Test solution— Transfer a volume of Injection, equivalent to about 40 mg of yohimbine, to a separator, add 5 mL of a sodium carbonate solution (1 in 20), and extract with four 10-mL portions of chloroform, combining the chloroform extracts in a beaker and evaporating to dryness. Add 20 mL of methanol to the beaker, and swirl to dissolve the residue.

Standard solution— Prepare a solution of USP Yohimbine Hydrochloride RS in methanol containing 2 mg per mL.

Mixed solution: a mixture of the Test solution and the Standard solution (1:1).

Application volume: 1 µL.

Developing solvent system: methylene chloride, methanol, and ammonium hydroxide (90:14:1), in a saturated chamber.

Procedure— Allow the plate to air-dry in a hood. Expose the dry plate for 30 minutes to short-wavelength UV light, then examine under long-wavelength UV light: the size, intensity, and RF value of the principal spots in the chromatograms obtained from the Test solution and the Mixed solution correspond to those characteristics of the principal spot in the chromatogram obtained from the Standard solution.

Diluent— Prepare a mixture of acetonitrile, water, and glacial acetic acid (49:49:2).

Mobile phase— Prepare a mixture of water, acetonitrile, and glacial acetic acid (603:377:20) containing 3.5 g of sodium 1-decanesulfonate in each 1000 mL. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 55 mg of USP Yohimbine Hydrochloride RS, accurately weighed, to a 25-mL volumetric flask, add 20 mL of water, warm, and swirl to dissolve. Add 84 mg of anhydrous citric acid, and swirl to dissolve. Allow the solution to cool, adjust with 1 N sodium hydroxide to a pH of 4.0, dilute with water to volume, and mix. Transfer 125.0 µL of this stock solution to a second 25-mL volumetric flask, dilute with Diluent to volume, and mix. This solution contains about 0.011 mg of USP Yohimbine Hydrochloride RS per mL.

Resolution solution— Prepare a solution in methanol containing about 0.56 mg of methylparaben and 0.06 mg of propylparaben per mL. Transfer 200 µL of this solution to a 25-mL volumetric flask, add 125.0 µL of the stock solution used to prepare the Standard preparation, dilute with Diluent to volume, and mix.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 0.25 mg of yohimbine, to a 25-mL volumetric flask, dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.4 for methylparaben, 0.7 for propylparaben, and 1.0 for yohimbine; and the resolution, R, between methylparaben and propylparaben and between propylparaben and yohimbine is not less than 2.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of yohimbine (C21H26N2O3) in each mL of the Injection taken by the formula: in which 354.45 and 390.90 are the molecular weights of yohimbine and yohimbine hydrochloride, respectively; C is the concentration, in mg per mL, of USP Yohimbine Hydrochloride RS in the Standard preparation; V is the volume, in µL, of Injection taken to prepare the Assay preparation; and rU and rS are the yohimbine peak responses obtained from the Assay preparation and the Standard preparation, respectively.