A: Dissolve a quantity of Cream,equivalent to about 50mg of pramoxine hydrochloride,in a mixture of 25mLof methanol and 75mLof ether,and extract with three 25-mLportions of a mixture of equal volumes of 3Nhydrochloric acid and water.Discard the methanol-ether solution,render the combined extracts alkaline with 25mLof 5Nsodium hydroxide,and extract the pramoxine with 50mLof chloroform.Evaporate the clear chloroform extract with the aid of a current of air to dryness:the UVabsorption spectrum of a 1in 100,000solution of the residue so obtained,in 0.1Nhydrochloric acid,exhibits maxima and minima at the same wavelengths as that of a similar solution of the residue similarly obtained from USP Pramoxine Hydrochloride RS,concomitantly measured.

B: To a 5-mg portion of the pramoxine obtained in Identificationtest Aadd 1drop of nitric acid.To the yellow solution cautiously add 5drops of ammonium hydroxide:a red-brown precipitate is formed.

pH7.5phosphate buffer— Dissolve 3.5g of dibasic potassium phosphate in 100mLof water,and adjust the solution by the addition of phosphoric acid solution (1:1)to a pHof 7.5±0.1.

Mobile phase— Prepare a suitable degassed and filtered mixture of acetonitrile,water,and pH7.5phosphate buffer(22:17:1).

Internal standard solution— Prepare a solution of dibutyl phthalate in methanol having a final concentration of about 4µLper mL.

Standard preparation— Prepare a solution of USP Pramoxine Hydrochloride RSin methanol having a known concentration of about 2mg per mL.Pipet 10mLof this solution and 5mLof Internal standard solutioninto a 100-mLvolumetric flask,dilute with methanol to volume,mix,and filter.

Assay preparation— Transfer an accurately weighed portion of Cream,equivalent to about 18mg of pramoxine hydrochloride,to a glass-stoppered,250-mLconical flask.Add 15.0mLof isopropyl alcohol and 40.0mLof methanol,heat on a steam bath,with swirling,to dissolve the Cream,add 40.0mLof methanol and 5.0mLof Internal standard solution,and mix.Cool the flask to a temperature of 10or less to precipitate the waxes,and filter the solution.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 224-nm detector,a 4.6-mm ×3-cm guard column that contains packing L1,and a 4-mm ×30-cm analytical column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph three replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%,and the resolution factor between pramoxine hydrochloride and dibutyl phthalate is not less than 2.4.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.8for pramoxine hydrochloride and 1.0for dibutyl phthalate.Calculate the quantity,in mg,of pramoxine hydrochloride in the portion of Cream taken by the formula: in which Cis the concentration,in mg per mL,of USP Pramoxine Hydrochloride RSin the Standard preparation,and RUand RSare the peak response ratios of pramoxine hydrochloride and internal standard obtained from the Assay preparationand the Standard preparation,respectively.