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 15 mm in diameter. The roots are also pale to yellowish brown, cylindrical or slightly tapering, sometimes spirally twisted, longitudinally wrinkled and deeply furrowed, up to 4 to 10 mm 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, 25 to 75 µm 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 300 µm long and 20 to 40 µm wide, with intercellular spaces forming schizogenous oleoresin canals that are 80 to 150 µm 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, 300 to 800 µm 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 100 to 300 µm long, with phytomelanin often absent).

A: Thin-Layer Chromatographic Identification Test 201—

B: Thin-Layer Chromatographic Identification Test 201—

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

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

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

Solution B— Use filtered and degassed acetonitrile.

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— Dissolve an accurately weighed quantity of USP Powdered Echinacea angustifolia Extract RS in Solvent, shaking and heating in a water bath. Dilute with Solvent to obtain a solution having a known concentration of about 1 mg 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 RS in Solvent, shaking for 1 minute. Dilute quantitatively, and stepwise if necessary, with Solvent to obtain a solution having a known concentration of about 40 µg per mL. Pass through a membrane filter having a 0.45-µm or finer porosity.

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

Chromatographic system (see Chromatography 621)— 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 1.5 mL per 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 RS and the resolution, R, between the 1,3-dicaffeoylquinic acid isomer and echinacoside is not less than 1.0. Chromatograph Standard solution 2, and record the peak responses as directed for Procedure: the capacity factor, k¢, is not less than 3.0; 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 5 µL) of Standard solution 1, Standard solution 2, and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the relevant peaks. Identify the relevant analytes in the chromatogram obtained from the Test 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 angustifolia taken by the formula: in which F is the response factor and is equal to 0.695 for chicoric acid, 0.729 for dicaffeoylquinic acids, 0.881 for caftaric acid, 1.000 for chlorogenic acid, and 2.220 for echinacoside; C is the concentration, in mg per mL, of USP Chlorogenic Acid RS in Standard solution 2; W is the weight, in mg, of Echinacea angustifolia taken; and ri and rS are the peak responses for the relevant analyte obtained from the Test solution and Standard solution 2, respectively. Calculate the percentage of total phenols in the portion of Echinacea 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 angustifolia Extract RS in methanol, shaking for 10 minutes, and dilute with methanol to obtain a solution having a concentration of about 5 mg 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 2E,4E-Hexadienoic Acid Isobutylamide RS in methanol, shaking for 1 minute. Dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 10 µg per mL. Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 2.5 g of finely powdered Echinacea angustifolia (capable of passing through a 40-mesh sieve), accurately weighed, into a round-bottom flask. Add 80 mL of methanol, and reflux for 30 minutes. Cool to room temperature, and filter into a 100-mL volumetric 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 621)— 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 1.5 mL per 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 angustifolia Extract 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 25 µL) of Standard solution 1, Standard solution 2, and the Test solution into 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 solution by comparison with the chromatogram obtained from Standard solution 1. Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Echinacea angustifolia taken by the formula: in which 1.353 is the response factor for 2E,4E-hexadienoic acid isobutylamide; C is the concentration, in mg per mL, of USP 2E,4E-Hexadienoic Acid Isobutylamide RS in Standard solution 2; W is the weight, in mg, of the portion of Echinacea angustifolia taken; ri is the sum of the peak responses of the relevant analytes obtained from the Test solution; and rS is the peak response obtained from Standard solution 2.