A:Infrared Absorption á197Kñ—

B: The crystals obtained in Identificationtest Amelt between 132and 136(see Melting Range or Temperature á741ñ).

C: To 0.05mLof Solution add 5mLof a solution of cetyltrimethylammonium bromide (1in 100),1mLof 10Nsodium hydroxide,and 1mLof bromine TS:a deep red color is produced.

D: Prepare a test solution by diluting 10mLof Solution to 50mLwith water.This solution meets the requirements for Identificationtest Bunder Calcium Gluconate,except that the chromatogram is developed until the solvent front has moved about 10cm from the point of spotting.

Solution A,Solution B,Mobile phase,and Diluent— Proceed as directed in the Assay.

Test solution— Transfer 5.0mLof Solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 25-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 2mg of chlorhexidine gluconate per mL.

Reference solution A— Transfer 3.0mLof the Test solutionto a 100-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 0.06mg of chlorhexidine gluconate per mL.

Reference solution B— Transfer 2.0mLof Reference solution Ato a 100-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 0.0012mg of chlorhexidine gluconate per mL.

Resolution solution— Transfer about 10mg of USP Chlorhexidine Related Compounds RSto a 10-mLvolumetric flask,dissolve in 2mLof acetonitrile,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— Proceed as directed in the Assay,except the chromatograph is programmed as follows.

Procedure— Separately inject equal volumes (about 20µL)of the Test solution,Reference solution A,and Reference solution Binto the chromatograph,record the chromatograms,and measure the areas for the peaks.Examine the chromatogram obtained from the Test solution:the sum of the peak areas,other than chlorhexidine and any peak areas less than that obtained for chlorhexidine in the chromatogram obtained from Reference solution B,is not more than the peak area for chlorhexidine in the chromatogram obtained from Reference solution A(3.0%).

Solution A,Solution B,Mobile phase,Diluent,System suitability solution,and Chromatographic system— Proceed as directed in the Assay.

Standard solutions— Transfer about 10mg of p-chloroaniline,accurately weighed,to a 100-mLvolumetric flask,add 2mLof acetonitrile,swirl to dissolve,dilute with Diluentto volume,and mix.Dilute accurately measured volumes of this solution quantitatively,and stepwise if necessary,with Diluentto obtain Standard solutionshaving known concentrations of about 1.5,1.2,0.6,and 0.3µg of p-chloroaniline per mL.

Test solution— Transfer 5.0mLof Solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 250-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 0.4mg of chlorhexidine gluconate per mL.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionsand the Test solutioninto the chromatograph,record the chromatograms,and measure the areas for the p-chloroaniline peaks.Plot the peak responses obtained from the Standard solutionsversus the relevant concentrations,in µg per mL.Draw the straight line best fitting the four plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of p-chloroaniline in the Test solution.Calculate the quantity,in µg per mL,of p-chloroaniline in the portion of Solution taken by the formula: Not more than 500µg per mLis found.If the quantity so obtained is less than 150µg per mL,the tests may be repeated using a more appropriate dilution in the preparation of the Test solution.

Solution A— Prepare a solution of 27.6g of monobasic sodium phosphate and 10mLof triethylamine in about 1.5liters of water.Adjust with phosphoric acid to a pHof 3.0,dilute with water to 2000mL,and mix.Prepare a mixture of this solution and acetonitrile (70:30).[NOTE—Small adjustments in the acetonitrile content may be made to meet acceptable resolution criteria (see System Suitabilityunder Chromatography á621ñ).]Degas before use and sparge with helium during the analysis.

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.

Diluent— Prepare a solution of 27.6g of monobasic sodium phosphate in about 1.5liters of water.Adjust with phosphoric acid to a pHof 3.0,dilute with water to 2000mL,and mix.

System suitability solution— Prepare a solution in Diluentcontaining about 50µg of USP Chlorhexidine Acetate RSand 1µg of p-chloroaniline per mL.

Standard preparation— Prepare a solution of USP Chlorhexidine Acetate RSin water having a known concentration of about 1mg per mL.Dilute an accurately measured volume of this stock solution quantitatively with Diluentto obtain a Standard preparationhaving a known concentration of about 50µg per mL.

Assay preparation— Transfer 5.0mLof Solution to a 250-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 250-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 239-nm detector and a 4.6-mm ×25-cm column that contains base-deactivated 5-µm packing L1and is maintained at a constant temperature of about 40.The flow rate is about 1.5mLper minute.The chromatograph is programmed as follows.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for chlorhexidine.Calculate the percentage (w/v)of C22H30Cl2N10·2C6H12O7in the portion of Solution taken by the formula: in which 897.77and 625.66are the molecular weights of chlorhexidine gluconate and chlorhexidine acetate,respectively;Cis the concentration,in µg per mL,of USP Chlorhexidine Acetate RSin the Standard preparation;and rUand rSare the peak areas for chlorhexidine obtained from the Assay preparationand the Standard preparation,respectively.