Identification—
A:
Apply 10µLof Ophthalmic Solution and 10µLof a Standard solution containing 5mg per mLof
USP Gentamicin Sulfate RSin water to a thin-layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and in a paper-lined tank develop the chromatogram in a solvent system consisting of the lower phase mixture of dichloromethane,methanol,and ammonium hydroxide (1:1:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the plate to air-dry.Locate the spots on the plate by placing it in a tank containing about 15g of iodine crystals for 15minutes:the
RFvalues of the three principal spots obtained from the test solution correspond to those obtained from the Standard solution.
B:
The retention time of the major peak obtained in the chromatogram of the
Assay preparationcorresponds to that of the
Standard preparation,both relative to the internal standard,as obtained in the
Assay for betamethasone acetate.
Assay for gentamicin—
Proceed as directed for gentamicin under
Antibiotics—Microbial Assays á81ñ,using an accurately measured volume of Ophthalmic Solution diluted quantitatively and stepwise with
Buffer No.3to obtain a
Test Dilutionhaving a concentration assumed to be equal to the median dose level of the Standard.
Assay for betamethasone acetate—
Mobile phase—
Prepare a filtered and degassed mixture of water and acetonitrile (8:7).Make adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Internal standard solution—
Dissolve a quantity of o-phenylphenol in methanol to obtain a solution containing about 0.55mg per mL.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Betamethasone Acetate RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 0.45mg per mL.Transfer 2.0mLof this solution to a 10-mLvolumetric flask,add 1.0mLof
Internal standard solution,dilute with methanol to volume,and mix to obtain a solution having a known concentration of about 0.09mg of
USP Betamethasone Acetate RSper mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 2mg of betamethasone acetate,to a 10-mLvolumetric flask.Dilute with methanol to volume,and mix.Transfer a portion of this solution to a centrifuge tube,and centrifuge.Transfer 4.0mLof the clear supernatant to a 10-mLvolumetric flask.Add 1.0mLof Internal standard solution,dilute with a mixture of methanol and water (1:1)to volume,and mix.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-mm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the
Standard preparation,and record the peak responses as directed under
Procedure:the relative retention times are about 1.3for
o-phenylphenol and 1.0for betamethasone acetate;the resolution,
R,between the betamethasone acetate and
o-phenylphenol peaks is not less than 3.9;and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of betamethasone acetate (C
24H
31FO
6)in each mLof the Ophthalmic Solution taken by the formula:
25(C/V)(RU/RS),
in which
Cis the concentration,in mg per mL,of
USP Betamethasone Acetate RS,calculated on the anhydrous basis,in the
Standard preparation;
Vis the volume,in mL,of Ophthalmic Solution taken to prepare the
Assay preparation;and
RUand
RSare the ratios of the betamethasone acetate peak response to the internal standard peak response obtained from the
Assay preparationand the
Standard preparation,respectively.