Assay for polymyxin—
Proceed with Ophthalmic Ointment as directed for polymyxin under
Antibiotics—Microbial Assays 
81
, using an accurately weighed portion of Ophthalmic Ointment, equivalent to about 5000 Polymyxin B Units, shaken in a separator containing about 50 mL of ether and extracted with four 20-mL portions of
Buffer No. 6. Combine the aqueous extracts in a 100-mL volumetric flask, dilute, if necessary, with
Buffer No. 6 to volume, and mix. Dilute an accurately measured portion of the resulting solution quantitatively with
Buffer No. 6 to obtain a
Test Dilution having a concentration assumed to be equal to the median dose level of the Standard.
Assay for chloramphenicol and hydrocortisone acetate—
Standard preparation—
Transfer about 25 mg of
USP Chloramphenicol RS and 25
J mg of
USP Hydrocortisone Acetate RS, both accurately weighed,
J being the ratio of the labeled amount, in mg, of hydrocortisone acetate to the labeled amount, in mg, of chloramphenicol per g of Ophthalmic Ointment, to a 100-mL volumetric flask, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 10.0 mL of the resulting solution to a 25-mL volumetric flask, dilute with
Mobile phase to volume, and mix. Filter a portion of this solution through a 0.5-µm or finer porosity filter, and use the clear filtrate as the
Standard preparation.
Procedure—
Proceed as directed for
Procedure in the
Assay under
Chloramphenicol. Calculate the quantity, in mg, of chloramphenicol (C
11H
12Cl
2N
2O
5) in the portion of Ophthalmic Ointment taken by the formula:
0.25C(rU / rS),
in which the terms are as defined therein. Calculate the quantity, in mg, of hydrocortisone acetate (C
23H
32O
6) in the portion of Ophthalmic Ointment taken by the formula:
250C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Hydrocortisone Acetate RS in the
Standard preparation, and the other terms are as defined therein.
