Identification—
A:
It meets the requirements for polymyxin B under
Thin-Layer Chromatographic Identification Test 
201BNP
.
B:
The retention time of the trimethoprim peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for trimethoprim.
Assay for polymyxin B—
Proceed as directed for polymyxin B under
Antibiotics—Microbial Assays 81, using an accurately measured volume of Ophthalmic Solution, diluted quantitatively and stepwise with
Buffer No. 6, to obtain a
Test Dilution having a concentration of polymyxin B assumed to be equal to the median dose level of the Standard.
Assay for trimethoprim—
Diluent—
Prepare a mixture of 0.01 N hydrochloric acid and acetonitrile (870:130).
Mobile phase—
Dissolve 1.65 g of ethanesulfonic acid in 1000 mL of a mixture of water and acetonitrile (870:130). Adjust with 10 N sodium hydroxide or 0.1 N hydrochloric acid to a pH of 3.5. Pass this solution through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Trimethoprim RS in
Diluent to obtain a solution having a known concentration of about 0.04 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 1 mg of trimethoprim, to a 25-mL volumetric flask, dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L11. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 1.5, when calculated at 10% height of the peak; and the relative standard deviation for replicate injections is not more than 2.0%.
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 peaks. Calculate the quantity, in mg, of trimethoprim (C
14H
18N
4O
3) in each mL of the Ophthalmic Solution taken by the formula:
25(C/V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Trimethoprim RS in the
Standard preparation; V is the volume, in mL, of Ophthalmic Solution taken to prepare the
Assay preparation; and
rU and
rS are the trimethoprim peak area responses obtained from the
Assay preparation and the
Standard preparation, respectively.
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