OMB Meeting Book_9-11-14

Vitamin A & E - MLT For ERP Use Only DO NOT DISTRIBUTE

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 . Vitamin A acetate (A1) Accurately weigh 35mg retinol acetate into a 50mL volumetric flask. Dissolve in and dilute to volume with ethanol . 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. dl-α-Tocopherol (E2) Accurately weigh 100mg dl-α-tocopherol into a 50mL volumetric flask. Dissolve in and dilute to volume with iso-octane.

b.

c.

d.

Page 3