Diluent— Use pH 6.6 phosphate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions).

Mobile phase— Prepare a mixture of water, acetonitrile, and glacial acetic acid (65:35:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of phenoxyacetic acid quantitatively in Diluent to obtain a solution having a known concentration of about 0.1 mg per mL.

Test solution— Dissolve an accurately weighed quantity of Penicillin V quantitatively in Diluent to obtain a solution containing 20.0 mg per mL. [NOTE—Use this solution on the day prepared.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column containing 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the responses as directed for Procedure: the tailing factor is not more than 1.5, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the phenoxyacetic acid peaks. Calculate the percentage of phenoxyacetic acid in the portion of Penicillin V taken by the formula: in which C is the concentration, in mg per mL, of phenoxyacetic acid in the Standard solution, and rU and rS are the phenoxyacetic acid peak responses obtained from the Test solution and the Standard solution, respectively. Not more than 0.5% is found.

100rp / rs,

Mobile phase— Prepare a suitable filtered and degassed mixture of water, acetonitrile, and glacial acetic acid (650:350:5.75). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Penicillin V Potassium RS in Mobile phase to obtain a solution having a known concentration of about 2.5 mg per mL.

Assay preparation— Transfer about 125 mg of Penicillin V, accurately weighed, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Resolution solution— Prepare a solution in Mobile phase containing about 2.5 mg of penicillin G potassium and 2.5 mg of penicillin V potassium per mL.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.8 for penicillin G and 1.0 for penicillin V, the column efficiency determined from the penicillin V peak is not less than 1800 theoretical plates, and the resolution, R, between penicillin G and penicillin V is not less than 3.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major penicillin V peaks and any p-hydroxypenicillin V peaks with a retention time of about 0.4 relative to that of the main penicillin V peak. Calculate the quantity, in USP Penicillin V Units, in each mg of the Penicillin V taken by the formula: in which C is the concentration, in mg per mL, of USP Penicillin V Potassium RS in the Standard preparation, P is the designated potency, in USP Penicillin V Units per mg, of USP Penicillin V Potassium RS, WU is the weight, in mg, of Penicillin V taken to prepare the Assay preparation, and rU and rS are the sums of the p-hydroxypenicillin V and penicillin V peak responses obtained from the Assay preparation and the Standard preparation, respectively.

(Official January 1, 2007)