Continue refluxing for 20 min and, without cooling, add 2 mL of hydrochloric acid dropwise through the condenser.

Cool, and transfer the contents of the flask to a 500-mL separator, rinsing the flask with 100 mL each of water and of ether, and adding the rinsings to the separator. Shake vigorously, allow the layers to separate, and collect each of the two layers in individual separators. Extract the aqueous layer with two 50-mL portions of ether, and add these extracts to the main ether extract. Wash the combined ether extracts with four 100-mL portions of water, then evaporate the ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until about 7 or 8 mL remain. Complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in dilute sulfuric acid in alcohol (1 in 72), transfer to a 200-mL volumetric flask, and dilute with dilute sulfuric acid in alcohol (1 in 72) to volume.

Add 2 mL of nitric acid with swirling to 10 mL of Sample solution, and heat at about 75 for 15 min.

To the entire volume of a Sample solution, as prepared above, add 20 mL of a solution (1 in 10) of potassium ferricyanide in sodium hydroxide solution (1 in 125), and shake for 3 min. Wash the ether solution with four 50-mL portions of water, discard the washings, and dry over anhydrous sodium sulfate. Evaporate the dried ether solution on a water bath under reduced pressure or in an atmosphere of nitrogen until 7–8 mL remain, then complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in 5.0 mL of isooctane, and determine the optical rotation using as c the number of g of total tocopherols, determined in the Assay, in each 100 mL of solution used for the test.

Chromatograph the Interference check solution so that the principal peak exhibits NLT 50% of maximum recorder response. Similarly chromatograph the Internal standard solution. If a peak observed in the Interference check solution has the same retention time as that for hexadecyl hexadecanoate, make any necessary correction for factors of dilution or attenuation, and determine the area due to the interfering component that must be subtracted from the area of the internal standard peak appearing in the Sample solution.