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© 2013 AOAC INTERNATIONAL
AOAC Official Method 2012.10
Simultaneous Determination of 13-
cis
and
all
-
trans
Vitamin A Palmitate (Retinyl Palmitate),
Vitamin A Acetate (Retinyl Acetate),
and Total Vitamin E (dl-α-Tocopherol
and dl-α-Tocopherol Acetate)
in Infant Formula and Adult Nutritionals
Normal-Phase HPLC
First Action 2012
[Applicable to the concurrent quantitative analysis of total
vitamin E (dl-α-tocopherol and dl-α-tocopherol acetate), vitamin A
palmitate, and vitamin A acetate (
cis
- and
trans
-isomers) present
in milk- and soy-based infant formula and adult nutritionals and
formulas containing hydrolyzed protein. Vitamin A is defined
as 13-
cis
and
all-trans
retinol (CAS No. 68-26-8), retinyl esters
(retinyl palmitate; CAS No. 79-81-2), and retinyl acetate (CAS
No, 127-47-9). The determination of vitamin E focuses on d-α-
tocopherol (CAS No. 59-02-9),
all
-racemic α-tocopherol (CAS
No. 1406-18-4), and their esters. α-Tocopherol and esters can be
reported separately.]
The analytical range of the method is as follows:
Vitamin A, retinyl palmitate
.—2–450 μg/100 g reconstituted.
Vitamin A acetate
.—2–450 μg/100 g reconstituted.
dl-α-Tocopherol acetate
.—0.02–9.4 mg/100 g reconstituted.
dl-α-Tocopherol
.—0.03–8.0 mg/100 g reconstituted.
Caution:
Correct personal and environmental safety standards
shall be used while performing this analytical method.
Laboratory personnel handling solvents, acids, and
reagents should be knowledgeable of their potential
hazards. Consult the Material Safety Data Sheets
(MSDSs) for information on the hazards and take proper
precautions. Transfer solvents and acids inside efficient
fume hoods and extractors. Ensure all glassware is free
from chipping and hairline cracks.
A. Principle
This procedureutilizes theproteolytic enzymepapain tohydrolyze
the hydrophilic protein coating of fat micelles in milk- or soy-
based infant formulations in an aqueous solution. The hydrophobic
contents of the micelles are then extracted quantitatively into iso-
octane in a single extraction and chromatographed by normal-
phase HPLC using a Zorbax NH2 analytical column. The analytes
are eluted with a gradient and dl-α-tocopherol and dl-α-tocopherol
acetate quantified using fluorescence detection, excitation/emission,
280/310 nm. Vitamin A palmitate (
cis
and
trans
) and vitamin A
acetate (
cis
and
trans
) are quantified using UV detection. In order
to account for the different biopotency values of the isomers, if
required, all analytes are quantified in IUs and converted to μg or
mg/100 g reconstituted final product following summation of the
isomers.
B. Apparatus
(
a
)
HPLC system.—
Contains pump, autosampler, and
programmable UV and fluorescence detectors (FLD), controlled by
applicable software, Agilent (Santa Clara, CA) 1200, or equivalent.
A Zorbax NH
2
, 150 × 4.6 mm id, 5 μm particle size column
(Agilent), or equivalent with equal performance was used.
(
b
)
Water bath
.—Capable of 37 ± 2°C.
(
c
)
Centrifuge
.—With adapters for 50 mL centrifuge tubes
capable of 4000 rpm.
(
d
)
Laboratory mechanical test tube shaker (optional)
.
(
e
)
UV-Vis spectrophotometer
.—With 1 cm quartz cells.
(
f
)
Standard laboratory glassware
.
(
g
)
Vials
.— 2 mL amber fitted with PTFE liners (Agilent).
(
h
)
Duran bottles
.—1 and 2 L, for the mobile phase (Wertheim/
Main, Germany).
(
i
)
Disposable centrifuge tubes
.—50 mL Falcon tubes, or
equivalent (Fisher, Pittsburgh, PA).
(
j
)
Disposable Pasteur pipets.
C. Standards
(
a
)
Vitamin A palmitate.—
Reference standard, Sigma (St. Louis,
MO) Cat. No. R3375, or equivalent.
(
b
)
Vitamin A acetate.—
Reference standard, Sigma Cat. No.
46958, or equivalent.
(
c
)
dl-α-Tocopherol acetate.—
Reference standard, Sigma Cat.
No. T3376, or equivalent.
(
d
)
dl-α-Tocopherol.—
Reference standard, Sigma Cat. No.
95240, or equivalent.
D. Chemicals and Reagents
(
a
)
Deionized water
.—>18 MΩ resistance (EMD Millipore,
Billerica, MA, or equivalent).
(
b
)
Methyl-t-butyl ether
.—HPLC grade (also known as
tert
-
butyl methyl ether).
(
c
)
Hexane, ethanol, methanol, and iso-octane (2,2,4-
trimethylpentane)
.—HPLC grade.
(
d
)
Papain (from Carica Papain), 3 units/mg
.—Sigma Cat. No.
76220, or equivalent.
(
e
)
Hydroquinone
.—Sigma H9003, or equivalent.
(
f
)
Anhydrous sodium acetate
.—BDH (Visela, CA) 10236, or
equivalent.
E. Solutions
(
a
)
2% Papain solution
.—Dissolve 100 mg hydroquinone and
4 g sodium acetate in approximately 80 mL water in a 100 mL
volumetric flask. Adjust the pH to 5.0 with dilute hydrochloric acid.
Add 2 g papain and make up to volume. Prepare fresh on day of
use.
(
b
)
Glacial acetic acid
.—Reagent grade.
(
c
)
Dilute hydrochloric acid
.—100 mL of 37% HCl diluted to
200 mL with distilled water.
(
d
)
Acidified methanol
.—Add 20 mL acetic acid to 1 Lmethanol
and mix. Prepare fresh on day of use.
(
e
)
Mobile phase A
.—Hexane, filtered, and deaerated for 10 min
in an ultrasonic bath.
(
f
)
Mobile phase B.
—Hexane–methyl-
t
-butyl ether (75 + 25,
v/v). Add 3 mL methanol, filter, and deaerate for 10 min in an
ultrasonic bath.
F. Calibration Standards
Note
: Class A certified glassware is recommended for the
preparation of stock reference standards.
(
a
)
Vitamin A palmitate stock standard (P1)
.—Weigh (to
0.01 mg) approximately 70 mg retinyl palmitate into a 50 mL
volumetric flask. Dissolve in and dilute to volume with iso-octane.
(
b
)
Vitamin A acetate stock standard (A1)
.—Weigh (to 0.01 mg)
approximately 35 mg retinol acetate into a 50 mL volumetric flask.
Dissolve in and dilute to volume with ethanol.
Candidates for 2016 Method of the Year
357