SIMULTANEOUS DETERMINATION OF 13-CIS AND ALL-TRANS VITAMIN A PALMITATE (RETINYL
PALMITATE), VITAMIN A ACETATE (RETINYL ACETATE), AND TOTAL VITAMIN E (Α-TOCOPHEROL
AND DL-Α-TOCOPHEROL ACETATE) IN INFANT FORMULA AND ADULT NUTRITIONALS BY
NORMAL PHASE HPLC
CODE:
DATE:
PAGE:
VER:
Purpose:
AOAC 2012.10
Jan 2014
1 of 11
04
AOAC MLT Study
Page: 2 of 11
D. WARNINGS AND SAFETY PRECAUTIONS
Analysts using this procedure should be familiar with good laboratory practice. It is the
responsibility of the analyst to ensure they are familiar with the appropriate SDSs and safety
practices for all the equipment utilized.
Transfer all solvents and acids inside efficient fume hoods and extractors. Organic solvents are, in
general flammable. Do not dispense or transfer solvents near any strong heat source or naked
flame, e.g. Bunsen burner flame. Ensure all glassware to be used are free from chipping and
hairline cracks.
E. PRINCIPLE
This procedure utilizes the proteolytic enzyme, papain, to hydrolyze the hydrophilic protein coating
of fat miscelles in milk- or soy-based infant formulations in an aqueous solution. The hydrophobic
contents of the miscelles are then extracted quantitatively into iso-octane in a single extraction.
The extract is analysed by normal phase HPLC using a Zorbax NH
2
analytical column with gradient
elution. dl-α-Tocopherol and dl-α-Tocopherol Acetate are quantified using fluorescence detection
with excitation and emission wavelengths as 280/310nm. Vitamin A Palmitate (cis and trans) and
vitamin A acetate (cis and trans) are quantified using UV detection at 325nm.
F. APPARATUS
1. HPLC equipped with UV detector operating at 325nm for vitamin A and Fluoresence
detector, operating at (excitation/emission) 280/310nm for Vitamin E.
2. HPLC Column: Zorbax NH2 150mm x 4.6mm x 5µm or equivalent
3. Water bath se at 37±2
⁰
C
4. Centrifuge with adaptors for 50mL centrifuge tubes capable of 4000rpm
5. UV-Visible Spectrophotometer with 1cm quartz cells
6. Analytical balance, weighing to four decimal places.
7. Amber HPLC vials, 2mL, with plastic push caps and PTFE seals.
8. Falcon tubes, 50mL
9. Laboratory mechanical test tube shaker
10. Sonicator bath
11. Volumetric flasks, 50mL, 100mL calibrated.
12. Vacuum filtration apparatus with 0.45μm nylon membrane.
13. Schott Duran Bottles, 1L and 2L for mobile phase
G. STANDARDS
a.
Vitamin A Palmitate reference standard: Sigma R3375 or equivalent (note:
the standard
must contain antioxidant)
Vitamin A & E - MLT
For ERP Use Only
DO NOT DISTRIBUTE
Page 2
