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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AOAC 2012.10

Jan 2014

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AOAC MLT Study

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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)

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