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: 3 of 11
b.
Vitamin A Acetate reference standard: Sigma 46958 or equivalent
c.
dl-α-Tocopherol Acetate reference standard: Sigma T3376 or equivalent
d.
dl-α-Tocopherol reference standard: Sigma T3251, Fluka 95240 or equivalent
H. CHEMICALS AND REAGENTS
a.
Laboratory Reagent Grade Water
b.
Methyl-t-butyl ether – HPLC grade (also known as tert-butylmethylether)
c.
Hexane, ethanol, methanol and iso-octane (2,2,4-trimethylpentane) HPLC grade
d.
Papain (from
Carica papaya
) ≥3units/mg; Sigma/Fluka 76220 or equivalent
e.
Hydroquinone: Sigma H9003 or equivalent
f.
Glacial acetic acid: Analytical reagent grade
g.
Sodium acetate anhydrous: BDH 10236 or equivalent
I. SOLUTIONS
a.
Papain solution (2%): Dissolve 100mg hydroquinone and 4g sodium acetate in
approximately 80mL water in a 100mL volumetric flask. Adjust pH to 5.0 with dilute
hydrochloric acid. Add 2g papain and make up to volume (prepare fresh prior to use).
b.
Dilute hydrochloric acid: 100mL of 36% HCL diluted to 200mL with water
c.
Acidified methanol: Add 20mL of glacial acetic acid to 1L methanol and mix (prepare
fresh).
d.
Mobile Phase A: Hexane, filtered, and degassed for 15 minutes in an ultrasonic bath
e.
Mobile Phase B: Mix 750mL of Hexane with 250mL of Methyl-t-butyl ether. Add 3mL of
methanol, filter, and de-aerate for 15minute in an ultrasonic bath.
J. CALIBRATION STANDARDS
a.
Vitamin A Palmitate (P1)
Accurately weigh 70mg retinyl palmitate into a 50mL volumetric flask. Dissolve in and
dilute to volume with
iso-octane
.
b.
Vitamin A acetate (A1)
Accurately weigh 35mg retinol acetate into a 50mL volumetric flask. Dissolve in and
dilute to volume with
ethanol
.
c.
dl-α-Tocopherol Acetate (E1)
Accurately weigh 180mg DL-α-tocopherol acetate into a 50mL volumetric flask.
Dissolve in and dilute to volume with
iso-octane.
d.
dl-α-Tocopherol (E2)
Accurately weigh 100mg dl-α-tocopherol into a 50mL volumetric flask. Dissolve in and
dilute to volume with
iso-octane.
Vitamin A & E - MLT
For ERP Use Only
DO NOT DISTRIBUTE
Page 3
