(Official January 1, 2007)

Cyclohexane— Spectrophotometric grade, or material that has been purified by being passed through a column of activated silica gel and distilled.

Iodine solution— Transfer about 10 mg of iodine into a 100-mL volumetric flask. Dissolve in cyclohexane, dilute with cyclohexane to volume, and mix. Dilute 10.0 mL of this solution with cyclohexane to 100 mL, and mix. Prepare this solution fresh daily.

Assay preparations— Combine the contents of not fewer than 20 Capsules, and grind, using a freezer mill, to a fine powder of uniform color. Transfer an accurately weighed quantity of the finely ground capsule contents, equivalent to about 75 mg of beta carotene, to a 1000-mL volumetric flask, add 500 mL of water, and heat at 60 for 15 minutes. Cool to ambient temperature, dilute with water to volume, and mix (Assay preparation 1). Transfer 5.0 mL of Assay preparation 1 to a glass-stoppered, 50-mL centrifuge tube. Add 3 g of sodium sulfate decahydrate, 2 mL of 1 N hydrochloric acid, and 20.0 mL of chloroform. Shake by mechanical means for 10 minutes, centrifuge for 5 minutes, and remove the aqueous layer without disturbing the chloroform layer. Add 2 g of anhydrous sodium sulfate to the chloroform layer, shake vigorously, and allow to settle (Assay preparation 2). Transfer 5.0 mL of Assay preparation 2 to a 50-mL volumetric flask, add 30 mL of cyclohexane, and mix. Add 0.05 mL of Iodine solution, dilute with cyclohexane to volume, mix, and allow to stand for 3 hours (Assay preparation 3). Transfer 20 mL of Assay preparation 3 to a centrifuge tube, and centrifuge for 2 minutes.

Standard preparations— Accurately weigh about 17 mg of Beta Carotene, previously subjected to the Assay and previously dried in vacuum over phosphorus pentoxide at 40 for 4 hours, and transfer to a 1000-mL volumetric flask. Add 10 mL of water, heat at 60 for 15 minutes, and cool to room temperature. Add 3 g of sodium sulfate decahydrate and 2 mL of 1 N hydrochloric acid, and shake by mechanical means for 10 minutes. Add 200 mL of chloroform, and shake for 10 minutes. Add 750 mL of chloroform, shake, and dilute with chloroform to volume, disregarding the aqueous layer (Standard preparation 1). Shake vigorously, and allow the layers to separate completely. Transfer about 20 mL of the chloroform layer to a centrifuge tube, add 2 g of anhydrous sodium sulfate, shake vigorously, and allow to settle. Transfer 5.0 mL to a 50-mL volumetric flask, add 30 mL of cyclohexane, and mix. Add 0.05 mL of Iodine solution, dilute with cyclohexane to volume, mix, and allow to stand for 3 hours (Standard preparation 2). Transfer about 20 mL of Standard preparation 2 to a centrifuge tube, and centrifuge for 2 minutes.

Procedure— Concomitantly measure the absorbances of Assay preparation 3 and Standard preparation 2 at 452 nm, with a suitable spectrophotometer, using cyclohexane as the blank. Calculate the quantity, in mg, of C40H56 in the portion of Capsule contents taken by the formula: in which C is the concentration, in µg per mL, of Beta Carotene in the Standard preparation; and A U and A S are the absorbances of Assay preparation 3 and Standard preparation 2, respectively.