AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

( 1 ) Into four separate 100 mL volumetric flasks transfer by pipet 1 mL of each of the stock standard solutions, retinyl palmitate, retinyl acetate, α-tocopherol acetate, and α-tocopherol. Label each flask with the individual analyte names. ( 2 ) Mix and dilute each to volume with iso-octane. ( 3 ) Into four separate labeled 2 mL autosampler vials transfer by autopipettor 60 µL retinyl palmitate solution, 30 µL retinyl acetate solution, 100 µL α-tocopherol acetate solution, and 400 µL α-tocopherol. Fill vial with iso-octane to approximately 2 mL. ( 4 ) Vortex briefly and inject into the LC system according to the method parameters described in G . Analyze vitamin A palmitate and vitamin A acetate by UV at 325 nm. For α-tocopherol acetate, analyze by UV at 284 nm and for α-tocopherol, analyze at 292 nm. ( 5 ) Calculate the chromatographic purity (CP) as a decimal for each peak of interest after integration of all the peaks appearing on each chromatogram, using Equation 5: CP = A/100 (5) where CP = area of peak of interest/total peak area excluding solvent. ( f )  Calculation of the concentrations of working standard solutions .—Calculate the concentration, ρ w , of each vitamin in the working standard solutions from the stock solution concentration using the appropriate dilution factor as shown in Equations 6 to 9 in µg/mL for retinyl palmitate (RP) and retinyl acetate (RA) and mg/mL for α-tocopherol (T) and α-tocopherol acetate (TA).

Figure 2012.10C. HPLC chromatogram of vitaminA acetate calibration standard. Peak 1, 13 cis- isomer; peak 3, trans - isomer.

( d )  Spectrometric purity of α-tocopherol stock solution .— ( 1 ) Pipet 3 mL α-tocopherol stock standard solution into a l00 mL volumetric flask and make up to volume with ethanol. ( 2 ) Determine the absorption at 292 nm, zeroed against ethanol in a 1 cm quartz cell. Repeat the reading twice, rinsing the sample cuvet with the solution before each reading. ( 3 ) Calculate the average absorbance reading. Calculate the spectrometric purity as a decimal, SP T , of α-tocopherol using Equation 4: SP T = 01 3 100 05 8.57 × × × ts T m A ρ (4) where A = average absorbance reading, determined above; 75.8 = extinction coefficient of tocopherol at 292 nm; and m st = mass of the reference standard in mg. ( e )  Chromatographic purity of stock standard solutions .— Prepare each stock standard solution separately as follows:

ts m PC PS PR

4

8

V

000 1 × × × × × × (6)

ρ

=

a

wRP

PR

05

05

05 100

ts m PC PS AR

V

4

8

000 1 × × × × × × = a (7)

ρ

wRA

AR

05

05

05 100

Figure 2012.10E. HPLC chromatogram of α-tocopherol acetate and α-tocopherol calibration standard. Peak 1, α-tocopherol acetate; peak 2, α-tocopherol.

Figure 2012.10D. HPLC chromatogram of vitaminA acetate test sample. Peak 1, 13 cis -isomer; peak 2, cis -isomer; peak 3, trans -isomer.

© 2016 AOAC INTERNATIONAL