A: Thin-Layer Chromatographic Identification Test 201—

B: The retention time for the major peak in the chromatogram of the Test solution corresponds to that for the echinacoside peak in the chromatogram of Standard solution 1, as obtained in the test for Content of total phenols.

Solvent, Solution A, Solution B, Mobile phase, Standard solution 1, Standard solution 2, and Chromatographic system— Proceed as directed for Content of total phenols under Echinacea angustifolia.

Test solution— Transfer about 60 mg of Powdered Extract, accurately weighed, to an appropriate 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.

Procedure— Proceed as directed for Content of total phenols under Echinacea angustifolia. Calculate the percentage of each relevant component of total phenols in the portion of Powdered Extract 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 the portion of Powdered Extract 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 Powdered Extract taken by adding the individual percentages.

Mobile phase and Standard solution 2— Proceed as directed for Content of dodecatetraenoic acid isobutylamides under Echinacea angustifolia.

Standard solution 1— Dissolve an accurately weighed quantity of USP Powdered Echinacea angustifolia Extract RS in methanol, shaking for 1 minute, and dilute with methanol to volume to obtain a solution having a known concentration of about 1 mg per mL. Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 500 mg of Powdered Extract, accurately weighed, to a 100-mL volumetric flask. Add 80 mL of methanol, and sonicate for 30 minutes. Dilute with methanol to volume, and pass through a membrane filter having a 0.45-µm or finer porosity.

Chromatographic system— Prepare as directed for Content of dodecatetraenoic acid isobutylamides under Echinacea angustifolia. 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 the USP Powdered Echinacea angustifolia Extract RS, and the resolution, R, between the two isomers of dodecatetraenoic acid isobutylamides 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 3.5; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.5%.

Procedure— Proceed as directed for Content of dodecatetraenoic acid isobutylamides under Echinacea angustifolia. Calculate the percentage of dodecatetraenoic acid isobutylamides in the portion of Powdered Extract 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, in the portion of Powdered Extract 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.