Gentamicin and Prednisolone Acetate Ophthalmic Ointment
» Gentamicin and Prednisolone Acetate Ophthalmic Ointment contains the equivalent of not less than 90.0 percent and not more than 120.0 percent of the labeled amount of gentamicin, and not less than 90.0 percent and not more than 110.0 percent of the labeled amount of prednisolone acetate (C23H30O6).
Packaging and storage—
Preserve in collapsible ophthalmic ointment tubes, and avoid exposure to excessive heat.
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Identification—
A:
Shake a quantity of Ophthalmic Ointment, equivalent to about 5 mg of gentamicin, with a mixture of 200 mL of chloroform and 5 mL of water. Allow to separate, and filter the aqueous layer: the filtrate so obtained meets the requirements of the Identification test under Gentamicin Sulfate Injection.
B:
The chromatogram of the Assay preparation obtained as directed in the Assay for prednisolone acetate exhibits a major peak for prednisolone acetate, the retention time of which corresponds to that obtained in the chromatogram of the Standard preparation obtained as directed in the Assay for prednisolone acetate.
Sterility 
71
:
meets the requirements.
71:
meets the requirements.
Minimum fill 755:
meets the requirements.
755:
meets the requirements.
Water, Method I 921:
not more than 2.0%, 20 mL of a mixture of toluene and methanol (7:3) being used in place of methanol in the titration vessel.
921:
not more than 2.0%, 20 mL of a mixture of toluene and methanol (7:3) being used in place of methanol in the titration vessel.
Metal particles—
It meets the requirements of the test for Metal Particles in Ophthalmic Ointments 751.
751.
Assay for gentamicin—
Proceed with Ophthalmic Ointment as directed for gentamicin under Antibiotics—Microbial Assays 81, using an accurately weighed quantity of Ophthalmic Ointment, equivalent to about 1 mg of gentamicin, shaken with about 50 mL of ether in a separator, and extracted with four 20-mL portions of Buffer No. 3. Combine the aqueous extracts, and dilute quantitatively and stepwise with Buffer No. 3 to obtain a Test Dilution having a concentration of gentamicin assumed to be equal to the median dose level of the Standard.
81, using an accurately weighed quantity of Ophthalmic Ointment, equivalent to about 1 mg of gentamicin, shaken with about 50 mL of ether in a separator, and extracted with four 20-mL portions of Buffer No. 3. Combine the aqueous extracts, and dilute quantitatively and stepwise with Buffer No. 3 to obtain a Test Dilution having a concentration of gentamicin assumed to be equal to the median dose level of the Standard.
Assay for prednisolone acetate—
Mobile phase—
Prepare a suitable mixture of water and acetonitrile (60:40), and pass through a suitable filter having a porosity of 1 µm or less. Make adjustments if necessary (see System Suitability under Chromatography 621).
621).
Internal standard solution—
Prepare a solution in methanol containing about 2.7 mg of fluorometholone acetate per mL.
Standard preparation—
Transfer about 38 mg of USP Prednisolone Acetate RS, accurately weighed, to a 100-mL volumetric flask, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 8.0 mL of this solution to a 50-mL volumetric flask, add 25 mL of n-hexane, and shake. Add 2.0 mL of Internal standard solution, dilute with methanol to volume, and shake vigorously for 30 seconds. Allow the layers to separate, remove the upper n-hexane layer by aspiration, and discard the aspirate. Dilute the solution in the volumetric flask with methanol to volume, and mix. Centrifuge a portion of this solution, and use the clear liquid as the Standard preparation. This solution contains about 0.06 mg of USP Prednisolone Acetate RS per mL.
Assay preparation—
Transfer an accurately weighed portion of Ophthalmic Ointment, equivalent to about 3 mg of prednisolone acetate, to a 50-mL volumetric flask, add 25 mL of n-hexane, and shake. Add 2.0 mL of Internal standard solution, dilute with methanol to volume, and shake vigorously for 30 seconds. Allow the layers to separate, remove the upper n-hexane layer by aspiration, and discard the aspirate. Dilute the solution in the volumetric flask with methanol to volume, and mix. Centrifuge a portion of this solution, and use the clear liquid as the Assay preparation.
Chromatographic system
(see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 20-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and measure the peak responses as directed for Procedure: the tailing factor is not more than 1.5; the resolution, R, between the prednisolone acetate peak and the fluorometholone acetate peak is not less than 2.0; the column efficiency is not less than 2500 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.
621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 20-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and measure the peak responses as directed for Procedure: the tailing factor is not more than 1.5; the resolution, R, between the prednisolone acetate peak and the fluorometholone acetate peak is not less than 2.0; the column efficiency is not less than 2500 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 30 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 prednisolone acetate (C23H30O6) in the portion of Ophthalmic Ointment taken by the formula:
50C(RU / RS)
in which C is the concentration, in mg per mL, of USP Prednisolone Acetate RS in the Standard preparation; and RU and RS are the ratios of the response of the prednisolone acetate peak area to that of the fluorometholone acetate peak obtained from the Assay preparation and the Standard preparation, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Ahalya Wise, M.S.
Senior Scientific Liaison 1-301-816-8161 |
(SM12010) Monographs - Small Molecules 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
|
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 3331