A: Thin-Layer Chromatographic Identification Test 201—

B: The chromatogram of the Test solution exhibits peaks for daidzein, genistein, formononetin, and biochanin A at retention times that correspond to those in the chromatogram of Standard solution 1, as obtained in the test for Content of isoflavones. Calculate the ratio of 5,7-dihydroxyisoflavones to 7-hydroxyisoflavones by the formula: in which B, G, D, and F are the percentages of biochanin A, genistein, daidzein, and formononetin, respectively, as obtained in the test for Content of isoflavones: the ratio is between 0.1 and 10.0.

Solvent, Solution A, Solution B, Standard solution 1, Standard solution 2, and Chromatographic system— Proceed as directed under Red Clover.

Test solution— Transfer an accurately weighed quantity of Powdered Extract, equivalent to about 30 mg of the labeled content of isoflavones, to a 250-mL volumetric flask. Add 15 mL of dehydrated alcohol, sonicate until dissolved, dilute with Solvent to volume, and mix. Transfer 50.0 mL of this solution to a round-bottom flask, and evaporate to dryness under vacuum. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 minutes. Quantitatively transfer the resulting solution with the aid of about 15 mL of alcohol to a 50-mL volumetric flask, and dilute with Solvent to volume. Centrifuge, or pass through a filter having a 0.45-µm or finer porosity.

Procedure— Proceed as directed under Red Clover. Separately calculate the percentage of each relevant isoflavone component in the portion of Powdered Extract taken by the formula: in which W is the weight, in g, of Powdered Extract taken to prepare the Test solution; and the other terms are as defined therein. Calculate the percentage of isoflavones in the Powdered Extract taken by adding the individual quantities calculated.