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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Note: All the above stock standards are stable in a refrigerator at 4 – 8 O C for up to 7 days e. Combined working standards (S2) Transfer by pipette 4mL of P1, 4mL of A1, 7mL of E1 and 20mL of E2 into a 50mL volumetric flask. Dilute to volume with iso-octane. f. Combined working standards (S3) Transfer by pipette 8mL of S2 into a 100mL volumetric flask and dilute to volume with iso-octane. Note: Above combined standards should be freshly prepared prior to use. Standard Calibration curve: Into separate 50mL volumetric flasks, transfer by pipette 0.5mL, 2mL, 4mL, 8mL, 16mL, 32mL of S3 and dilute to volume with iso-octane. These solutions are used to construct a multipoint calibration curve. These should be prepared daily prior to use. g.

K. CONCENTRATION OF STOCK STANDARDS

Vitamin A Palmitate

a. Pipette 3 mL of stock solution P1 into a 100mL volumetric flask and make up to volume with ethanol. Pipette 3mL of this solution into a 100mL volumetric flask and dilute to volume with ethanol .

b. Determine the absorption at 325 nm, zeroed against ethanol in a 1cm quartz cell.

c. Repeat the reading a further two times, rinsing the sample cuvette with the solution before each reading. Calculate the average absorbance reading.

d. The potency of the vitamin A palmitate stock solution is then calculated as follows:

Vitamin A Palmitate (µg/mL) = ABS × 100 × 100 × 10000 975 3 3

Where: ABS = average absorbance reading, determined above; 975 = extinction coefficient of retinyl palmitate at 325nm; 10000 = conversion of percentage to µg/mL.

Vitamin A Acetate

e. Pipette 3mL of stock solution A1 into a 100mL volumetric flask and make up to volume with ethanol. Pipette 3mL of this solution into a 100mL volumetric flask and dilute to volume with ethanol .

b. Determine the absorption at 325 nm, zeroed against ethanol in a 1cm quartz cell.

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