Identification—
A:
Thin-Layer Chromatographic Identification Test 
201
—
Test solution—
Transfer a quantity of Injection, equivalent to about 5 mg of dexamethasone, to a 50-mL separator, add 10 mL of water, and extract with two 20-mL portions of chloroform. Filter the lower layers through chloroform-saturated cotton into a 50-mL conical flask, and evaporate to dryness. Dissolve the residue in 10 mL of chloroform.
Developing solvent system:
a mixture of methylene chloride and methanol (180:16).
Procedure—
Visualize the spots using a 1 in 5 solution of p-toluenesulfonic acid in a mixture of alcohol and propylene glycol (9:1), followed by heat.
B:
The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of water and acetonitrile (70:30). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
System suitability solution—
Prepare a solution in
Mobile phase containing in each mL about 0.3 mg of
USP Dexamethasone RS, 1.35 mg of benzyl alcohol, 0.27 mg of methylparaben, and 0.03 mg of propylparaben.
Standard preparation—
Quantitatively dissolve an accurately weighed amount of
USP Dexamethasone RS in methanol to obtain a stock solution having a known concentration of about 7.5 mg per mL. Transfer 4.0 mL to a 100-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain a solution having a known concentration of about 0.3 mg of
USP Dexamethasone RS per mL.
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 30 mg of dexamethasone, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 2 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.4 for benzyl alcohol, 0.5 for methylparaben, 1.0 for dexamethasone, and 1.4 for propylparaben; and the resolution,
R, between the neighboring peaks for benzyl alcohol and methylparaben, methylparaben and dexamethasone, and dexamethasone and propylparaben is not less than 3. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses for dexamethasone. Calculate the quantity, in mg, of dexamethasone (C
22H
29FO
5) in each mL of the Injection taken by the formula:
100(C / V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Dexamethasone RS in the
Standard preparation; V is the volume, in mL, of Injection taken; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
