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

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

Mobile phase— Prepare a suitable mixture of Potassium phosphate buffer and methanol (550:450), filter through a filter of 0.5 µm or finer porosity, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

Assay preparation 1 (where it is represented as being in a single-dose container)—Allow 1 container 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 200 Penicillin G Units per mL.

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

Chromatographic system (see Chromatography 621)— 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 1 mL per minute. Chromatograph the Standard preparation having a concentration of about 200 Penicillin G Units per mL, and record the peak responses as directed for Procedure: the tailing factor for the penicillin G peak is not more than 2, and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparations and the appropriate Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Plot the peak responses obtained from the Standard preparations versus 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 mL of the appropriate Assay preparation. Calculate the number of Penicillin G Units in the container, or in each mL of the Injection taken by the formula: in which L is the labeled number of Penicillin G Units in the container, or in each mL of Injection taken, and D is the number of Penicillin G Units in each mL of 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 mL of the Injection taken, and the extent of dilution.