Standard Preparation—
Dissolve in the solvent specified in the table of
Solvents and Final Concentrationsa 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 2.0mLof this solution into each of two 125-mLglass-stoppered conical flasks.
Solvents and Final Concentrations
| Antibiotic |
Solvent* |
Final
Concentration |
| Amoxicillin |
Water |
1.0mg per mL |
| Ampicillin |
Water |
1.25mg per mL |
| Ampicillin Sodium |
Buffer No.1 |
1.25mg per mL |
| Cloxacillin Sodium |
Water |
1.25mg per mL |
| Cyclacillin |
Water |
1.0mg per mL |
| Dicloxacillin Sodium |
Buffer No.1 |
1.25mg per mL |
| Methicillin Sodium |
Buffer No.1 |
1.25mg per mL |
| Nafcillin Sodium |
Buffer No.1 |
1.25mg per mL |
| Oxacillin Sodium |
Buffer No.1 |
1.25mg per mL |
Penicillin G
Potassium |
Buffer No.1 |
2,000units per mL |
| Penicillin G Sodium |
Buffer No.1 |
2,000units per mL |
Penicillin V
Potassium |
Buffer No.1 |
2,000units per mL |
Phenethicillin
Potassium |
Buffer No.1 |
2,000units per mL |
*
Unless otherwise noted,the Buffersare the potassium phosphate buffers defined in the section Media and Diluentsunder Antibiotics—Microbial Assays á81ñ,except that sterilization is not required before use.
|
Procedure—
Inactivationand Titration—
To 2.0mLof the Standard Preparationand of the Assay Preparation,in respective flasks,add 2.0mLof 1.0Nsodium hydroxide,mix by swirling,and allow to stand for 15minutes.To each flask add 2.0mLof 1.2Nhydrochloric acid,add 10.0mLof 0.01Niodine VS,immediately insert the stopper,and allow to stand for 15minutes.Titrate with 0.01Nsodium thiosulfate VS.As the endpoint is approached,add 1drop of starch iodide paste TS,and continue the titration to the discharge of the blue color.
Blank Determination—
To a flask containing 2.0mLof the Standard Preparationadd 10.0mLof 0.01Niodine VS.If the Standard Preparationcontains amoxicillin or ampicillin,immediately add 0.1mLof 1.2Nhydrochloric acid.Immediately titrate with 0.01Nsodium thiosulfate VS.As the endpoint is approached,add 1drop of starch iodide paste TS,and continue the titration to the discharge of the blue color.Similarly treat a flask containing 2.0mLof the Assay Preparation.
Calculations—
Calculate the microgram (or unit)equivalent (
F)of each mLof 0.01Nsodium thiosulfate consumed by the
Standard Preparationby the formula:
(2CP)/(B-I),
in which
Cis the concentration,in mg per mL,of Reference Standard in the
Standard Preparation,Pis the potency,in µg (or units)per mg,of the Reference Standard,
Bis the volume,in mL,of 0.01Nsodium thiosulfate consumed in the
Blank determination,and
Iis the volume,in mL,of 0.01Nsodium thiosulfate consumed in the
Inactivation and titration.Calculate the potency of the specimen under test by the formula given in the individual monograph.