Sterility 
71
—
It meets the requirements when tested as directed for
Antibiotic Solids, Bulks, and Blends in the section
Membrane Filtration under
Test for Sterility of the Product to be Examined, except to use
Fluid D, to which has been added sufficient sterile penicillinase to inactivate the ampicillin and to swirl the vessel until solution is complete before filtering. If it does not dissolve completely, proceed as directed for
Solids in the section
Direct Inoculation of the Culture Medium under
Test for Sterility of the Product to be Examined, except to use Fluid Thioglycollate Medium and Soybean–Casein Digest Medium containing sufficient penicillinase to inactivate the ampicillin in each vessel.
Assay—
Phosphate buffer solution—
Accurately weigh 68 g of monobasic potassium phosphate, and transfer to a 500-mL volumetric flask. Dissolve in and dilute with water to volume.
Mobile phase—
Prepare a suitable mixture of water, acetonitrile, Phosphate buffer solution, and glacial acetic acid (3600:360:40:4). Pass through a 0.45-µm nylon filter, and degas.
Standard preparation—
Dissolve, with sonication, an accurately weighed quantity of
USP Ampicillin RS in water to prepare a solution having 0.5 mg per mL. Pass through a 0.45-µm PTFE filter, discarding the first 3 mL of the filtrate.
Caffeine solution—
Transfer about 30 mg of caffeine, accurately weighed, to a 50-mL volumetric flask. Add 25 mL of water, sonicate to dissolve, and dilute with water to volume. Pass through a 0.45-µm PTFE filter, discarding the first 3 mL of the filtrate.
System suitability solution—
Prepare a solution of 1.0 mL of Caffeine solution and 9.0 mL of Standard preparation, and mix.
Assay preparation—
Quantitatively dilute an accurately measured volume of Ampicillin for Injectable Suspension, constituted as directed in the labeling, with water to obtain a solution containing about 0.5 mg per mL. Pass through a 0.45-µm PTFE filter, discarding the first 3 mL of the filtrate.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm analytical column that contains 10-µm packing L1. The flow rate is about 2.0 mL per minute. The column temperature is maintained at 40
. Chromatograph the
System suitability solution and the
Standard preparation, and record the peak responses as directed for
Procedure: the order of elution is ampicillin followed by caffeine; the resolution,
R, between ampicillin and caffeine is greater than 2; the column efficiency is not less than 2000 theoretical plates for the ampicillin peak; the tailing factor is not greater than 1.4; and the relative standard deviation for replicate injections of the
Standard preparation is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas of the major peaks. Calculate the quantity, in mg, of ampicillin (C
16H
19N
3O
4S) in each mL of the constituted solution of Ampicillin for Injectable Suspension taken by the formula:
CD(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Ampicillin RS in the
Standard preparation; D is the dilution factor used in preparing the
Assay preparation; and
rU and
rS are the average peak responses of the ampicillin peaks obtained from the
Assay preparation and the
Standard preparation, respectively.
