Sodium citrate buffer— Dissolve 0.8g of sodium citrate (dihydrate)in about 150mLof water,adjust with 0.1Nhydrochloric acid to a pHof 6.8,dilute with water to 200mL,and mix.

Potassium phosphate buffer— Dissolve 10g of monobasic potassium phosphate in 900mLof water,adjust with phosphoric acid to a pHof 4.15,dilute with water to 1000mL,and mix.

Mobile phase— Prepare a suitable mixture of Potassium phosphate bufferand methanol (550:450),filter through a filter of 0.5µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparations— Prepare a standard stock solution of USP Penicillin G Potassium RSin Sodium citrate buffercontaining a known concentration of about 2000Penicillin G Units per mL.To three separate 100-mLvolumetric flasks transfer 5.0,10.0,and 15.0mLof the standard stock solution,dilute with water to volume,and mix.These solutions contain about 100,200,and 300Penicillin G Units per mL,respectively.

Assay preparation 1 (where it is represented as being in a single-dose container)—Allow 1container of Injection to thaw,and mix.Withdraw all of the withdrawable contents,using a suitable hypodermic needle and syringe,and dilute quantitatively with water to obtain a solution containing about 200Penicillin G Units per mL.

Assay preparation 2 (where the label states the quantity of penicillin Gin a given volume of Injection)—Allow 1container of Injection to thaw,and mix.Dilute an accurately measured volume of the Injection quantitatively with water to obtain a solution containing about 200Penicillin G Units per mL.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×10-cm column containing 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparationhaving a concentration of about 200Penicillin G Units per mL,and record the peak responses as directed for Procedure:the tailing factor for the penicillin Gpeak is not more than 2,and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationsand the appropriate Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Plot the peak responses obtained from the Standard preparationsversus concentration,in Penicillin G Units per mL,and draw the straight line best fitting the three plotted points.From the graph so obtained,determine the number,N,of Penicillin G Units in each mLof the appropriate Assay preparation.Calculate the number of Penicillin G Units in the container,or in each mLof the Injection taken by the formula: in which Lis the labeled number of Penicillin G Units in the container,or in each mLof Injection taken,and Dis the number of Penicillin G Units in each mLof Assay preparation 1,or of Assay preparation 2,as appropriate,on the basis of the labeled number of Penicillin G Units in the container,or in each mLof the Injection taken,and the extent of dilution.