Macroscopic— The Black Cohosh rhizome is dark brown, longitudinally grooved, rough, strongly knotty, and somewhat curled and irregular. It is 15 cm long and up to 2.5 cm thick. The upper surface is covered with numerous round scars of the earlier stalks; laterally, it is clearly curled, and the lower surface is covered with thin, longitudinally grooved, dark brown, easily breakable roots. The fracture is horny and fibrous. The transverse surface shows a thin outer bark surrounding a ring of numerous pale, narrow wedges of vascular tissue alternating with dark medullary rays and a large central pith. Black Cohosh roots are dark brown, between 1 and 3 mm in diameter, brittle, nearly cylindrical or obtusely quadrangular, and longitudinally wrinkled. The fracture is short. The transverse surface shows a distinct cambium line separating a wide outer bark from a central region composed of three to six wedges of lignified xylem tissue united by their apices and separated by broad nonlignified medullary rays.

Microscopic— In a surface view, suberous epidermal cells are tabular with moderately thickened walls. The parenchymatous cortex is filled with starch. Xylem wedges are lignified and composed of numerous small vessels with bordered pits or reticulately thickened walls, thin-walled fibers, and xylem parenchyma. The parenchyma of the pith is unlignified. Medullary rays are filled with starch granules, which are spherical or polygonal and are mostly simple or two to three compounded but can be up to six compounded. Individual starch granules are between 3 and 15 µm in diameter, each with a somewhat central slit-shaped hilum.

A: Thin-Layer Chromatographic Identification Test 201—

B: Thin-Layer Chromatographic Identification Test 201—

C: The chromatogram of the Test solution exhibits peaks for cimiracemoside A, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, cimigenol–arabinoside, and cimigenol–xyloside at retention times corresponding to those compounds in the chromatogram of the Standard solution, as obtained in the test for Content of triterpene glycosides. The ratio of the peak areas of cimigenol–arabinoside to cimigenol–xyloside is not less than 0.4 (distinction from Cimicifuga foetida).

Solution A— Use filtered and degassed 0.05% trifluoroacetic acid in water.

Solution B— Use filtered and degassed acetonitrile.

Mobile phase— Use variable quantities of Solution A, Solution B, and water as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Dissolve amounts of USP Actein RS and USP 23-epi-26-Deoxyactein RS in methanol to obtain a solution having concentrations of about 0.1 mg per mL of each.

Standard solution— Dissolve an accurately weighed quantity of USP Powdered Black Cohosh Extract RS in methanol with shaking for 1 minute, and dilute with methanol to obtain a solution having a known concentration of about 30 mg per mL. Pass through a membrane filter having a 0.45-µm or finer porosity.

23-epi-26-Deoxyactein standard solutions— Dissolve an accurately weighed quantity of USP 23-epi-26-Deoxyactein RS in methanol with shaking for 1 minute. Dilute quantitatively, and stepwise if necessary, to obtain solutions having known concentrations of about 500, 100, 50, 25, and 12.5 µg per mL. Pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Accurately weigh approximately 750 mg of ground plant material, and place into a 20-mL polytef-capped centrifuge tube. Pipet 15 mL of methanol, mix, sonicate for 30 minutes, centrifuge, and transfer the supernatant to an evaporation flask. Repeat the extraction twice. Evaporate the combined extracts under vacuum at 45 to 50. Dissolve the residue in methanol, and quantitatively transfer to a 10-mL volumetric flask. Dilute with methanol to volume, and pass through a membrane filter having a 0.45-µm or finer porosity.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with an evaporative light-scattering detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.6 mL per minute. The column temperature is maintained at 35. The detector is set up according to the manufacturer's instruction in order to achieve a signal-to-noise ratio of not less than 10 for the 12.5 µg per mL 23-epi-26-Deoxyactein standard solution. The chromatograph is programmed as follows. Chromatograph the Standard solution, the 100 µg per mL 23-epi-26-Deoxyactein standard solution, and the System suitability solution, and record the peak responses as directed for Procedure: the chromatogram of the Standard solution is similar to the Reference Chromatogram provided with the lot of USP Powdered Black Cohosh Extract RS; the resolution, R, between the (26S)-actein and the 23-epi-26-deoxyactein peaks in the chromatogram of the System suitability solution is not less than 1.0; the tailing factor for the 23-epi-26-deoxyactein peak in the chromatogram of the 100 µg per mL 23-epi-26-Deoxyactein standard solution is not more than 2.0; and the relative standard deviation of the logarithm of the area responses for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the System suitability solution, the 23-epi-26-Deoxyactein standard solutions, the Standard solution, and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the analyte peaks. Using the chromatogram of the Standard solution and the Reference Chromatogram provided with the lot of USP Powdered Black Cohosh Extract RS, identify the retention times of the peaks corresponding to the triterpene glycosides. The approximate relative retention times of the triterpene glycosides are provided in the following table. Plot the logarithms of the peak area responses versus the logarithms of the concentrations, in µg per mL, of the 23-epi-26-Deoxyactein standard solutions, and determine the regression line using a least-squares analysis. The correlation coefficient for the regression line is not less than 0.995. From the graphs so obtained, determine the concentration, C, in µg per mL, of the relevant analyte in the Test solution. Separately calculate the percentages of cimicifugoside H-1, cimiracemoside A, (26R)-actein, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxyactein, acetyl-shengmanol–xyloside, cimigenol–arabinoside, cimigenol–xyloside (cimicifugoside), 26-deoxyactein, 25-acetyl-cimigenol–arabinoside, (24S)-25-acetyl-cimigenol–xyloside, 25-O-methyl-cimigenol–arabinoside, and 25-O-methyl-cimigenol–xyloside as 23-epi-26-deoxyactein (C37H56O10) in the portion of Black Cohosh taken by the formula: in which C is the concentration, in µg per mL, of the relevant analyte in the Test solution, as obtained above; and W is the weight, in mg, of Black Cohosh taken to prepare the Test solution. Calculate the content of triterpene glycosides, in percentage, in the portion of Black Cohosh taken by adding all of the percentages calculated for the individual analytes.