A: Place an amount of the finely powdered contents of Capsules,equivalent to about 65mg of propoxyphene hydrochloride,in a test tube,add 5mLof methanol,shake for 5minutes,and centrifuge.The clear supernatant is the test solution.Dissolve weighed amounts of USP Propoxyphene Hydrochloride RS,USP Aspirin RS,and USP Caffeine RScorresponding,proportionately,to the amounts of propoxyphene hydrochloride,aspirin,and caffeine in the Capsules to obtain a Standard solution having a known concentration of about 13mg of propoxyphene hydrochloride per mL.Apply 10µLeach 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.Develop the chromatograms in a solvent system consisting of a mixture of chloroform,butyl acetate,and formic acid (30:20:10)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,allow it to dry in a fume hood,and examine the plate under short-wavelength UVlight:the chromatogram of the test solution exhibits 3principal spots that correspond in RFvalue and intensity with those obtained from the Standard solution.

B: The Assay preparationprepared as directed in the Assay for propoxyphene hydrochloride and caffeineis dextrorotatory.

Medium:pH4.5acetate buffer; 500mL.

Apparatus 1: 100rpm.

Time: 60minutes.

pH4.5acetate buffer— Dissolve 2.99g of sodium acetate trihydrate in 200mLof water,add 1.66mLof glacial acetic acid,dilute with water to 1000mL,and mix.

Determination of dissolved aspirin— Transfer 5.0mLof a filtered portion of the solution under test to a 25-mLvolumetric flask.Concurrently pipet 5mLof a standard solution,prepared by dissolving 35mg of accurately weighed USP Aspirin RSin 50.0mLof pH4.5acetate buffer,into a second 25-mLvolumetric flask.Proceed as directed for Procedurein the Assay for aspirin,beginning with “Into each flask pipet 5mLof Sodium hydroxide reagent.”Concomitantly determine the absorbances of both solutions against a similarly treated blank of 5.0mLof pH4.5acetate buffer.Determine the amount of aspirin (C9H8O4)in solution by comparison with the Standard solution.

Determination of dissolved propoxyphene hydrochloride—

INTERNALSTANDARD SOLUTION —Dissolve n-tricosane in chloroform to obtain a solution containing about 0.06mg per mL.

STANDARDPREPARATION —Dissolve an accurately weighed quantity of USP Propoxyphene Hydrochloride RSin pH4.5acetate bufferto obtain a solution having a known concentration (between 0.06and 0.13mg per mL)that corresponds to the concentration of propoxyphene hydrochloride that is estimated for the solution under test,based on the labeled content of the Capsules and the extent of dissolution.

PROCEDURE —Transfer equal,accurately measured,volumes (between 5.0and 10.0mL)of filtered portions of the solution under test and the Standard preparationinto separate,screw-capped centrifuge tubes.To each tube add 5.0mLof sodium carbonate solution (1in 5),and shake.Extract with one 5.0-mLportion of Internal standard solution,and one 5.0-mLportion of chloroform,and shake each extract for 5minutes.Pass the organic layers through phase-separating paper.Evaporate to about 1mL,and inject a 10-µLportion into a suitable gas chromatograph equipped with a flame-ionization detector.Proceed as directed for Procedurein the Assay for propoxyphene hydrochloride and caffeine,beginning with “The column is typically 60cm ×4mm.”Determine the amount of propoxyphene hydrochloride (C22H29NO2·HCl)in solution by comparison with the Standard preparation.

Tolerances— Not less than 75%(Q)of the labeled amount of C9H8O4is dissolved in 60minutes,and not less than 85%(Q)of the labeled amount of C22H29NO2·HCl is dissolved in 60minutes.

Ferric chloride-urea reagent— Dissolve by swirling,without the aid of heat,60g of urea in a mixture of 8mLof ferric chloride solution (6in 10)and 42mLof 0.05Nhydrochloric acid.Adjust this solution,if necessary,with 6Nhydrochloric acid to a pHof 3.2.

Standard preparation— Transfer 15.0mg of USP Salicylic Acid RS,accurately weighed,to a 100-mLvolumetric flask,add chloroform to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask containing 20mLof methanol,4drops of hydrochloric acid,and 20mLof a 1in 10solution of glacial acetic acid in ether,dilute with chloroform to volume,and mix.

Test preparation— Pack a pledget of glass wool in the base of a 25-×200-mm chromatographic tube.In a beaker,prepare a mixture of 6g of chromatographic siliceous earth,2mLof freshly prepared Ferric chloride-urea reagent,and 40mLof chloroform.Transfer the mixture to the chromatographic tube.Rinse the beaker with 15mLof chloroform,transfer to the column,and pack tightly.Place a small amount of glass wool at the top of the column.Weigh accurately a quantity of the finely powdered contents of Capsules,equivalent to about 50mg of aspirin,mix with 10mLof chloroform by stirring for 3minutes,and transfer to the chromatographic column with the aid of 10mLof chloroform.Pass 40mLof chloroform through the column,rinse the tip of the chromatographic tube with chloroform,and discard the eluate.Prepare as a receiver a 100-mLvolumetric flask containing 20mLof methanol and 4drops of hydrochloric acid,and elute any salicylic acid from the column with 20mLof a 1in 10solution of glacial acetic acid in ether that recently has been saturated with water,followed by 30mLof chloroform.Dilute the eluate with chloroform to volume,and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparationand the Test preparationin 1-cm cells at the wavelength of maximum absorbance at about 306nm,with a suitable spectrophotometer,using as the blank a solvent mixture of the same composition as that used for the Standard preparation:the absorbance of the Test preparationdoes not exceed that of the Standard preparation(3.0%,calculated on the basis of the labeled content of aspirin).

