Identification—
Transfer a volume of Ophthalmic Suspension, equivalent to about 2.5 mg of dexamethasone, to a test tube, add 5 mL of chloroform, and shake. Centrifuge, and apply 10 µL of the chloroform layer and 10 µL of a Standard solution of
USP Dexamethasone RS in chloroform containing 500 µg per mL on a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatogram in
Solvent A as directed under
Single-steroid Assay 511. Mark the solvent front, and locate the spots on the plate by spraying with a 1 in 5 solution of
p-toluenesulfonic acid in a mixture of 9 volumes of alcohol and 1 volume of propylene glycol, and heating until spots appear. The
RF value of the principal spot obtained from the solution under test corresponds to that obtained from the Standard solution.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of water and acetonitrile (60:40). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Dexamethasone RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 0.12 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Suspension, freshly mixed and free from air bubbles, equivalent to about 3 mg of dexamethasone, to a 25-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 packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak response as directed for
Procedure: the column efficiency determined from the analyte peak is not less than 1750 theoretical plates; the tailing factor for the analyte peak is not more than 3.0; and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure—
Separately inject equal volumes (about 10 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 C
22H
29FO
5 in each mL of the Ophthalmic Suspension taken by the formula:
25(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 Ophthalmic Suspension taken; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
