A: The retention time exhibited by clonidine hydrochloride in the chromatogram of the Assay preparationcorresponds to that of clonidine hydrochloride in the chromatogram of the Standard preparationas obtained in the Assay.

B: Transfer a quantity of finely powdered Tablets,equivalent to about 1mg of clonidine hydrochloride,to a separator containing 30mLof water and 5mLof 1Nsodium hydroxide.Swirl gently to dissolve the test specimen,and extract with 20mLof chloroform.Allow the layers to separate,and filter the chloroform extract.Evaporate the filtrate to dryness,and dissolve the residue in 0.1mLof methanol to obtain the test solution.Prepare a Standard solution in methanol containing 10mg of USP Clonidine Hydrochloride RSper mL.Apply separately 2-µLportions of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Position the plate in a chromatographic chamber,and develop the chromatograms in a solvent system consisting of a mixture of methanol and ammonium hydroxide (200:3)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Examine the plate under short-wavelength UVlight:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Medium: 0.01Nhydrochloric acid;500mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Procedure— Using Dissolution Mediuminstead of Mobile phaseto prepare the Standard clonidine hydrochloride stock solutionand the Standard preparation,determine the amount of C9H9Cl2N3·HCl dissolved,employing the procedure set forth in the Assay,making any necessary modifications.

Tolerances— Not less than 75%(Q)of the labeled amount of C9H9Cl2N3·HCl is dissolved in 30minutes.

Mobile phase— Dissolve 1.1g of sodium 1-octanesulfonate in 500mLof water,and add 500mLof methanol and 1mLof phosphoric acid.Mix,and adjust with 1Nsodium hydroxide to a pHof 3.0.Mix,filter through a 0.45-µm or finer porosity filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard clonidine hydrochloride stock solution— Dissolve an accurately weighed quantity of USP Clonidine Hydrochloride RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 100µg per mL.

2 ,6-Dichloroaniline stock solution—Transfer about 12mg of 2,6-dichloroaniline to a 100-mLvolumetric flask,add Mobile phaseto volume,and mix.Dilute an accurately measured volume of this solution quantitatively with Mobile phaseto obtain 2,6-Dichloroaniline stock solutionhaving a known concentration of about 12µg per mL.

Standard preparation— Transfer 2.0mLof Standard clonidine hydrochloride stock solutionto a 200-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.This solution contains about 1µg of clonidine hydrochloride.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 0.1mg of clonidine hydrochloride,to a 100-mLvolumetric flask.Add about 60mLof Mobile phase,shake by mechanical means for 15to 30minutes,dilute with Mobile phaseto volume,and mix.Centrifuge a portion of this solution to obtain a clear solution (Assay preparation).

System suitability preparation— Transfer 2.0mLof Standard clonidine hydrochloride stock solutionand 20.0mLof 2,6-Dichloroaniline stock solutionto a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×15-cm column that contains packing L7,which has been deactivated for basic compounds.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the relative standard deviation for replicate injections is not more than 2.0%for the clonidine peak.Chromatograph the System suitability preparation,and record the peak responses as directed under Procedure:the column efficiency determined from the clonidine peak is not less than 3500theoretical plates and the tailing factor for the clonidine peak is not more than 1.5.The relative retention times are about 0.5for clonidine and 1.0for 2,6-dichloroaniline.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C9H9Cl2N3·HCl in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Clonidine Hydrochloride RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.