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

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Purpose:

AOAC 2012.10

Jan 2014

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04

AOAC MLT Study

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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.

= 40±2 ⁰ C

Column oven

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

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