Macroscopic— Flower head is hemispherical,about 6mm in diameter,composed of a few ray florets and numerous disk florets (distinction from Matricaria discoidea,which has disk florets only),carried on a receptacle surrounded by an involucre.Involucre is green,formed of two to three rows of lanceolate,glabrous,and imbricated bracts with blunt apices and scarious whitish edges.Ray florets,which usually have fallen off,have 10to 20pistils;corolla is ligulate,white,but darkens at a length of 6mm and a width of 2mm,3-toothed,and traversed by four main veins.Disk florets are yellow,perfect,about 2mm in length;corolla is tubular with five teeth;five stamens are epipetalous and syngenesious.Receptacle is hollow (distinction from Chrysanthemumand Anthemisspecies),hemispherical in the young and conical in the old flower head,3to 10mm in width,and lacking paleae.Achene is ovoid and has three to five longitudinal ribs.

Microscopic— Separate the capitulum into its parts and examine under a microscope.The outer,abaxial epidermis of the involucral bracts shows a scarious margin with a single layer of radially elongated cells and a central part made up of chlorophyll tissue covered by elongated epidermal cells with sinuous lateral walls,stomata,and secretory trichomes.The vascular bundles are surrounded by numerous elongated,pitted sclereids with fairly large lumens.In surface view,ligulate and tubular corollas show isodiametric or elongated cells with more or less wavy walls and a few glandular trichomes.The outer part of the epidermis of the ligulate florets consists of papillary cells with cuticular striations radiating from their tips.In the mesophyll,very small clusters of calcium oxalate are sometimes seen.Four main veins run lengthwise through the entire mesophyll,sometimes accompanied by one or two other veins,which are shorter and run parallel to the main veins.Each of the two main median veins splits into two near the tip and,with the lateral veins,anastomose two by two to form three arcs at the three terminal teeth of the ligule.The ovaries,oval to spherical,of both kinds of florets have at their base a sclerous ring consisting of a single row of cells.The epidermis of the ovary is made up of elongated cells with sinuous walls between which secretory trichomes are situated.The ovaries contain numerous very small clusters of calcium oxalate.In the tubular florets,the low part of each stamen filament is surrounded by thick-walled cells.The ends of the two stigmata have papillose epidermal cells.The pollen grains have a diameter of about 30µm and are rounded and triangular,with three germinal pores and a spiny exine.

A: Thin-Layer Chromatographic Identification Test á201ñ—

B: Dissolve 0.25g of dimethylaminobenzaldehyde in a mixture of 5mLof phosphoric acid,45mLof acetic acid,and 45mLof water.Transfer 2.5mLof this solution and 0.1mLof the test solution,prepared as directed for Identificationtest A,to a test tube.Heat on a water bath for 2minutes,and allow to cool.Add 5mLof solvent hexane,and shake.The aqueous layer has a distinct greenish blue or blue color.

NOTE—Retain the volatile oils for use in the test for Content of bisabolan derivatives.

Dilute phosphoric acid— Transfer 5.0mLof phosphoric acid to a 100-mLvolumetric flask containing about 50mLof water,dilute with water to volume,and mix.

Solution A— Prepare a 0.005Msolution of monobasic potassium phosphate.Adjust with Dilute phosphoric acidto a pHof 2.55±0.05.

Solution B— Prepare a mixture of acetonitrile and methanol (65:35).

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system(see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve accurately weighed quantities of USP Apigenin-7-glucoside RSand 7-methoxycoumarin in methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having known concentrations of about 25.0µg of USP Apigenin-7-glucoside RSand 10.0µg of 7-methoxycoumarin per mL.

Test solution— Transfer about 1.0g of Chamomile,accurately weighed,to a suitable flask fitted with a reflux condenser and a stirrer,add 80.0mLof methanol,and heat and reflux the mixture with stirring for 1hour.Cool the flask to room temperature,pass the methanolic distillate through a folded filter paper,and collect the filtrate in a 100-mLvolumetric flask.Rinse the flask with 3mLof methanol,pour the methanolic rinsings through the filter paper,and add the filtrate to the volumetric flask.Dilute with methanol to volume,mix,and filter.Transfer 25.0mLof the filtered solution to a round-bottom flask fitted with a reflux condenser and a stirrer,add 5.0mLof sodium hydroxide solution,prepared by dissolving 0.4g of sodium hydroxide in 5.0mLof water,and heat and reflux the mixture for 25minutes.Cool the flask,and adjust the solution with hydrochloric acid to a pHof 5.0to 6.2.Quantitatively transfer the solution to a 50-mLvolumetric flask,dilute with methanol to volume,mix,and filter,discarding the first 10mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 335-nm detector and a 4-mm ×12.5-cm column that contains packing L1.The flow rate is about 1mLper minute.The chromatograph is programmed as follows: Make adjustments,if necessary,to obtain relative retention times of about 0.63and 1.0for apigenin-7-glucoside and 7-methoxycoumarin,respectively.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.30,0.47,0.66,0.89,and 1.0for apigenin-7-glucoside,7-methoxycoumarin,apigenin,trans-spiroether,and cis-spiroether,respectively;the resolution,R,between apigenin-7-glucoside and 7-methoxycoumarin is not less than 3.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 15µL)of the Standard solutionand the Test solutioninto the chromatograph,and allow the Test solutionto elute for not less than six times the retention time of apigenin-7-glucoside.Record the chromatograms,and measure the responses for all the peaks observed in the chromatogram of the Test solution.Calculate the percentage of apigenin-7-glucoside in the portion of Chamomile taken by the formula: in which Cis the concentration,in mg per mL,of USP Apigenin-7-glucoside RSin the Standard solution;Wis the weight,in g,of Chamomile taken for the Test solution;and rUand rSare the apigenin-7-glucoside peak responses obtained from the Test solutionand the Standard solution,respectively:not less than 0.3%is found.

Standard solution— Prepare a solution of USP Levomenol RSin cyclohexane having a known concentration of about 1mg per mL.

Test solution— Transfer the volatile oils obtained in the test for Volatile oil contentto a 25-mLvolumetric flask,rinse the graduated tube of the apparatus with a small portion of cyclohexane,transfer the rinsing to the 25-mLvolumetric flask,add cyclohexane to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 0.32-mm ×30-m fused-silica capillary column coated with a 0.25-µm film of phase G16.The carrier gas is helium,flowing at a rate of about 1.0mLper minute.The chromatograph is programmed as follows.Initially the column temperature is maintained at 70,then it is increased at a rate of 4per minute to 230,and maintained at 230for 10minutes.The detector is maintained at a temperature of 250,and the injection port is maintained at 220.Chromatograph the Standard solution,and record the peak areas as directed for Procedure:the tailing factor for levomenol is not more than 1.8;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas.Identify levomenol,bisabol oxide B,bisabol oxide,and bisabol oxide Ain the Test solutionusing the retention time of levomenol in the Standard solutionand the chromatogram that is provided with USP Levomenol RS.Calculate the percentage of levomenol in the portion of Chamomile taken by the formula: in which rUis the sum of the peak areas for bisabol oxide B,bisabol oxide,levomenol,and bisabol oxide Aobtained from the Test solution;rSis the levomenol peak area obtained from the Standard solution;CSis the concentration,in mg per mL,of USP Levomenol RSin the Standard solution;and CUis the concentration,in mg per mL,of chamomile in the Test solution:not less than 0.15%of bisabolan derivatives is found.