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: 4 of 11
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.
g.
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.
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:
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.
Vitamin A Palmitate (µg/mL) = ABS × 100 × 100 × 10000
975
3
3
Vitamin A & E - MLT
For ERP Use Only
DO NOT DISTRIBUTE
Page 4
