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: 7 of 11
b.
For liquid products, transfer 5.0g of thoroughly homogenized sample directly to a
screw-top 50mL centrifuge tube.
c.
To the above weighed solutions, add 5mL of a 2% papain solution.
d.
Mix to disperse each sample, cap, and place the tubes in a 37±2
0
C water bath for 20 to
25 minutes. Remove the samples from the water bath and cool. (Place in freezer for
about 5 minutes or refrigerate for about 20 minutes).
e.
Add 20mL acidified methanol to each sample tube and shake the tubes for 10minutes
preferably with a mechanical shaker.
f.
Accurately pipet 10mL iso-octane to each sample tube. Close tightly to avoid leakage
and shake the tubes for 10mins preferably with a mechanical shaker.
g.
Centrifuge for 10minutes at 4000rpm to obtain a clear iso-octane layer. Transfer the
clear iso-octane layer into amber vial for HPLC analysis.
M. HPLC ANALYSIS
Column
= Zorbax NH
2
150 mm x 4.6 mm x 5 µm x 100Å
Mobile phase A
= n-Hexane
Mobile Phase B
= n-Hexane + Methyl-tert-butyl ether + Methanol (750 mL + 250 mL + 3 mL)
Flow rate
= 1.5 mL/min.
Injection volume
= 50 µL.
Column oven
= 40±2
⁰
C
Run time
= 20 min.
Detector Settings
Set PDA / UV detector at 325nm for Vitamin A palmitate and Vitamin A acetate. Set the
Fluorescence detector, (excitation/emission) 280/310nm for DL-α-Tocopherol Acetate and DL-α-
Tocopherol.
Pump Gradient Elution Cycle
Time (min)
Flow, mL/min
% Mobile phase A
% Mobile phase B
0.0
1.5
95
5
3.0
1.5
95
5
Vitamin A & E - MLT
For ERP Use Only
DO NOT DISTRIBUTE
Page 7
