A: It responds to the Identificationtest under Penicillin G Benzathine Oral Suspension:the spot obtained from the test solution,corresponding in RFvalue to that obtained from the Standard solution,is completely resolved from a second spot,produced by penicillin Gprocaine.

B: It responds to the Identificationtest under Penicillin G Procaine.

Mobile phase— Dissolve 4g of sodium 1-hexanesulfonate and 5.44g of monobasic potassium phosphate in 760mLof water,adjust with phosphoric acid to a pHof 2.5,dilute with acetonitrile to 1000mL,and mix.Pass through a filter having a 0.5-µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

pH6.0Phosphate buffer— Dissolve 16g of monobasic potassium phosphate and 4g of dibasic sodium phosphate in water,dilute with water to 200mL,adjust with phosphoric acid or 1Nsodium hydroxide to a pHof 6.0.

Diluent— Transfer 60mLof butyl alcohol,100mLof acetonitrile,and 10mLof pH6.0Phosphate bufferto a 500-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution— Transfer about 24mg of USP Penicillin G Potassium RS,accurately weighed,and about 8mg of USP Procaine Hydrochloride RS,accurately weighed,to a 100-mLvolumetric flask,add 12mLof butyl alcohol and 20mLof acetonitrile,and shake to dissolve.Add 2mLof pH6.0Phosphate bufferto volume,and mix.

Test solution— Centrifuge about 20mLof the Suspension.Remove the supernatant fluid,and pass it through a filter having a 5-µm or finer porosity.Transfer 5.0mLof the clear filtrate to a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 204-nm detector and a 4-mm ×15-cm column that contains 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 0.3%.

Procedure— Separately inject equal volumes (about 5µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of penicillin G(C16H18N2O4S)in the Test solutionby the formula: in which 334.40and 372.48are the molecular weights of penicillin Gand penicillin Gpotassium respectively;Cis the concentration,in mg per mL,of USP Penicillin G Potassium RSin the Standard solution;and rUand rSare the responses of the penicillin Gpeaks in the chromatograms of the Test solutionand the Standard solution,respectively:not more than 1%is found.Calculate the percentage of procaine (C13H20N2O2)in the Test solutionby the formula: in which 236.32and 272.78are the molecular weights of procaine and procaine hydrochloride,respectively;Cis the concentration,in mg per mL,of USP Procaine Hydrochloride RSin the Standard solution;and rUand rSare the responses of the procaine peaks in the chromatograms of the Test solutionand the Standard solution,respectively:not more than 1%is found.

Standard preparations— Transfer about 14kmg of USP Procaine Hydrochloride RS,accurately weighed,to a 500-mLvolumetric flask,and dissolve in 2mLof 0.5Nsodium hydroxide,kbeing the ratio of the labeled number of Penicillin G Procaine Units to the labeled number of Penicillin G Benzathine Units in the Injectable Suspension.After 15minutes,add 1mLof 1.2Nhydrochloric acid,dilute with water to volume,and mix.This stock solution contains about 28kµg of USP Procaine Hydrochloride RSper mL.Transfer 1.0,2.0,3.0,4.0,and 5.0mL,respectively,of this stock solution to each of five 25-mLvolumetric flasks.Transfer 4.0,3.0,2.0,and 1.0mLof water to the first four flasks,respectively.

Assay preparation— Where the Injectable Suspension is represented as being in a single-dose container,withdraw all of the withdrawable contents,using a suitable hypodermic needle and syringe.Where the label states the quantities of penicillin Gbenzathine and penicillin Gprocaine in a given volume of Injectable Suspension,remove an accurately measured volume of the Injectable Suspension.For each 300,000Penicillin G Benzathine Units in the specimen of Injectable Suspension taken,add 20mLof 0.5Nsodium hydroxide,and mix.After 15minutes,add 0.5mLof 1.2Nhydrochloric acid for each mLof 0.5Nsodium hydroxide used,and dilute quantitatively with water to obtain a solution containing 36Penicillin G Procaine Units per mL.Transfer 5.0mLof this solution to a 50-mLvolumetric flask.

Procedure— To each of the flasks containing the Standard preparationsand the Assay preparation,and to a seventh 50-mLvolumetric flask containing 5.0mLof water to provide the blank,add 0.5mLof 4Nhydrochloric acid,1.0mLof sodium nitrite solution (1in 1000),1.0mLof ammonium sulfamate solution (1in 200),and 1.0mLof N-(1-naphthyl)ethylenediamine dihydrochloride solution (1in 1000),mixing and allowing 2minutes to elapse after each addition.Dilute the contents of each flask with water to volume,and mix.Concomitantly determine the absorbances of the solutions from the Standard preparationsand the Assay preparationat the wavelength of maximum absorbance at about 550nm,with a suitable spectrophotometer,using the blank to set the instrument at zero.Plot the absorbance values of solutions from the Standard preparationsversus concentration,in mg per mL,of procaine hydrochloride in the solutions from the Standard preparations,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration (C),in mg per mL,of procaine hydrochloride in the solution from the Assay preparation.Calculate the quantity,in Penicillin G Procaine Units in each container or in each mLof the Injectable Suspension taken by the formula: in which 588.73and 272.78are the molecular weights of penicillin Gprocaine monohydrate and procaine hydrochloride,respectively;1009.1is the theoretical potency,in Penicillin G Units,in each mg of penicillin Gprocaine;Lis the labeled amount,in Penicillin G Procaine Units in each container or in each mLof Injectable Suspension taken;and Dis the concentration,in Penicillin G Procaine Units per mL,of the solution from the Assay preparation,on the basis of the labeled amount in each container or in each mLof Injectable Suspension taken and the extent of dilution.

Standard preparation— Using USP Penicillin G Potassium RS,prepare as directed for Standard preparationunderIodometric Assay—Antibiotics á425ñ.

Assay preparation— Where the Injectable Suspension is represented as being in a single-dose container,withdraw all of the withdrawable contents,using a suitable hypodermic needle and syringe.Where the label states the quantities of penicillin Gbenzathine and penicillin Gprocaine in a given volume of Injectable Suspension,remove an accurately measured volume of Injectable Suspension,freshly mixed but free from air bubbles.Dilute the specimen of Injectable Suspension taken quantitatively with 1Nsodium hydroxide to obtain a solution containing about 2000Penicillin G Units per mL.Pipet 2mLof this solution into each of two glass-stoppered,125-mLconical flasks.

Blank preparation— Prepare as directed for Assay preparation,except to use Buffer No.1instead of 1.0Nsodium hydroxide.

Procedure— Proceed as directed for Procedureunder Iodometric Assay—Antibiotics á425ñ,except in performing the Inactivation and Titrationto omit the addition of 1.0Nsodium hydroxide to the Assay preparation,and in performing the Blank Determinationto use the Blank preparationin place of the Assay preparation.Calculate the total quantity,T,in Penicillin G Units,in each mLof the Injectable Suspension taken by the formula: in which Lis the labeled quantity,in Penicillin G Units in each container,or per mL,in the Injectable Suspension taken;and Dis the concentration,in Penicillin G Units per mL,in the Assay preparationon the basis of the labeled quantity in the Injectable Suspension and the extent of dilution.Calculate the quantity,in Penicillin G Benzathine Units,in each container,or in each mL,of the Injectable Suspension taken by the formula: in which Pis the quantity,in Penicillin G Procaine Units,in each container,or in each mL,of Injectable Suspension taken,as determined in the Assay for penicillin Gprocaine.