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

( b )  Spectrometric purity of retinyl acetate stock solution .— ( 1 ) Pipet 1 mL retinyl acetate stock standard solution into a l00 mL volumetric flask and make up to volume with ethanol. ( 2 ) Determine the absorption at 325 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 AA , of retinyl acetate using Equation 2: SP AA = 01 1 100 05 560 1 × × × ts m A ρ (2) where A = average absorbance reading, determined above; 1560 = extinction coefficient of retinyl acetate at 325 nm; and m st = mass of the reference standard in mg. ( c )  Spectrometric purity of α-tocopherol acetate stock solution.— ( 1 ) Pipet 3 mL α-tocopherol acetate stock standard solution into a l00 mL volumetric flask and make up to volume with ethanol. ( 2 ) Determine the absorption at 284 nm, zeroed against ethanol, in a 1 cm quartz cell. Repeat the reading twice, rinsing the sample cuvet with the solution before each reading. Calculate the average absorbance reading. ( 3 ) Calculate the spectrometric purity as a decimal, SP TA , of α-tocopherol acetate using Equation 3: SP TA = 01 3 100 05 6.34 × × × ts AT m A ρ (3) where A = average absorbance reading, determined above; 43.6 = extinction coefficient of tocopherol acetate at 284 nm; and m st = mass of the reference standard in mg.

Figure 2012.10A. HPLC chromatogram of vitaminA palmitate calibration standard. Peak 1, 13 cis -isomer; peak 2, trans -isomer.

( g )  Calibration standard solutions .—Into separate 50 mL volumetric flasks, transfer by pipet 0.5, 2, 4, 8, 16, and 32 mL combined working standard solution 2, and dilute to volume with iso-octane. These solutions are used to construct a multipoint calibration curve. Prepare these solutions daily prior to use. Note : For routine testing and depending on the concentration range of the analytes in the test samples, a 3- or 4-point standard curve can be used, provided the ranges are within the lowest and highest points of the 6-point curve listed above. If the result of any analyte is outside the calibration range, standard weights and/or dilutions can be adjusted accordingly. G. Stock Standard Purity Determinations ( a )  Spectrometric purity of retinyl palmitate stock solution .— ( 1 ) Pipet 1 mL retinyl palmitate stock standard solution into a l00 mL volumetric flask and make up to volume with ethanol. ( 2 ) Determine the absorption at 325 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 AP , of retinyl palmitate using Equation 1:

1 100 05

A

01 × × × = (1)

ρ

SP

=

PA

975

m

AP

ts

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

where A = average absorbance reading, determined above; 975 = extinction coefficient of retinyl palmitate at 325 nm; and m st = mass of the reference standard in mg.

© 2016 AOAC INTERNATIONAL