A: The retention times of the major peaks in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

B: Developing solvent—Prepare a mixture of acetonitrile,methylene chloride,and n-propylamine (56:40:4).

Standard solution— Dissolve about 12.5mg of USP Diphenhydramine Hydrochloride RSand 30mg of USP Pseudoephedrine Hydrochloride RSin 5.0mLof water in a 50-mLcentrifuge tube.

Test solution— Transfer an amount of Capsule contents,equivalent to about 12.5mg of diphenhydramine hydrochloride and about 30.0mg of pseudoephedrine hydrochloride,to a 50-mLcentrifuge tube.Add 5.0mLof water,and shake.

Procedure— To each centrifuge tube add 5.0mLof a saturated solution of sodium carbonate and 10mLof methylene chloride.Insert the stoppers into the tubes and shake for about 1minute.Centrifuge at about 2000rpm for about 10minutes until the layers are well separated.Draw off and discard the top layer.Add 5g of anhydrous sodium sulfate to the remaining solution,insert the stopper tightly,and shake.Separately apply 30µL(3×10µL)each of the Test solutionand the Standard solutionto a suitable unactivated thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture (see Chromatography á621ñ)and allow the spots to dry.Develop the chromatograms in a paper lined chromatographic chamber equilibrated with the Developing solventuntil the solvent front has moved about three-fourths of the length of the plate.Remove the plate,air-dry,place the plate in an iodine chamber,and allow to develop over several hours:the RFvalues of the principal spots obtained from the Test solutioncorrespond to those obtained from the Standard solution.

Medium: water,900mL.

Apparatus 1: 100rpm.

Time: 30minutes.

Procedure— Inject a measured volume (about 50µL)of a filtered portion of the solution under test into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Determine the quantities of C17H21NO·HCl and of C10H15NO·HCl dissolved by employing the procedures set forth in the Assay,making any necessary modifications.

Tolerances— Not less than 75%(Q)of the labeled amounts of C17H21NO·HCl and of C10H15NO·HCl are dissolved in 30minutes.

Mobile phase— Prepare as directed in the Assay.

Diluting solution— Prepare a mixture of water,acetonitrile and methanol (64:26:10).

Standard solution— Transfer accurately weighed quantities of about 5mg each of benzhydrol and benzophenone to a 500-mLvolumetric flask,dissolve in Diluting solution,using heat and sonication if necessary,dilute with Diluting solutionto volume,and mix.Dilute 5.0mLof this solution quantitatively with Diluting solutionto 25.0mL,mix,and filter.

Test solution— Transfer 10Capsules to a 500-mLvolumetric flask,add about 350mLof Diluting solution,sonicate in warm water at about 40to effect dissolution,and cool.Dilute with Diluting solutionto volume,and mix.Dilute 5.0mLof this solution with Diluting solutionto 25.0mL,mix,and filter.

Resolution solution— Prepare a solution in Diluting solutioncontaining about 2µg each of benzhydrol and benzophenone,and 100µg of USP Diphenhydramine Hydrochloride RS,per mL.

Chromatographic system— Proceed as directed for Chromatographic systemunder Assay.Chromatograph 50µLof the Resolution solution:the resolution,R,between benzhydrol and diphenhydramine is not less than 1.3and the resolution,R,between diphenhydramine and benzophenone is not less than 2.0.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solution,and record the areas of the benzhydrol and benzophenone peaks.Calculate the amounts of benzhydrol and benzophenone in the Capsules taken by the formula: in which Cis the concentration of either benzhydrol or benzophenone,in µg per mL,in the Standard solution,and rUand rSare the areas of the corresponding analyte peaks obtained from the Test solutionand the Standard solution,respectively:the sum of the amounts of benzhydrol and benzophenone does not exceed 2%(w/w)of the diphenhydramine hydrochloride.

Aqueous solution— Dissolve 1.7g of sodium 1-heptanesulfonate and 0.8mLof triethylamine in about 800mLof water,adjust with glacial acetic acid to a pHof 3.3±0.05,dilute with water to 1L,mix,and filter.

Mobile phase— Prepare a filtered and degassed mixture of Aqueous solution,acetonitrile,and methanol (64:26:10).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 25mg of USP Diphenhydramine Hydrochloride RS,accurately weighed,to a 100-mLvolumetric flask.Add 25Jmg of USP Pseudoephedrine Hydrochloride RS,accurately weighed,Jbeing the ratio of the labeled amount,in mg,of pseudoephedrine hydrochloride to the labeled amount,in mg,of diphenhydramine hydrochloride per capsule,dissolve in 0.5%glacial acetic acid,dilute with 0.5%glacial acetic acid to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with 0.5%glacial acetic acid to volume,and mix.

Assay preparation— Remove as completely as possible,the contents of not less than 10Capsules,and weigh accurately.Mix the combined contents,and transfer an accurately weighed portion of the powder,equivalent to about 25mg of diphenhydramine hydrochloride,to a 100-mLvolumetric flask.Dissolve in 0.5%glacial acetic acid,dilute with 0.5%glacial acetic acid to volume,mix,and filter.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with 0.5%glacial acetic acid to volume,and mix.

Chromatographic system— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L10.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak areas as directed for Procedure:the resolution,R,between the pseudoephedrine and diphenhydramine peaks is not less than 3.0.For each analyte peak,the tailing factor is not greater than 2.0,and the relative standard deviation for replicate injections is not greater than 2.0%.

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 the two analytes.The relative retention times are about 1.0for pseudoephedrine and 3.0for diphenhydramine.Calculate the quantity,in mg,of diphenhydramine hydrochloride (C17H21NO·HCl)in the portion of Capsules taken by formula: in which Cis the concentration,in µg per mL,of USP Diphenhydramine Hydrochloride RSin the Standard preparation;and rUand rSare the peak areas obtained from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of pseudoephedrine hydrochloride (C10H15NO·HCl)in the portion of Capsules taken by the same formula changing the terms to refer to pseudoephedrine hydrochloride.