Macroscopic— The rhizome is knotty and of irregular cylindrical form with a diameter of 15to 40mm.The heartwood area is light brown,and the connecting splint wood is pale yellow.The bark is approximately 2mm thick and is firmly affixed to the xylem.The surface is gray-brown or black-brown,coarse,and longitudinally valleculate and plicate.Abroken rhizome is coarse and fibrous,particularly inside of the xylem.The fractured surface of the bark shows short thin fibers.Numerous roots spring from the underside of the rhizome.These roots are 35to 150mm long,cylindrical,and knotty,with a diameter of 3to 15mm.The surface of the roots is gray-brown to black-brown,is smoother than the rhizome,and has longitudinal stripes.A0.5-mm thin bark is tightly affixed to the pale yellow xylem.Abroken root is sparsely fibrous and appears yellowish gray where the thin epidermis is flaked off.

Histology— The roots have five to seven rows of brown cork cells.Secretory canals with brown contents appear in groups of four or five and are not greater than 20µm in diameter.Phloem fibers with thick lignified walls occur singly or in small groups;crystals of calcium oxalate cluster in the phloem parenchyma.Parenchymatous cells surround the secretory cells,and medullary ray cells contain small starch granules.The xylem shows reticulately thickened and pitted vessels.The rhizome is similar to the roots except for its larger secretory canals,up to 25µm in diameter,and the presence of a pith with parenchymatous cells containing starch granules.

Test solution— Comminute about 10g of Eleuthero,add about 50mLof alcohol 30%(v/v),and heat under reflux in a water bath for 30minutes.Cool to room temperature,filter,gently evaporate the solvent,and suspend the residue in 5mLof methanol.

Standard solution— Prepare a solution of eleutheroside Bin methanol containing about 1mg per mL.

Developing solvent system: a mixture of chloroform,methanol,and water (70:30:4).[NOTE—Saturate the chamber with Developing solvent systembefore the development of the chromatogram.]

Spray reagent— Prepare a solution of antimony trichloride in chloroform having a concentration of about 200mg per mL.

Procedure— Develop the chromatogram until the solvent front has moved to 15cm,dry,and spray the plate with Spray reagent.Heat the plate at 120for 10minutes,and examine it under UVlight at 365nm and in daylight.The chromatogram of the Test solutionshows a brownish to red zone due to eleutheroside B,corresponding in color and RFvalue to the zone exhibited by the chromatogram of the Standard solution.Ablue zone appears directly above,and a yellow zone appears directly below the red zone.In daylight,a violet band is visible in the lower half sector.Some brownish to yellowish bands occur in the upper sector.

Solvent— Use a mixture of methanol and water (1:1).

Solution A— Prepare a filtered and degassed mixture of water and acetonitrile (95:5).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and water (60:40).

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— Transfer an accurately weighed quantity of USP Powdered Eleuthero Extract RSto a suitable volumetric flask,and add Solventto obtain a solution having a known concentration of about 5.0mg of Powdered Extract per mL.Sonicate for 30minutes,cool to room temperature,decant,and pass through a nylon filter having a 0.45-µm or finer porosity.

Test solution— Transfer about 5.0g of finely ground Eleuthero,accurately weighed,to a round-bottom flask equipped with a condenser,add 50.0mLof Solvent,and heat under reflux for 30minutes.Decant the suspension,and filter the supernatant through cotton wool into a 100-mLvolumetric flask.Transfer the cotton wool to the round bottom-flask,and repeat the extraction twice,using 22mLof Solventfor each extraction.Filter through cotton wool into the volumetric flask,wash the residue and the cotton wool with Solvent,cool to room temperature,and dilute with Solventto volume.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4.0-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 1.0mLper minute.The chromatograph is programmed as follows. Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram provided with USP Powdered Eleuthero Extract RS;and the relative standard deviation for replicate injections determined from the eleutheroside Bpeak is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatogram,identify the eleutheroside Band eleutheroside Epeaks in the chromatogram of the Test solutionby comparison with the Reference Chromatogram,and measure the peak responses.Separately calculate the percentages of eleutheroside Band eleutheroside Ein the portion of Eleuthero taken by the formula: in which Pis the percentage of eleutheroside Bor eleutheroside Ein USP Powdered Eleuthero Extract RS,respectively;Cis the concentration,in mg per mL,of USP Powdered Eleuthero Extract RSin the Standard solution;Wis the weight,in mg,of Eleuthero taken to prepare the Test solution;rUis the peak response of the relevant analyte obtained from the Test solution;and rSis the eleutheroside Bor eleutheroside Epeak response obtained from the Standard solution.Calculate the percentage of eleutheroside Band eleutheroside Ein the portion of Eleuthero taken by adding the individual percentages.