Test specimen— Dry an amount of Injection,equivalent to about 2mg of alprostadil,on about 500mg of spectroscopic grade potassium bromide at about 40to 50under vacuum.Prepare a pellet from this mixture.

Standard specimen: a similar preparation of USP Alprostadil RS,in dehydrated alcohol.

Mobile phase— Prepare a filtered and degassed mixture of methylene chloride,1,3-butanediol,and water (1000:6:0.5).Make adjustments if necessary (see System Suitability under Chromatography á621ñ).

Internal standard solution— Dissolve ethylparaben in methylene chloride to obtain a solution containing about 0.05mg per mL.

Standard stock preparation— Dissolve an accurately weighed quantity of USP Alprostadil RSin dehydrated alcohol,and quantitatively dilute with dehydrated alcohol to obtain a solution having a known concentration of about 0.5mg per mL.

Standard preparation— Gently evaporate a 0.5-mLportion of the Standard stock preparation to dryness with a stream of nitrogen.Proceed as directed for theAssay preparation beginning with “Add 150µLof a 1in 25freshly prepared solution.”

Assay preparation— Pool the contents of several containers of the Injection,and gently evaporate an accurately measured volume,equivalent to about 0.25mg of alprostadil,to dryness using a stream of nitrogen.Add 150µLof a 1in 25freshly prepared solution of a-bromo-2¢-acetonaphthone in acetonitrile,rinse the inside of the container with this solution,and swirl.Add 150µLof a freshly prepared 1in 200solution of diisopropylethylamine in acetonitrile to the container,rinse the inside of the container with this solution,and swirl.Cap and sonicate to dissolve.Heat the container at 45for 45minutes,swirling occasionally.Sonicate again after heating is complete.[NOTE—If the entire sample does not dissolve,the specimen should be discarded.]Evaporate the solution using a stream of nitrogen,add 2.0mLof Internal standard solution,and mix.Sonicate to dissolve.[NOTE—If incomplete dissolution is still observed,discard the specimen.]

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.4-mm ×25-cm column that contains packing L18.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.4and 1.0,for ethylparaben and alprostadil,respectively;the resolution,R,between alprostadil and the internal standard is not less than 9.0;and the relative standard deviation for replicate injections is not more than 2.5%.

Procedure— Separately inject equal volumes of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of alprostadil (C20H34O5)in the volume of Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Alprostadil RSin the Standard stock preparation;and RUand RSare the peak response ratios of alprostadil to the internal standard obtained from the Assay preparationand the Standard preparation,respectively.