Macroscopic— Red Clover inflorescences are ovoid with a rounded summit, mostly from 12 to 34 mm in length and width, usually on a very short stalk, shriveled, purplish, and more or less brown from drying, consisting of many papilionaceous flowers, crowded together and clothed at the base with broad, pointed, pale green ciliate stipules with darker veins. The flowers, which may or may not be accompanied by diminutive trifoliate leaves, are up to 15 mm in length and have the following: five green, hairy, subulate calyx teeth, one longer than the other four; petals united into a more or less campanulate tube, somewhat recurved, and colorless with pinkish purple veins; diadelphous stamens; slender style; a faintly aromatic, somewhat tea-like odor; and a sweetish, then slightly bitter taste.

Microscopic— Epidermis of calyx composed of polygonal cells with faintly striated cuticle and occasional anomocytic stomata on the outer epidermis only; abundant, uniseriate, covering trichomes with two small, thin-walled basal cells and a thick-walled tapering end cell, up to 1 mm in length with a warty cuticle. Glandular trichomes are also present, particularly on the lower epidermis, each with a one- or two-celled stalk and a large, cylindrical head composed of several cells arranged in two rows. Epidermal cells of the corolla, papillose at the tip, are elongated with slightly wavy walls and a strongly striated cuticle; vascular strands of corolla and calyx are surrounded by a crystal sheath containing prismatic crystals of calcium oxalate. The following are also present: fibrous layer of anthers; subspherical pollen grains, 20 to 48 µm in diameter with smooth exine, three distinct pores, and three furrows; upper epidermal cells of leaflets with sinuous and slightly beaded anticlinal walls; lower epidermis with sinuous to wavy walls; anomocytic stomata on both surfaces, but more frequent on the lower surface; abundant covering trichomes on both surfaces and on the margins; and fibrovascular strands surrounded by a crystal sheath containing prismatic crystals of calcium oxalate.

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.

Solvent: a mixture of alcohol and water (1:1).

Solution A— Prepare a filtered and degassed mixture of water and acetonitrile (75:25) containing 0.05% trifluoroacetic acid.

Solution B— Use filtered and degassed acetonitrile containing 0.05% trifluoroacetic acid.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution 1— Transfer an accurately weighed quantity of USP Powdered Red Clover Extract RS, 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 filter through a membrane having a 0.45-µm or finer porosity.

Standard solution 2— Dissolve an accurately weighed quantity of USP Formononetin RS in a mixture of n-propanol and water (1:1) with sonication. Dilute quantitatively, and stepwise if necessary, with the mixture of n-propanol and water (1:1) to obtain a solution having a known concentration of about 0.1 mg per mL. Filter through a membrane having a 0.45-µm or finer porosity.

Test solution— Accurately weigh approximately 2500 mg of ground plant material, and place in a 120-mL flask with a stopper. Add exactly 100 mL of Solvent, close the flask, and shake on an orbital or wrist-action shaker for not less than 12 hours. Transfer 50.0 mL of this solution to a round-bottom flask, and evaporate to dryness under vacuum at about 40. Add 15 mL of 2 N hydrochloric acid, and heat in a water bath for 30 minutes. Quantitatively transfer this solution, with the aid of about 15 mL of alcohol, to a 50-mL volumetric flask, and dilute with Solvent to volume. Filter through a membrane having a 0.45-µm or finer porosity, discarding the first 4 mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains end-packed 5-µm packing L1. The flow rate is about 1.0 mL per minute. The column temperature is maintained at 45. The chromatograph is programmed as follows. Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the chromatograms obtained are similar to the Reference Chromatogram provided with the USP Powdered Red Clover Extract RS; the tailing factor for formononetin is not more than 2.0; and the relative standard deviation for replicate injections of Standard solution 1 is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of Standard solution 1, Standard solution 2, and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the analyte peaks. Identify the retention times of the peaks corresponding to daidzein, genistein, formononetin, and biochanin A by comparison of the chromatogram of Standard solution 1 with that obtained from the Reference Chromatogram. Separately calculate the percentages of daidzein, genistein, formononetin, and biochanin A in the portion of Red Clover taken by the formula: in which F is the conversion factor for each analyte (0.97 for daidzein, 1.13 for genistein, 1.00 for formononetin, and 1.05 for biochanin A); C is the concentration, in mg per mL, of USP Formononetin RS in Standard solution 2; W is the weight, in g, of Red Clover taken to prepare the Test solution; rU is the peak response for each relevant isoflavone obtained from the Test solution; and rS is the peak response for formononetin obtained from Standard solution 2.