Internal standard solution— Dissolve n-tricosane in chloroform to obtain a solution having a concentration of about 0.6mg per mL.

Standard preparation— Transfer to a 50-mLvolumetric flask accurately weighed quantities of about 32mg of USP Propoxyphene Hydrochloride RS,about 32Jmg of USP Aspirin RS,and about 32J¢mg of USP Caffeine RS,where Jis the ratio of the labeled amount,in mg,of aspirin to the labeled amount,in mg,of propoxyphene hydrochloride per Tablet,and J¢is the ratio of the labeled amount,in mg,of caffeine to the labeled amount,in mg,of propoxyphene hydrochloride per Tablet.Add 10mLof acetone,and swirl to dissolve the reference standards completely.Dilute with water to volume,and mix.

Assay preparation— Remove as completely as possible the contents of 20Capsules,and transfer an accurately weighed portion of the powder,equivalent to 65mg of propoxyphene hydrochloride,to a 100-mLvolumetric flask containing 20mLof acetone.If the Capsules contain a pellet (propoxyphene hydrochloride)as well as a powder,finely grind the pellets,then mix with the powder before proceeding.Sonicate for about 1minute.Dilute the milky solution with water to volume,and mix.Filter,discarding the first 20mLof the filtrate.

Procedure for propoxyphene hydrochlorideand caffeine— Transfer 5.0-mLaliquots of the Assay preparationand the Standard preparationto separate 60-mLseparators.To each add 5.0mLof sodium carbonate solution (1in 5)and 5.0mLof Internal standard solution.Shake vigorously for 5minutes,and allow the layers to separate.Drain the chloroform layer through phase-separating paper,suitably supported in a funnel,into a screw-capped test tube.Extract with one 5-mLportion of chloroform,and drain the chloroform layer through phase-separating paper.Evaporate the combined chloroform extracts,using a stream of dry nitrogen,to a final volume of about 2mL.Inject separately a suitable volume,equivalent to about 6.4µg of propoxyphene,of the chloroform extracts from the Assay preparationand the Standard preparationinto a suitable gas chromatograph equipped with a flame-ionization detector.The column is typically 60cm ×3mm and is packed with 3%methyl phenyl silicone,liquid phase on 80-to 100-mesh chromatographic siliceous earth.The temperature of the injection port is 200,the column temperature is 175,and the carrier gas,nitrogen,has a flow rate of about 60mLper minute.Relative retention times are about 0.65for caffeine,1.0for the internal standard,and 1.7for propoxyphene.In a suitable chromatogram,the resolution factor is not less than 1.0between any two peaks,the relative standard deviation for five replicate injections of the Standard preparationis not more than 2.0,and the tailing factor for caffeine is not greater than 1.5.Calculate the quantities,in mg,of propoxyphene hydrochloride (C22H29NO2·HCl)and caffeine (C8H10N4O2),respectively,in the portion taken for the Assay preparationby the same formula: in which Cis the concentration,in mg per mL,of the appropriate USP Reference Standard in the Standard preparation,and RUand RSare the ratios of the peak areas of the corresponding analyte to those of the internal standard obtained from the Assay preparationand the Standard preparation,respectively.

Sodium hydroxide reagent— Dissolve 1g of polyoxyethylene (23)lauryl ether in about 100mLof hot water contained in a 1000-mLvolumetric flask.Dilute with water to about 600mL,and dissolve 10g of sodium hydroxide in this solution.Dilute with water to volume,and mix.

Ferric nitrate reagent— Mix 70mLof nitric acid with about 600mLof water contained in a 1000-mLvolumetric flask.Dissolve 40g of ferric nitrate [Fe(NO3)3·9H2O]in this solution,dilute with water to volume,and mix.

Standard preparation and Assay preparation—Prepare as directed in the Assay for propoxyphene hydrochloride and caffeineto obtain solutions having concentrations of about 4mg of aspirin per mL.

Procedure— Into separate 25-mLvolumetric flasks pipet 2mLeach of the Standard preparationand the Assay preparation,and 2mLof dilute acetone (1in 5)to provide the blank.Into each flask pipet 5mLof Sodium hydroxide reagent,mix by gentle swirling,and allow to stand at room temperature for 8minutes.Dilute with Ferric nitrate reagentto volume,and mix.Concomitantly determine the absorbances of both solutions against the blank in 1-cm cells at the wavelength of maximum absorbance at about 530nm,taking care to allow the solutions to reach an equilibrium temperature in the cell compartment.The color intensity is temperature-dependent.Calculate the quantity,in mg,of aspirin (C9H8O4)in the portion taken for the Assay preparationby the formula: in which Cis the concentration,in mg per mL,of USP Aspirin RSin the Standard preparation,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.