Macroscopic— The rhizome is knotty, subcylindrical, and occasionally has an aerial stem. It is 1 to 5 cm in length and 2 to 10 mm in diameter. Externally, the rhizome is brown to dusky yellowish orange, deeply furrowed, and marked by numerous stem and bud scale scars. Numerous brittle roots arise roughly from the same side of the main axis. Fractures are short and resinous with a dark yellow to yellowish-brown bark, greenish-yellow margins, and a yellowish-orange center that is waxy in appearance. An interrupted circle of small, radially elongated fibrovascular bundles are also present. The roots are filiform, up to 35 cm in length and 1 mm in diameter, and are either curved or twisted, tangled together, or broken. Fractures are short and brittle, and show an internal color of yellowish orange to greenish yellow.

Histology—

Test solution— Finely powder the rhizome and the root, transfer 0.5 g of the powder to a suitable glass vial, add 0.5 mL of 10% sodium carbonate, and mix. Add 5 mL of methanol, and heat for 10 minutes in a water bath at 60. Cool to room temperature, filter, and dry under a stream of nitrogen. Add 0.5 mL of methanol to dissolve the residue.

Standard solution— Dissolve accurately weighed quantities of USP Berberine Chloride RS and USP Hydrastine Hydrochloride RS in methanol to obtain a solution having a concentration of 0.5 mg of each USP Reference Standard per mL.

Application volume: 10 µL to 20 µL, as bands.

Developing solvent system: a mixture of ethyl acetate, butyl alcohol, formic acid, and water (50:30:10:10).

Procedure— Proceed as directed in the chapter, except to air-dry the plates, and examine them under UV light at about 365 nm. The chromatograms show zones having a lemon-yellow fluorescence due to berberine at an RF value of about 0.53 and a blue-white fluorescence due to hydrastine at an RF value of about 0.42.

Mobile phase— Dissolve 9.93 g of monobasic potassium phosphate in 730 mL of distilled water. Add 270 mL of acetonitrile, mix, filter, and degas. Make other adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve accurately weighed quantities of USP Berberine Chloride RS and USP Hydrastine Hydrochloride RS in a mixture of water and methanol (1:1), and dilute quantitatively, and stepwise if necessary, to obtain a solution containing about 0.05 mg of each USP Reference Standard per mL. [NOTE—Concentrations of berberine and hydrastine in this solution are calculated by multiplying the concentration of each of the USP Reference Standards by correction factors of 0.905 and 0.913, respectively.]

System suitability solution— Prepare a solution of palmatine in a mixture of water and methanol (1:1) having a known concentration of about 0.05 mg per mL. Mix equal volumes of this solution and the Standard solution.

Test solution— Finely powder a quantity of Goldenseal, and transfer 0.12 g, accurately weighed, to a 50-mL volumetric flask. Add 40 mL of a mixture of 0.1 M monobasic potassium phosphate and acetonitrile (60:40). Sonicate for 5 minutes, and shake for 10 minutes on a rotation shaker. Dilute with the mixture of 0.1 M monobasic potassium phosphate and acetonitrile (60:40), mix, and filter.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 235-nm detector and a 4.6-mm × 150-mm column that contains packing L1. The flow rate is about 1.8 mL per minute. Inject the Standard solution into the chromatograph, and record the peak responses as directed for Procedure: the capacity factor, k¢, determined from the hydrastine and berberine peaks is not less than 3.0; the column efficiency is not less than 5000 theoretical plates; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.5%. Inject the System suitability solution into the chromatograph, and record the peak responses as directed for Procedure: identify the locus for palmatine, and calculate the resolution, R, with respect to hydrastine and berberine: the resolution, R, between berberine and palmatine is not less than 1.5, and the resolution, R, between hydrastine and palmatine is not less than 1.5.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the percentages of berberine and hydrastine in the portion of Goldenseal taken by the formula: in which C is the concentration, in mg per mL, of berberine or hydrastine in the respective USP Reference Standard in the Standard solution, using the correction factors as noted above; V is the final volume, in mL, of the Test solution; W is the weight, in mg, of Goldenseal taken; and rU and rS are the peak areas for berberine and hydrastine obtained from the Test solution and the Standard solution, respectively. Using the values obtained from the chromatogram of the Test solution, divide the peak area of berberine by the peak area of any peak at the locus for palmatine (if present): the ratio is more than 50:1.