A: Infrared Absorption á197Kñ.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

NOTE—Prepare all solutions in the tests for Related compoundsand Chromatographic purityimmediately prior to use,and apply to plates as quickly as possible.

Spray reagent— [Caution—Avoid contact with o-tolidine.Prepare and use this Spray reagent in a well-ventilated hood.]Dissolve 50mg of o-tolidine in 100mLof alcohol,and mix.

Chlorine chamber— Transfer 1.5g of potassium permanganate to a 100-mLbeaker,dissolve in and dilute with water to volume,and mix.Transfer 25mLof this solution to a beaker,and place the beaker inside a chromatographic chamber.Pipet 10mLof hydrochloric acid into the beaker,and cover the chamber.

Developing solvent system— Prepare a fresh mixture of ethyl acetate,glacial acetic acid,and acetonitrile (70:25:3).

Standard solutions— Dissolve accurately weighed quantities of USP Guanfacine Hydrochloride RSand guanidine hydrochloride in methanol to obtain a solution having a known concentration of 0.4mg each of USP Guanfacine Hydrochloride RSand guanidine hydrochloride per mL.Quantitatively dilute this solution with methanol to obtain Standard solutionshaving the following compositions:

Test solution— Dissolve an accurately weighed quantity of Guanfacine Hydrochloride in methanol to obtain a solution having a concentration of about 20mg per mL.

Procedure— Use a thin-layer chromatographic plate (see Chromatography á621ñcoated with a 0.25-mm layer of chromatographic silica gel.Prewash the plates by placing in a chromatographic chamber saturated with Developing solvent system.Remove the plates from the chamber,and allow to dry.Separately apply 10µLeach of the Standard solutionsand the Test solutionto the chromatographic plate.Allow the spots to dry,and develop the chromatogram in Developing solventuntil the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,mark the solvent front,and allow the plate to air-dry for about 1hour.Examine the plate under short-wavelength UVlight.Place the dried plate in the Chlorine chamberfor 15minutes,remove,and allow the excess chlorine to evaporate by air drying for 5minutes.Spray the plate with Spray reagent,and examine:any spot due to guanidine hydrochloride observed in the chromatogram of the Test solutionis not greater in size or intensity than the guanidine hydrochloride spot obtained from Standard solution 3(0.5%);no other individual impurity spot observed in the chromatogram of the Test solutionis greater in size or intensity than the guanfacine hydrochloride spot obtained from Standard solution 4(0.25%);and the sum of all impurities found,including guanidine hydrochloride,is not more than 1.0%.

Spray reagent 1— Prepare a mixture of tertiary butyl alcohol and water (9:1).

Spray reagent 2— Dissolve 5g of 4,4¢-tetramethyldiaminodiphenylmethane in 20mLof glacial acetic acid,add 10mLof water,and mix (Solution 1).Dissolve 6g of potassium iodide in 120mLof water,and mix (Solution 2).Dissolve 0.3g of ninhydrin in 10mLof glacial acetic acid,dilute with water to 100mL,and mix (Solution 3).Mix Solution 1and Solution 2,and add 9mLof Solution 3.

Developing solvent system— Prepare a fresh mixture of hexanes,diisopropyl ether,toluene,and glacial acetic acid (60:30:5:3).

Reference solutions— Dissolve an accurately weighed quantity of 2,6-dichlorophenylacetic acid in a mixture of methanol and water (9:1)to obtain a solution having a concentration of 1mg per mL(Reference solution 1).Quantitatively dilute this solution with a mixture of methanol and water (9:1)to obtain Reference solution 2and Reference solution 3having known concentrations of 0.5and 0.25mg per mLof 2,6-dichlorophenylacetic acid,respectively.

Test solution— Prepare a solution of Guanfacine Hydrochloride in a mixture of methanol and water (9:1),containing 100mg per mL.

Procedure— Use a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Prewash the plates by placing in a chromatographic chamber saturated with Developing solvent system.Remove the plates from the chamber,and allow to dry.Separately apply 25µLof each of the Reference solutionsand the Test solutionto the chromatographic plate.Allow the spots to dry,and develop the chromatograms in the Developing solvent systemuntil the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,mark the solvent front,and allow the plate to air-dry for 30minutes.Examine the plate under short-wavelength UVlight.Spray the plate with Spray reagent 1,wait for 1minute,and then spray with Spray reagent 2.Place the wet plate under short-wavelength UVlight for 10minutes,remove,and observe under white light:no spot observed in the chromatogram of the Test solution,other than that due to guanfacine hydrochloride,is greater in size or intensity than the principal spot obtained from Reference solution 2(0.5%);and the sum of all impurities found is not more than 1.0%.

Dilute phosphoric acid— Prepare a mixture of water and phosphoric acid (4:1).

Buffer solution— Dissolve 68g of monobasic potassium phosphate in water,dilute with water to 1000mL,and mix.Dilute 100mLof this solution with water to 1000mL,add 5mLof triethylamine,mix,and adjust with Dilute phosphoric acidto a pHof 3.0.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand acetonitrile (79:21).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Guanfacine Hydrochloride RSin a mixture of acetonitrile and water (3:1)to obtain a solution having a known concentration of about 1mg of USP Guanfacine Hydrochloride RSper mL.Transfer 2.0mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer an accurately weighed quantity of about 50mg of Guanfacine Hydrochloride to a 50-mLvolumetric flask,dissolve in and dilute with a mixture of acetonitrile and water (3:1)to volume,and mix.Transfer 2.0mLof this solution to a 50-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 L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the responses as directed for Procedure:the capacity factor,k¢,is between 2and 5;the column efficiency is not less than 1500theoretical plates;the tailing factor is not more than 2;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)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 C9H9Cl2N3O·HCl in the portion taken by the formula: in which Cis the concentration,in µg per mL,of USP Guanfacine Hydrochloride RSin the Standard preparation;and rUand rSare the guanfacine hydrochloride peaks obtained from the Assay preparationand the Standard preparation,respectively.