A:Infrared Absorption 197K—

B: The crystals obtained in Identification test A melt between 132 and 136 (see Melting Range or Temperature 741).

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

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

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

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

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

Reference solution B— Transfer 2.0 mL of Reference solution A to a 100-mL volumetric flask, dilute with Diluent to volume, and mix. This solution contains about 0.0012 mg of chlorhexidine gluconate per mL.

Resolution solution— Transfer about 10 mg of USP Chlorhexidine Related Compounds RS to a 10-mL volumetric flask, dissolve in 2 mL of acetonitrile, dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay, except the chromatograph is programmed as follows.

Procedure— Separately inject equal volumes (about 20 µL) of the Test solution, Reference solution A, and Reference solution B into 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 10 mg of p-chloroaniline, accurately weighed, to a 100-mL volumetric flask, add 2 mL of acetonitrile, swirl to dissolve, dilute with Diluent to volume, and mix. Dilute accurately measured volumes of this solution quantitatively, and stepwise if necessary, with Diluent to obtain Standard solutions having known concentrations of about 1.5, 1.2, 0.6, and 0.3 µg of p-chloroaniline per mL.

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

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solutions and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the p-chloroaniline peaks. Plot the peak responses obtained from the Standard solutions versus 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 500 µg per mL is found. If the quantity so obtained is less than 150 µg per mL, the tests may be repeated using a more appropriate dilution in the preparation of the Test solution.

(Official January 1, 2007)

Solution A— Prepare a solution of 27.6 g of monobasic sodium phosphate and 10 mL of triethylamine in about 1.5 liters of water. Adjust with phosphoric acid to a pH of 3.0, dilute with water to 2000 mL, 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 Suitability under Chromatography 621).] Degas before use and sparge with helium during the analysis.

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.

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

System suitability solution— Prepare a solution in Diluent containing about 50 µg of USP Chlorhexidine Acetate RS and 1 µg of p-chloroaniline per mL.

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

Assay preparation— Transfer 5.0 mL of Solution to a 250-mL volumetric flask, dilute with water to volume, and mix. Transfer 5.0 mL of this solution to a 250-mL volumetric flask, dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)— 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 L1 and is maintained at a constant temperature of about 40. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for chlorhexidine. Calculate the percentage (w/v) of C22H30Cl2N10·2C6H12O7 in the portion of Solution taken by the formula: in which 897.77 and 625.66 are the molecular weights of chlorhexidine gluconate and chlorhexidine acetate, respectively; C is the concentration, in µg per mL, of USP Chlorhexidine Acetate RS in the Standard preparation; and rU and rS are the peak areas for chlorhexidine obtained from the Assay preparation and the Standard preparation, respectively.