Printed on: Mon Aug 02 2021, 06:45:19 PM Official Status: Currently Official on 02-Aug-2021 DocId: 1_GUID-9A5270B7-0F44-4340-B439-8246D888119E_2_en-US

(EST)
Printed by: Le Tran Official Date: Official Prior to 2013 Document Type: USP @2021 USPC
1

Sample solution: Nominally 0.12 mg/mL of tobramycin

Tobramycin Ophthalmic Solution from Ophthalmic Solution in water
DEFINITION Derivatized standard solution, Derivatized sample
Tobramycin Ophthalmic Solution contains the equivalent of solution, and Blank solution: Proceed as follows. Heat all
NLT 90.0% and NMT 120.0% of the labeled amount of solutions at the same temperature and for the same
tobramycin (C18H37N5O9). It may contain one or more duration of time as indicated. Move all flasks to and from
the 60° constant temperature bath at the same time.
suitable buffers, dispersants, preservatives, and tonicity
To separate 50-mL volumetric flasks transfer 5.0 mL of the
agents.
Standard solution, 5.0 mL of the Sample solution, and
IDENTIFICATION 5.0 mL of water. To each flask add 10 mL of Solution A and
• A. THIN-LAYER CHROMATOGRAPHY 10 mL of Solution C, shake, and insert the stopper. Place
Diluent: Butyl alcohol and pyridine (100:1) the flasks in a constant temperature bath at 60 ± 2°, and
Standard solution: 3 mg/mL of USP Tobramycin RS in heat for 50 ± 5 min. Remove the flasks from the bath, and
water allow to stand for 10 min. Add acetonitrile to about 2 mL
Sample solution: Ophthalmic Solution below the 50-mL mark, allow to cool to room
Solution A: Standard solution and Sample solution (1:1) temperature, then dilute with acetonitrile to volume. The
Chromatographic system solutions thus obtained are the Derivatized standard
(See Chromatography á621ñ, Thin-Layer Chromatography.) solution, the Derivatized sample solution, and the Blank
Adsorbent: 0.25-mm layer of chromatographic silica gel solution, respectively.
mixture System suitability stock solution: 0.24 mg/mL of
Application volume: 6 µL p-naphtholbenzein in acetonitrile. Prepare freshly.
Developing solvent system: Methanol, chloroform, and System suitability solution: Transfer 2 mL of the System
suitability stock solution to a 10-mL volumetric flask, dilute

al
ammonium hydroxide (60:25:30)
Spray reagent: 10 mg/mL of ninhydrin in Diluent with Derivatized standard solution to volume, and use
Analysis promptly.
Samples: Standard solution, Sample solution, and Solution A Chromatographic system
Apply the Standard solution, the Sample solution, and (See Chromatography á621ñ, System Suitability.)
Solution A to the plate. Place the plate in a suitable Mode: LC
chromatographic chamber, and develop the
chromatogram in the Developing solvent system until the
solvent front has moved about three-fourths of the
ci Detector: UV 365 nm
Column: 4-mm × 15-cm; packing L1
Column temperature: 40°
length of the plate. Remove the plate from the chamber, Flow rate: 1.2 mL/min
allow the solvent to evaporate, and heat the plate at 110° Injection volume: 20 µL
ffi
for 15 min. Immediately locate the spots on the plate by System suitability
spraying it with Spray reagent. Samples: Derivatized standard solution and System
Acceptance criteria: Tobramycin appears as a pink spot, suitability solution
and the RF values of the spots of the Sample solution and of [NOTE—The relative retention times for
Solution A, respectively, correspond to those of the Standard p-naphtholbenzein and tobramycin are about
solution. 0.6 and 1.0, respectively.]
O

• B. The retention time of the major peak of the Derivatized Suitability requirements
sample solution corresponds to that of the Derivatized Resolution: NLT 4.0 between p-naphtholbenzein and
standard solution obtained as directed in the Assay. tobramycin, System suitability solution
Relative standard deviation: NMT 2.0%, Derivatized
ASSAY standard solution
• PROCEDURE Analysis
Mobile phase: Dissolve 2.0 g of tris(hydroxymethyl) Samples: Derivatized standard solution, Derivatized sample
aminomethane in 800 mL of water. Add 20 mL of 1 N solution, and Blank solution
sulfuric acid, and dilute with acetonitrile to obtain 2000 mL Use the Blank solution to identify the solvent and reagent
of solution. Cool, and pass through a filter of 0.2-µm or finer peaks.
pore size. Calculate the percentage of the labeled amount of
Solution A: 10 mg/mL of 2,4-dinitrofluorobenzene in tobramycin (C18H37N5O9) in the portion of Ophthalmic
alcohol. This solution may be used for 5 days if refrigerated Solution taken:
when not in use.
Solution B: 15 mg/mL of tris(hydroxymethyl) Result = (rU/rS) × (CS/CU) × P × F × 100
aminomethane in water. This solution may be used for 1
month if refrigerated when not in use. rU = peak area of tobramycin from the Derivatized
Solution C: 3 mg/mL of tris(hydroxymethyl)aminomethane sample solution
prepared as follows. Transfer 40 mL of Solution B to a rS = peak area of tobramycin from the Derivatized
200-mL volumetric flask. Add dimethyl sulfoxide while standard solution
mixing, and dilute with dimethyl sulfoxide to volume. Use CS = concentration of USP Tobramycin RS in the
this reagent within 4 h. If kept immersed in an ice-water Standard solution (mg/mL)
bath below 10°, the reagent may be used for up to 8 h. CU = nominal concentration of tobramycin in the
Standard stock solution: 0.66 mg of USP Tobramycin RS Sample solution (mg/mL)
prepared as follows. Transfer 33 mg of USP Tobramycin RS P = potency of tobramycin in USP Tobramycin RS
into a 50-mL volumetric flask. Add 20 mL of water, 1 mL of (µg/mg)
1 N sulfuric acid, and mix. Dilute with water to volume. F = conversion factor, 0.001 mg/µg
Standard solution: 0.132 mg/mL of USP Tobramycin RS
from Standard stock solution in water Acceptance criteria: 90.0%–120.0%

https://online.uspnf.com/uspnf/document/1_GUID-9A5270B7-0F44-4340-B439-8246D888119E_2_en-US 1/2
Printed on: Mon Aug 02 2021, 06:45:19 PM Official Status: Currently Official on 02-Aug-2021 DocId: 1_GUID-9A5270B7-0F44-4340-B439-8246D888119E_2_en-US
(EST)
Printed by: Le Tran Official Date: Official Prior to 2013 Document Type: USP @2021 USPC
2

SPECIFIC TESTS • USP REFERENCE STANDARDS á11ñ

• STERILITY TESTS á71ñ: It meets the requirements in Test for USP Tobramycin RS
Sterility of the Product to Be Examined, Membrane Filtration.
• PH á791ñ: 7.0–8.0
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
avoid exposure to excessive heat.

al
ci
ffi
O

https://online.uspnf.com/uspnf/document/1_GUID-9A5270B7-0F44-4340-B439-8246D888119E_2_en-US 2/2