Macroscopic— Red Clover inflorescences are ovoid with a rounded summit,mostly from 12to 34mm 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 15mm 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 1mm 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,20to 48µm 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.

Change to read:

A:Thin-Layer Chromatographic Identification Test á201ñ—

B: The chromatogram of the Test solutionexhibits peaks for daidzein,genistein,formononetin,and biochanin Aat 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,andFare the percentages of biochanin A,genistein,daidzein,and formononetin,respectively,as obtained in the test forContent of isoflavones:the ratio is between 0.1and 10.

Change to read:

Change to read:

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 Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution 1— Transfer an accurately weighed quantity of USP Powdered Red Clover Extract RS,equivalent to about 30mg of the labeled content of isoflavones,to a 250-mLvolumetric flask.Add 15mLof dehydrated alcohol,sonicate until dissolved,dilute withSolvent to volume,and mix.Transfer 50.0mLof this solution to a round-bottom flask,and evaporate to dryness under vacuum.Add 15mLof 2Nhydrochloric acid,and heat in a water bath for 30minutes.Quantitatively transfer the resulting solution,with the aid of about 15mLof alcohol,to a 50-mLvolumetric flask,and dilute withSolvent to volume.Centrifuge,or filter through a membrane having a 0.45-µm or finer porosity.USP28

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

Test solution— Accurately weigh approximately 2500mg of ground plant material,and place in a 120-mLflask with a stopper.Add exactly 100mLof Solvent,close the flask,and shake on an orbital or wrist-action shaker for not less than 12hours.Transfer 50.0mLof this solution to a round-bottom flask,and evaporate to dryness under vacuum at about 40.USP28Add 15mLof 2Nhydrochloric acid,and heat in a water bath for 30minutes.Quantitatively transfer this solution,with the aid of about 15mLof alcohol,to a 50-mLvolumetric flask,and dilute with Solventto volume.Filter through a membrane having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.

Chromatographic system(see Chromatography á621ñ)— 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 1.0mLper 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 1is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)USP28of Standard solution 1,Standard solution 2,and theTest 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 Aby comparison of the chromatogram of Standard solution 1with that obtained from the Reference Chromatogram.Separately calculate the percentages of daidzein,genistein,formononetin,and biochanin Ain the portion of Red Clover taken by the formula: in which Fis the conversion factor for each analyte (0.97for daidzein,1.13for genistein,1.00for formononetin,and 1.05for biochanin A);Cis the concentration,in mg per mL,of USP Formononetin RSin Standard solution 2;Wis the weight,in g,of Red Clover taken to prepare the Test solution;rUis the peak response for each relevant isoflavone obtained from the Test solution;and rSis the peak response for formononetin obtained from Standard solution 2.