A: Dissolve 5g of ferric chloride in 50mLof 0.1Nhydrochloric acid.Transfer 2.5mLof this stock solution to a 100-mLvolumetric flask,dilute with 0.01Nhydrochloric acid to volume,and mix.Prepare Iron reagentat the time of use.Dissolve the specimen in water,and dilute with water to obtain a solution containing about 1mg of streptomycin per mL.To 5mLof this solution add 2.0mLof 1Nsodium hydroxide,and heat in a water bath for 10minutes.Cool in ice water for 3minutes,then add 2.0mLof 1.2Nhydrochloric acid,and mix.Add 5mLof Iron reagent,and mix:a violet color is produced.

B: It responds to the tests for Sulfate á191ñ.

Mobile phase— Use 70mMsodium hydroxide.During use store in a plastic bottle flushed with a blanket of helium above the liquid surface.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Streptomycin Sulfate RSin water,and quantitatively dilute with water to obtain a solution having a known concentration of about 0.03mg per mL.Sonicate for 1minute,and mix.

Assay preparation— Transfer about 30mg of Streptomycin Sulfate,accurately weighed,to a 100-mLvolumetric flask,dilute with water to volume,sonicate for 1minute,and mix.Transfer 10.0mLof this solution to a second 100-mLvolumetric flask,dilute with water to volume,and mix.

System suitability solution— Heat about 10mLof the Standard preparationat 75for 1hour.Allow to cool.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with an electrochemical detector,a gold working electrode,a pHsilver–silver chloride reference electrode,a 4-mm ×5-cm guard column that contains packing L48,and a 4-mm ×25-cm analytical column that contains packing L48.The electrochemical detector is used in the integrated amperometric mode with a range of 300nC,an output of 1Vfull scale,and a rise time of 0.5second,positive polarity.The potential is programmed as follows. The flow rate is about 0.5mLper minute.Chromatograph the System suitability solution,and measure the peak areas as directed for Procedure:the relative retention times are about 0.5for the main degradation product and 1.0for streptomycin;and the resolution,R,between the two peaks is not less than 3.Chromatograph the Standard preparation,and measure the peak areas as directed for Procedure:the tailing factor is not more than 2;the column efficiency is not less than 1000theoretical plates;and the relative standard deviation for replicate injections is not more than 5%.[NOTE—If variation of retention time or increase of tailing occurs,clean the columns with 0.2Msodium hydroxide.Carefully maintain the working and reference electrodes.]

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in µg,of streptomycin (C21H39N7O12)in each mg of Streptomycin Sulfate taken by the formula: in which Cis the concentration,in mg per mL,of USP Streptomycin Sulfate RSin the Standard preparation;Pis the designated streptomycin content,in µg per mg,of streptomycin (C21H39N7O12)in USP Streptomycin Sulfate RS;WUis the weight,in mg,of Streptomycin Sulfate taken to prepare the Assay preparation;and rUand rSare the streptomycin peak areas obtained from the Assay preparationand the Standard preparation,respectively.