425 IODOMETRIC ASSAY—ANTIBIOTICS
The following method is provided for the assay of most of the Pharmacopeial penicillin antibiotic drugs and their dosage forms, for which iodometric titration is particularly suitable.

Standard Preparation— Dissolve in the solvent specified in the table of Solvents and Final Concentrations a suitable quantity of the USP Reference Standard specified in the individual monograph, previously dried under the conditions specified in the individual monograph and accurately weighed, and dilute quantitatively and stepwise with the same solvent to obtain a solution having a known concentration of about that specified in the table. Pipet 2.0 mL of this solution into each of two 125-mL glass-stoppered conical flasks.
Solvents and Final Concentrations
Antibiotic Solvent* Final
Concentration
Amoxicillin Water 1.0 mg per mL
Ampicillin Water 1.25 mg per mL
Ampicillin Sodium Buffer No. 1 1.25 mg per mL
Cloxacillin Sodium Water 1.25 mg per mL
Cyclacillin Water 1.0 mg per mL
Dicloxacillin Sodium Buffer No. 1 1.25 mg per mL
Methicillin Sodium Buffer No. 1 1.25 mg per mL
Nafcillin Sodium Buffer No. 1 1.25 mg per mL
Oxacillin Sodium Buffer No. 1 1.25 mg per mL
Penicillin G
Potassium		 Buffer No. 1 2,000 units per mL
Penicillin G Sodium Buffer No. 1 2,000 units per mL
Penicillin V
Potassium		 Buffer No. 1 2,000 units per mL
Phenethicillin
Potassium		 Buffer No. 1 2,000 units per mL
*  Unless otherwise noted, the Buffers are the potassium phosphate buffers defined in the section Media and Diluents under Antibiotics—Microbial Assays 81, except that sterilization is not required before use.

Assay Preparation— Unless otherwise specified in the individual monograph, dissolve in the solvent specified in the table of Solvents and Final Concentrations a suitable quantity, accurately weighed, of the specimen under test, and dilute quantitatively with the same solvent to obtain a solution having a known final concentration of about that specified in the table. Pipet 2 mL of this solution into each of two 125-mL glass-stoppered conical flasks.

Procedure—
Inactivation and Titration— To 2.0 mL of the Standard Preparation and of the Assay Preparation, in respective flasks, add 2.0 mL of 1.0 N sodium hydroxide, mix by swirling, and allow to stand for 15 minutes. To each flask add 2.0 mL of 1.2 N hydrochloric acid, add 10.0 mL of 0.01 N iodine VS, immediately insert the stopper, and allow to stand for 15 minutes. Titrate with 0.01 N sodium thiosulfate VS. As the endpoint is approached, add 1 drop of starch iodide paste TS, and continue the titration to the discharge of the blue color.
Blank Determination— To a flask containing 2.0 mL of the Standard Preparation add 10.0 mL of 0.01 N iodine VS. If the Standard Preparation contains amoxicillin or ampicillin, immediately add 0.1 mL of 1.2 N hydrochloric acid. Immediately titrate with 0.01 N sodium thiosulfate VS. As the endpoint is approached, add 1 drop of starch iodide paste TS, and continue the titration to the discharge of the blue color. Similarly treat a flask containing 2.0 mL of the Assay Preparation.
Calculations— Calculate the microgram (or unit) equivalent (F) of each mL of 0.01 N sodium thiosulfate consumed by the Standard Preparation by the formula:
(2CP) / (B I)
in which C is the concentration, in mg per mL, of Reference Standard in the Standard Preparation, P is the potency, in µg (or units) per mg, of the Reference Standard, B is the volume, in mL, of 0.01 N sodium thiosulfate consumed in the Blank determination, and I is the volume, in mL, of 0.01 N sodium thiosulfate consumed in the Inactivation and titration. Calculate the potency of the specimen under test by the formula given in the individual monograph.

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Topic/Question Contact Expert Committee
General Chapter Ahalya Wise, M.S.
Scientist
1-301-816-8161		 (MDANT05) Monograph Development-Antibiotics
USP32–NF27 Page 154