A: Prepare a Standard solution of USP Tobramycin RScontaining 3mg per mL.Separately apply 6µLof Ophthalmic Solution,6µLof the Standard solution,and 6µLof a mixture consisting of equal volumes of the two solutions to a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Proceed as directed for Identificationtest Aunder Tobramycin,beginning with “Place the plate in a suitable chromatographic chamber.”The specified results are obtained.

B: The retention time of the major peak for tobramycin in the chromatogram of the Derivatized assay preparationcorresponds to that in the chromatogram of the Derivatized standard preparation,as obtained in the Assay.

Mobile phase ,2,4-Dinitrofluorobenzene reagent,Tris(hydroxymethyl)aminomethane reagent,and Resolution solution—Prepare as directed in the Assayunder Tobramycin.

Standard preparation— Transfer about 33mg of USP Tobramycin RS,accurately weighed,to a 50-mLvolumetric flask,add 20mLof water and 1mLof 1Nsulfuric acid,and swirl to dissolve.Dilute with water to volume,and mix.Transfer 10.0mLof this solution to a second 50-mLvolumetric flask,dilute with water to volume,and mix.This solution contains about 0.132mg of USP Tobramycin RSper mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 6mg of tobramycin,to a 50-mLvolumetric flask,dilute with water to volume,and mix.

Derivatization procedure— Proceed as directed in the Assayunder Tobramycin,except to use 5.0mLeach of the Standard preparationand the Assay preparation,instead of 4.0mLof each.

Chromatographic system— Proceed as directed in the Assayunder Tobramycin,except to use a 4-mm ×15-cm column and to maintain the column temperature at 40.

Procedure— Proceed as directed in the Assayunder Tobramycin.Calculate the quantity,in mg,of tobramycin (C18H37N5O9)in each mLof the Ophthalmic Solution taken by the formula: in which Vis the volume,in mL,of Ophthalmic Solution taken to prepare the Assay preparation;and the other terms are as defined therein.