8 mL 10 g/L ferrous sulfate solution; 1.0 mL 20 mg/mL acid
phosphatase solution.
Add four 6 mm glass beads to all test solutions to ensure mixing
during incubation.
Place solutions in a shaker bath at 37°C overnight (or at least 12 h)
to ensure complete dephosphorylation of the analytes. Remove the
incubated extracts from the shaker. Let cool and quantitatively
transfer to 50 mL volumetric flasks. Dilute to volume with deionized
water and mix. Filter extract through Whatman No. 40 filter paper,
or equivalent.
Discard first 10 mL of extract. Transfer 5.0 mL clear filtrate to a
small flask or beaker; add 4.5 mL 0.1M sodium borohydride

© 2005 AOAC INTERNATIONAL

solution. Mix by gently swirling for ca 20 s. Add 0.5 mL glacial Use the slope and intercept to calculate the concentration of
acetic acid. Gently swirl for 30 s. After effervescence has subsided, pyridoxine·HCl in the extract. This calculation is usually performed
filter ca 2 mL final extract through a 0.45 mm syringe filter. by the LC software.
(c) Chromatography.—Inject the test solutions, using same
volumes as selected for standards and run time of ca 25 min.
Pyridoxine×HCl, mg/g =
Re-inject standards at the end of the run to check for the presence of
long term drift. For very long runs, inject the standards at
intermediate periods. sample area - intercept 100 (mL)
´
Use the integrated area from the test extract to determine the slope test portion, wt (g)
concentration of vitamin B6 in the test samples.
H. Calculations
where 100 is the final volume of the extract.
Obtain the total vitamin B6 content of the test sample from the
linear regression plot of the pyridoxine hydrochloride standards. Reference: J. AOAC Int. 88, 30(2005).

© 2005 AOAC INTERNATIONAL