Macroscopic— The outer surface of the rhizome is pale to yellowish brown,crowned with remains of the aerial stem,and sometimes showing surface annulations up to 15mm in diameter.The roots are also pale to yellowish brown,cylindrical or slightly tapering,sometimes spirally twisted,longitudinally wrinkled and deeply furrowed,up to 4to 10mm in diameter,and passing imperceptibly into rhizome.The short fracture,when dry,becomes tough and pliable on exposure to air.

Microscopic— The rhizomes and roots in transverse section show a thin outer bark separated from a wide xylem by a distinct cambial line.The cork is composed of several rows of thin-walled cells containing yellowish brown pigment.The rhizome has a small circular pith,occasional small groups of thick-walled,lignified fibers in the pericycle,and a parenchymatous cortex.The phloem and xylem are composed of narrow strands of vascular tissue separated by wide,non-lignified medullary rays.Xylem vessels are lignified,25to 75µm in diameter,usually with reticulate thickening but occasionally with spiral or annular thickening.Sclereids occur singly or in small groups,varying considerably in size and shape from rounded to rectangular to elongated and fiber-like,up to 300µm long and 20to 40µm wide,with intercellular spaces forming schizogenous oleoresin canals that are 80to 150µm in diameter and contain a dense black deposit.The canals are present outside of the central cylinder only (unlike Echinacea pallida,where they are present both inside and outside of the central cylinder).Spherocrystalline masses of inulin occur throughout the parenchymatous tissues.Lignified fibers,300to 800µm long,are present in scattered groups,and are usually surrounded by phytomelanin (unlike fibers in Echinacea pallida,where they usually occur singly in the periphery of the cortex and are 100to 300µm long,with phytomelanin often absent).

Change to read:

A:Thin-Layer Chromatographic Identification Test á201ñ—

B:Thin-Layer Chromatographic Identification Test á201ñ—

C: The retention time of the major peak in the chromatogram of theTest solution corresponds to that of the echinacoside peak in the chromatogram ofStandard solution 1,as obtained in the test forContent of total phenols.The chromatogram of the Test solutionshows a peak for 1,3-dicaffeoylquinic acid corresponding in retention time to that obtained with Standard solution 1.USP28

Change to read:

Change to read:

Solvent— Prepare a mixture of alcohol and water (7:3).

Solution A— Prepare a filtered and degassed solution of phosphoric acid (0.1in 100).

Solution B— Use filtered and degassed acetonitrile.

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— Dissolve an accurately weighed quantity of USP PowderedEchinacea angustifolia Extract RSinSolvent,shaking and heating in a water bath.DiluteUSP28with Solvent to obtain a solution having a known concentration of about 1mg of per mL.Pass through a membrane filter having a 0.45-µm or finer porosity.

Standard solution 2— Dissolve an accurately weighed quantity of USP Chlorogenic Acid RSinSolvent,shaking for 1minute.Dilute quantitatively,and stepwise if necessary,with Solvent to obtain a solution having a known concentration of about 40µg per mL.Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 125mg of finely powdered Echinacea angustifolia (capable of passing through a 40-mesh sieve),accurately weighed,to a round bottom flask equipped with a condenser.USP28Add 25.0mLofSolvent,and heat under reflux,while shaking by mechanical means,for 15minutes.Centrifuge,or pass through a membrane filter having a 0.45-µm or finer porosity.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 330-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The column temperature is maintained at 35.The flow rate is about 1.5mLper minute.The chromatograph is programmed as follows. Chromatograph Standard solution 1,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram for total phenols provided with the USP Powdered Echinacea angustifolia Extract RSand the resolution,R,between the 1,3-dicaffeoylquinic acid isomer and echinacoside is not less than 1.0.USP28ChromatographStandard solution 2,and record the peak responses as directed forProcedure:the capacity factor,k¢,is not less than 3.0;USP28the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes (about 5µL)of Standard solution 1,Standard solution 2,and theTest solution into the chromatograph,record the chromatograms,and measure the areas for the relevant peaks.Identify the relevant analytes in the chromatogram obtained from theTest solution by comparison with the chromatogram obtained from Standard solution 1.Separately calculate the percentage of caftaric acid (C13H12O9),chicoric acid (C22H18O12),chlorogenic acid (C16H18O9),dicaffeoylquinic acids (C25H24O12),and echinacoside (C35H46O20)in the portion of Echinacea angustifoliataken by the formula: in whichFis the response factor and is equal to 0.695for chicoric acid,0.729for dicaffeoylquinic acids,USP280.881for caftaric acid,1.000for chlorogenic acid,and 2.220for echinacoside;Cis the concentration,in mg per mL,of USP Chlorogenic Acid RSin Standard solution 2;Wis the weight,in mg,ofEchinacea angustifolia taken;and riand rSare the peak responses for the relevant analyte obtained from theTest solution andStandard solution 2,respectively.Calculate the percentage of total phenols in the portion ofEchinacea angustifolia taken by adding the individual percentages calculated.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (55:45).

Standard solution 1— Dissolve,with sonication,an accurately weighed quantity of USP Powdered Echinacea angustifoliaExtract RSin methanol,shaking for 10minutes,and dilute with methanol to obtain a solution having a concentration of about 5mg per mL.Pass through a membrane filter having a 0.45-µm or finer porosity.

Standard solution 2— Dissolve an accurately weighed quantity of USP2E,4E-Hexadienoic Acid Isobutylamide RSin methanol,shaking for 1minute.Dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 10µg per mL.Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 2.5g of finely powdered Echinacea angustifolia(capable of passing through a 40-mesh sieve),accurately weighed,into a round-bottom flask.Add 80mLof methanol,and reflux for 30minutes.Cool to room temperature,and filter into a 100-mLvolumetric flask,using small portions of methanol to rinse the flask and the filter.Dilute with methanol to volume,and mix.Pass through a membrane filter having a 0.45-µm or finer porosity.

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 5-µm packing L1.The column temperature is maintained at 30.The flow rate is about 1.5mLper minute.Chromatograph Standard solution 1,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram for alkamides provided with USP Powdered Echinacea angustifoliaExtract RS;and the resolution,R,between dodecatetraenoic acid isobutylamide peaks is not less than 1.0.Chromatograph Standard solution 2,and record the peak responses as directed for Procedure:the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes (about 25µL)of Standard solution 1,Standard solution 2,and the Test solutioninto the chromatograph,record the chromatograms,and measure the areas for the relevant peaks.Identify the peaks due to 2E,4E,8Z,10E-dodecatetraenoic acid isobutylamide and 2E,4E,8Z,10Z-dodecatetraenoic acid isobutylamide in the chromatogram obtained from the Test solutionby comparison with the chromatogram obtained from Standard solution 1.Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Echinacea angustifoliataken by the formula: in which 1.353is the response factor for 2E,4E-hexadienoic acid isobutylamide;Cis the concentration,in mg per mL,of USP2E,4E-Hexadienoic Acid Isobutylamide RSin Standard solution 2;Wis the weight,in mg,of the portion of Echinacea angustifoliataken;riis the sum of the peak responses of the relevant analytes obtained from the Test solution;and rSis the peak response obtained from Standard solution 2.