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

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

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

Application volume: 1µL.

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 30minutes to short-wavelength UVlight,then examine under long-wavelength UVlight:the size,intensity,and RFvalue of the principal spots in the chromatograms obtained from the Test solutionand the Mixed solutioncorrespond 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 3.5g of sodium 1-decanesulfonate in each 1000mL.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 55mg of USP Yohimbine Hydrochloride RS,accurately weighed,to a 25-mLvolumetric flask,add 40mLof water,warm,and swirl to dissolve.Add 84mg of anhydrous citric acid,and swirl to dissolve.Allow the solution to cool,adjust with 1Nsodium hydroxide to a pHof 4.0,dilute with water to volume,and mix.Transfer 125.0µLof this stock solution to a second 25-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 0.011mg of USP Yohimbine Hydrochloride RSper mL.

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

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

Chromatographic system (see Chromatography á621ñ)— 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 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.4for methylparaben,0.7for propylparaben,and 1.0for 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 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of yohimbine (C21H26N2O3)in each mLof the Injection taken by the formula: in which 354.45and 390.91are the molecular weights of yohimbine and yohimbine hydrochloride,respectively;Cis the concentration,in mg per mL,of USP Yohimbine Hydrochloride RSin the Standard preparation;Vis the volume,in mL,of Injection taken to prepare the Assay preparation;and rUand rSare the yohimbine peak responses obtained from the Assay preparationand the Standard preparation,respectively.